Università degli studi di Cagliari Rettore: Prof. Giovanni ...
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Università degli Studi di Cagliari Dottorato di Ricerca in Chimica, XX ciclo
NUCLEAR MAGNETIC RESONANCE OF NUCLEAR MAGNETIC RESONANCE OF NUCLEAR MAGNETIC RESONANCE OF NUCLEAR MAGNETIC RESONANCE OF 129129129129XeXeXeXe
USED AS A PROBE USED AS A PROBE USED AS A PROBE USED AS A PROBE
FOR THE STRUCTURAL CHARACTERIZATION OFFOR THE STRUCTURAL CHARACTERIZATION OFFOR THE STRUCTURAL CHARACTERIZATION OFFOR THE STRUCTURAL CHARACTERIZATION OF
POROUS MATERIALS AND PROTEINSPOROUS MATERIALS AND PROTEINSPOROUS MATERIALS AND PROTEINSPOROUS MATERIALS AND PROTEINS
Supervisor Candidate
Prof. Mariano Casu Roberto Anedda
January 2008
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Acknowledgments
I would like to spend few words to thank all the people who have helped and
guided me throughout the doctorate. Each of the people I have worked with has given an
important contribution to the work described here.
First of all I would like to thank my supervisor, Prof. Mariano Casu, for his
constant encouragement, guidance and freedom. His continuous support and the diligent
reviewing of the manuscript have been much appreciated.
All the people I have worked with in Cagliari have patiently discussed with me all
the details of the experimental work and scientific concepts related to the systems
studied. In this regard, I have to sincerely thank the groups of biologists from the
Department of Applied Sciences in Biosystems of the University of Cagliari. In
particular, Drs. Antonella Fais, Benedetta Era, Simona Porcu and Prof. Marcella Corda
for their constant and meticulous efforts in explaining a number of complex concepts of
biochemistry of myoglobins and hemoproteins in general. Thanks to Antonella, Simona
and Benedetta also for their truthful friendship.
Prof. Giovanni Floris, Drs. Rosaria Medda, Alessandra Padiglia, Anna Mura,
Silvia Longu and Francesca Pintus for their collaboration, guidance and for the helpuful
discussions on Amine Oxidases.
A significant work has been done thanks to the collaboration with Prof. Paolo
Ruggerone and Dr. Matteo Ceccarelli, two researchers of CNR-INFM SLACS,
Department of Physics, University of Cagliari and CNR-INFM CRS DEMOCRITOS,
SISSA, Trieste. Their extensive knowledge of biophysical processes together with their
experience in molecular dynamics simulations and the very helpful comments and
discussions regarding the whole work has been essential to the writing of this final
manuscript.
The Materials Structure and Function Group within the Steacie Institute for
Molecular Science of the National Research Council of Canada is greatly acknowledged.
First of all I want to thank John Ripmeester for the opportunity he has given me to
work at NRC Canada and to activate a fruitful scientific collaboration.
Among the people I have worked with in Canada, Dima Soldatov was one of the
most important teachers and mentors.
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I also show my appreciation to Igor Moudrakovski who has always patiently
answered my questions concerning NMR, for the careful review of the manuscript on
Xenon and dipeptides, for his kind friendship while I was in Canada. My
acknowledgments go also to Chris Ratcliffe for his suggestions on solid state NMR
measurements.
Steve Lang and Gennady Ananchenko were great office mates and friends, their
help and hospitality has been important and really appreciated.
My thanks go also to Long Li Lai from Taiwan, for his help in studying
dendrimers and viologen inclusion compounds, a work which is still in progress.
I show my gratitude to Kostia Udatchin for helping me with X-ray
crystallographic measurements.
Robin, Satoshi and his family, Rasnish and his wife, Phil Brown, Shane Pawsey,
were pleasant and amusing buddies.
I would like also to express my gratitude to Michaela Pojarova for her guidance
and for introducing me to Canadian life.
Finally, I have to thank the people closest to me, with whom I spend most of my
lifetime; my parents for their constant support and encouragement, this work would not
have been possible without their gratifying words and continuous support; my brother
and my sister since they have been very supportive and I learnt a lot from them; all my
friends and collegues in Cagliari; at last but not the least my sweet love, she is the most
beautiful source of motivation and perseverance.
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Preface (Italiano)
L’utilizzo dello Xenon ha conosciuto un enorme sviluppo negli ultimi anni.
E’ particolarmente affascinante a mio avviso notare oggi quali grandi progressi
abbia fatto la tecnica Xenon-NMR successivamente agli studi preliminari risalenti ai
primi anni ‘80.
L’applicazione di questa tecnica abbraccia oggi numerosi campi della scienza e
della tecnica: la ricerca fondamentale sui composti organici, inorganici e biologici sia allo
stato solido che in soluzione, la caratterizzazione dei materiali solidi porosi che trovano
impiego nell’industria e nell’alta tecnologia, le applicazioni in campo medico su sistemi
in vitro e in vivo.
La scienza dei materiali, prima fra tutte, ha tratto numerosi vantaggi da questa
tecnica, come testimoniano le numerose pubblicazioni scientifiche di grande rilievo
riguardanti la caratterizzazione strutturale di catalizzatori, setacci molecolari, dispositivi
per l’immagazzinamento dei gas, idrati, clatrati e composti di inclusione, materiali
nanostrutturati e nanocompositi, materiali stimuli-responsive, sistemi per drug delivery.
Sebbene le dimensioni, il volume dei pori e l’area superficiale di un materiale poroso
sono determinabili mediante TEM e principalmente attraverso l’adsorbimento di azoto
BET, queste tecniche non forniscono sufficienti informazioni sulla connettività e struttura
delle superfici interne dei pori.
In campo biologico, l’utilizzo dello Xenon come sonda ha permesso
l’individuazione e la caratterizzazione strutturale di cavita’ all’interno di proteine ed
enzimi fornendo importanti indicazioni sul processo di diffusione dei ligandi e substrati
in bio-macromolecole e sulla relazione struttura-funzione in sistemi biologici
relativamente complessi.
Il settore medico diagnostico ha giovato soprattutto degli sviluppi della tecnica
Xenon-MRI per l’acquisizione di immagini. In particolare, l’uso di tecniche che
permettono di incrementare il segnale NMR dello Xenon di diversi ordini di grandezza
(iperpolarizzazione) ha permesso di ottenere significativi e promettenti risultati nello
studio dei polmoni, nell’acquisizione di immagini angiografiche, nello studio del cervello
e nella diagnosi precoce dei tumori. Inoltre, come e’ noto, lo Xenon viene usato come
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anestetico generale, ma tuttoggi il meccanismo molecolare di azione di questi agenti e’
elusivo e ulteriori studi a riguardo sono necessari.
Il mio dottorato e’ stato svolto prevalentemente all’Universita’ di Cagliari con la
supervisione di Prof Mariano Casu. Una collaborazione con il National Research Council
of Canada mi ha permesso di lavorare per un anno nei laboratori dello Steacie Institute
for Molecular Science di Ottawa. Questa importante esperienza di collaborazione
scientifica, ancora attiva, ha avuto per supervisore John Ripmeester, uno dei pionieri di
questa tecnica, che guida un gruppo (Materials Structure and Function Group) di oltre 30
ricercatori di varia estrazione scientifica.
Questa tesi e’ organizzata in quattro capitoli che trattano i diversi sistemi studiati
durante il dottorato. In particolare, il primo capitolo introduce i concetti generali sui quali
la tecnica Xenon-NMR si basa e elenca alcuni obiettivi del progetto. La descrizione dei
sistemi studiati e’ riportata nel secondo capitolo. Nel terzo capitolo, Risultati e
Discussione, sono descritti e commentati i risultati sperimentali ottenuti. Alla fine di ogni
sottocapitolo della sezione Risultati e Discussione si traggono alcune conclusioni e
considerazioni generali, in particolare cercando di sottolineare le novita’ introdotte da
questo lavoro e discutere possibili sviluppi futuri della tecnica Xenon-NMR. Il quarto
capitolo e’ lasciato alla descrizione dei metodi sperimentali usati per preparare i campioni
analizzati, per acquisire i dati e della tecnica di iperpolarizzazione dello Xenon.
La tesi e’ scritta in lingua inglese, ormai diventata la lingua ufficiale della
comunicazione scientifica, con la speranza di permettere la lettura del lavoro ad un
gruppo piu’ numeroso ed eterogeneo di ricercatori.
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Table of Contents
Chapter I
Introduction …………………………………………………………………………1
1.1 Generalities………………………………………………………....……2
1.2 Objectives…………………………………………………………..…….3
1.2.1 Proteins……………………………………………………….……..3
1.2.2 Microporous crystalline dipeptides……………….…………………5
1.3 NMR properties of Xenon…………………………….…………………6
1.4 Bibliography……………………………………………….……………11
Chapter II
The systems studied: void space in biomolecules……...……………14
2.1 Myoglobins: suitable model systems…………………………..………15
2.1.1 Function……………………………………………………………15
2.1.2 Structure……………………………………………………………16
2.1.3 Cavities in myoglobins…………………………………….………20
2.2 Copper-containing Amine Oxidases (AOs)……………..…………….24
2.2.1 Structure ………………………………………………..………….24
2.2.2 Hydrophobic cavities in AOs……………………………...……….26
2.2.3 Biological function: AOs’ catalytic process………………….……27
2.3 Biomaterials: microporous crystalline dipeptides……………...…….31
2.3.1 Developments of microporous materials……………………..……31
2.3.2 Characterization of bioorganic materials…………………………..32
2.3.3 Microporous dipeptides structure………………………….………36
2.3.4 129Xe NMR of dipeptides microporous crystals……………...…….39
2.4 Bibliography……………………………………………………….……41
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Chapter III
Results and discussion………………………………………………………….48
3.1 Myoglobins………………………………………………………...……49
3.1.1 129Xe NMR measurements in solutions of low-spin
(Fe3+ S=1/2) cyano-metmyoglobins…………………………….….52
3.1.2 129Xe NMR relaxation measurements
of CNMb solutions…………………………………………………62
3.1.3 1H NMR chemical shift in CNMbs from horse and pig……………70
3.1.4 NOE measurements used as a tool
to further assess His93 rotation relative to heme………………..…79
3.1.5 Thermodynamics of Xenon binding to
cyano-metmyoglobins from Xenon-induced 1H NMR
chemical shift variations……………………………………...……84
3.1.6 Myoglobins: CONCLUSIONS………………………………….…87
3.2 Copper containing Amine Oxidases enzymes:
Xenon-induced reactions………………………………………….……89
3.2.1 Lens Esculenta Amine oxidases (LSAO) in solution: 129Xe NMR chemical shifts …………………………………..……89
3.2.2 Spectral changes in the UV-vis region of LSAO solutions
induced by substrates and Xenon………………………………..…91
3.2.3 Involvement of a lysine residue
in the intra-molecular catalytic mechanism of LSAO………..……99
3.2.4 129Xe NMR of PKAO, ELAO and LSAO solutions…………...…105
3.2.5 Spectroscopic features induced by amine substrates
and Xenon in several AOs……………………………………..…107
3.2.6 Copper containing Amine Oxidases: CONCLUSION………...…113
3.3 Microporous Crystalline Dipeptides…………………………………115
3.3.1 Variable Temperature continuous flow HP 129Xe NMR:
General spectral features…………………………………….……115
3.3.2 Temperature dependence of the 129Xe CSA tensor……………..119
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3.3.2.1 Effect of channels loading on the CSA ………………...…124
3.3.2.2 Presence of specific sites (niches)……………………...…125
3.3.2.3 Effect of helicity and diameter of the channels on CSA…..125
3.3.2.4 Dynamics of Xe in the cross section of the pores
and CSA of 129Xe NMR signal…………………………….127
3.3.2 129Xe NMR isotropic chemical shifts
as a function of temperature………………………………………128
3.3.3 Thermodynamics of adsorption: the Langmuir model……………132
3.3.4 Signal intensities………………………………………………….138
3.3.5 Aging of dipeptide samples………………………………………143
3.3.6 Thermodynamics of adsorption in nanochannels.
General remarks (summary)…………………………………...…146
3.3.7 Dipeptides: CONCLUSIONS………………………………….…152
3.4 Bibliography……………………………………………………...……154
Chapter IV
Materials and methods……………………………………………….………164
4.1 Hyperpolarized Xenon: solving sensitivity problems………………...…165
4.1.1 Continuous-flow measurements………………………………..…167
4.1.2 Advantages (and drawbacks) implied in the use of
hyperpolarized and thermally polarized Xenon………………..…169
4.2 Myoglobins...................................................................................................171
4.3 Microporous Dipeptides..............................................................................174
4.4 Copper containing Amine Oxidases...........................................................175
4.5 Bibliography………………………………………………………….……178
Papers Published during the doctorate ............................................................180
CHAPTER I - INTRODUCTION
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1.1 Generalities
The scientific and technologic relevance of using Xenon atoms as probes for the
characterization of void spaces comprised within biological macromolecules and/or
porous materials appears clear when the extensive literature on this topic is considered.
It is even more evident from the most recent achievements the usefulness of combining
the sensitivity of Xenon and the versatility of a spectroscopic technique such as Nuclear
Magnetic Resonance in order to deeply characterize both structures and dynamics
involved in the host-guest systems.
Early studies proposed, discussed and demonstrated the usefulness of 129Xe NMR
in the characterization of void space in systems of different nature1,2. This technique is
useful in studying porous materials for gas sensing3, purification4, separation and
storage5,6, catalysis processes7,8. It has medical applications as well, as it allows for
acquisition of images of lungs, heart, kidneys and brain9-12 and helps in the challenging
studies that concern the understanding of the molecular mechanisms of the action of
general anesthetics13,14.
Recently, there has been renewed interest in Xenon NMR in view of using the
resonance of Xenon in the structural study of proteins15-24. In biochemistry, it is useful
for instance in characterizing ligand binding and diffusion within cavities of
biomolecules, just to mention one of its many applications in this field.
Moreover, among the most intriguing results, recent application of Xenon
biosensors made of conveniently functionalized Cryptophane-Xenon complexes has
allowed target-specific detections of specific proteins and oligonucleotide sequences by
means of Magnetic Resonance Spectroscopy and Imaging25-27.
Among the many advantages of combining Xenon as a biomolecular probe and
NMR as spectroscopic technique is that NMR parameters of nuclei belonging to both the
probe and the matrix can be studied. It has been demonstrated that, in fact, important
information on host-guest interactions in Xenon complexes can be derived both from
direct observation of 129Xe NMR signals and from Xenon-induced chemical shift changes
in 1H, 13C and 15N nuclei of the host compounds as well24,28-30. Moreover, polarization
transfer from hyperpolarized Xenon to protons by Spin Polarization-Induced Nuclear
Overhauser Effect (SPINOE) can be studied in suitable systems15,31-36. These latter
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experiments, beyond confirming the results obtained by directly observing the guest
Xenon atoms, provide site-specific information, which is particularly valuable especially
when exchange processes average 129Xe NMR results and also when particularly complex
systems, such as large biomolecules, are under study.
1.2 Objectives.
The studies that will be described in the following have been devoted to test the ability of
Xenon as an efficient probe of void spaces. We have decided to approach the problem by
analyzing both porous crystals in the solid state and very flexible molecules such as
proteins in solution.
1.2.1. - Proteins
a) Myoglobin (Mb), a globular protein, has been selected to probe the ability of Xenon to
extract important information on their structure and function. Studying Xenon binding to
a model compound such as myoglobin represents a useful approach but at the same time
challenging due to the presence of different interaction sites within the protein, beside the
heme iron. The presence of four cavities37 has been directly evidenced by X-ray
diffraction on sperm whale Mb crystals pressurized by Xenon.
However, this early studies were carried out only on Mb crystals, which suffer of
the drawbacks related to the lower flexibility of the overall protein compared to the
protein in solution. Previous studies of Xe-Mb complexes in solution have been
performed on horse Myoglobins, confirming the XRD studies19,.
Clearly, hints on the structure of cavities in solution and on Xe-protein affinity
can be substantiated by comparatively studying myoglobins of different species as in a
recent NMR investigation on the pig and horse metmyoglobins38 (MMbs). There, the
combined use of the 129Xe chemical shift and the 129Xe spin lattice relaxation rate as a
function of Xenon and protein concentration has unraveled the influence of the structure
and/or hydrophobicity of a cavity on its Xenon occupancy.
CHAPTER I - INTRODUCTION
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To exploit more completely the peculiar properties of the NMR technique aiming
at a deeper description of the Mb cavities, we combine in the present study the analysis of 129Xe NMR chemical shifts and relaxation rates to an accurate and appropriate 1H NMR
characterization of the proteins. In order to substantiate and complete the conclusions
made by Corda et al.38, we extend the comparison between pig and horse Mbs to two
more Mbs, those of sheep and rabbit. In particular, we focus our attention on the protein
in the low-spin cyano form (CNMb), of which several 1H signals of the residues in the
active site have been already assigned.
A final and promising issue of the present work concerns the possible use of
specific Xenon-cavities interactions as probe to monitor the displacements of the
individual protons induced by the Xenon binding. The host-guest interaction is a subtle
and not negligible aspect of a spectroscopic technique, since it determines the accuracy
and validity of the data analysis. In particular, we examine the behaviour of the residues
in the proximal and distal cavities of pig and horse Mb and verify the potential
occupation of Xenon in the cavities close to the myoglobin active site.
b) It is generally believed that Xenon atoms can induce structural changes in some
of the cavities or channels that they are bound to, both in solution39 and in the solid
state40. Xenon has been used as a probe for dioxygen-binding cavities in copper AOs by
recording XRD data under pressure of Xenon gas41-43. Here is discussed our investigation
on the binding of Xenon to purified lentil (Lens esculenta) seedling copper/TPQ-amine
oxidase in solution. Upon pressurization with 10 atm of Xenon gas the enzyme can
generate the free radical intermediate in the absence of substrates outlining a process that
probably involves a lysine residue at the active site. The study has been extended to
highly purifed AOs from various sources and our results strongly support the hypothesis
that a lysine residue is implicated in the catalytic mechanism of plant enzymes.
CHAPTER I - INTRODUCTION
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1.2.2. - Microporous crystalline dipeptides
This study was aimed at the detailed characterization of sorption in the
microporous dipeptides. In particular, fundamental thermodynamic parameters and
molecular-scale peculiarities of the sorption process were in the focus and how these
characteristics relate to the structure of the micropores. Standard approach including the
determination of sorption isotherms appeared to be hardly suitable for the materials under
study. Thermodynamic parameters may be extracted from such data provided a series of
sorption isotherms for each material is available. At the same time, the materials of this
study appeared to change upon aging and the long experimental times required for the use
of standard procedures would have resulted in unreliable results. A fast method was thus
necessary but which would give, at the same time, reliable quantitative information.
Another requirement to the method was to be able to monitor changes on the molecular
level occurring in the pores. As it was demonstrated previously40, the pores are very
flexible and the pore structure revealed in the crystal structure examination of an empty
sorbent will not account for its sorption behavior as the pores become loaded with the
guest species.
In order to overcome the above problems, we have developed and demonstrated
here an entirely different, new approach based on the determination of sorption isobars
using continuous-flow hyperpolarized 129Xe NMR. Using this approach made possible
the first systematic study of gas sorption in microporous peptides with quantitative
thermodynamic description of the process and detailed analysis of specificities occurring
on molecular level between the flexible host matrix and the included guest species.
In this work, variable-temperature 129Xe NMR experiments using a continuous
flow of hyperpolarized Xenon were conducted for the eight microporous dipeptides. It is
demonstrated that quantitative information on the thermodynamics of the sorption
process can be extracted from these experiments as well as comprehensive knowledge on
the sorption events occurring on the molecular scale level. The present study reveals the
relation of the observed NMR parameters of absorbed Xenon with the thermodynamics of
sorption, geometry and dynamics of the micropores, and the structural features of the
cavity-Xenon intraporous association.
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1.3 NMR properties of Xenon
Any distortion of the large electronic cloud of 129Xe is felt directly at the nucleus
and consequently affects the observed NMR parameters. The most sensitive parameter is
undoubtedly the chemical shift. The wide NMR spectral window typical of non-ligated 129Xe (∼350 ppm, which becomes ∼7500 ppm when also Xenon compounds are taken
into account) allows for detailed analysis of local environment around the observed
Xenon nuclei and facilitates simultaneous detection of 129Xe in different chemical
environments. Together with the ideal physico-chemical properties of Xenon, such as
inertness and large polarizability of the spherically symmetrical electronic cloud, it
should be pointed out that NMR sensitivity of naturally occurring 129Xe is quite good.
Due to the relatively high natural abundance of the isotope 129Xe, Xenon is approximately
32 times easier to observe than 13C, neglecting differences in relaxation times, and has
about 10-2 times the sensitivity of proton. Nevertheless, Xenon-NMR suffers of the
serious problem which is typical of all the nuclei that are traditionally studied by Nuclear
Magnetic Resonance: the low sensitivity that derives from low thermal polarization. In
order to face this problem, hyperpolarization techniques have been recently developed
which have allowed for obtainment of up to six orders of magnitude signal enhancement
by optical pumping [see Section 4.1].
The suitability of Xenon as a biomolecular probe is due to many of its physical
and chemical properties. Xenon is a monoatomic, non-toxic, chemically inert gas, small
enough (Van der Waals radius ∼ 2.2 Å) to be able to probe even very narrow pores and
cavities. Due to its hydrophobic properties, it is well suited to locate and explore
hydrophobic regions such as cavities and channels in biological systems; its large and
extremely polarizable electronic cloud makes it very sensitive to its local physical
environment: in particular, this sensitivity is readily detectable by analyzing NMR
spectroscopic parameters (chemical shift, relaxation times, line shapes and possibly
chemical shift anisotropy) which are mostly influenced by the atoms in the proximity of
observed nuclei.
Naturally occurring Xenon has nine stable isotopes, only two of which, 129Xe and 131Xe, have non-zero nuclear spin I, and are therefore detectable by means of Nuclear
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Magnetic Resonance spectroscopy. 129Xe has I=1/2 and natural abundance of 26.4%,
while 131Xe has I=3/2 and natural abundance of 21.2%.
The observed NMR parameters are simultaneously influenced by several
concurrent factors. The chemical shielding is basically influenced by two contributions: a
diamagnetic contribution σd, merely determined by the fundamental electronic state of
the atom and a paramagnetic part, which depends on the excited electronic states, i.e. on
the symmetry of the valence electronic shell.
The paramagnetic contribution is zero when a spherically symmetrical
distribution characterizes the valence electronic shell of Xe atoms, which can be observed
only for the ideal situation of isolated Xe atoms (Xe in gaseous state at a pressure
extrapolated to zero). Due to the large and easily polarizable Xe electronic cloud, the
paramagnetic contribution to the chemical shielding is expected to play a significant role
in determining the observed chemical shift of Xenon when it is interacting physically
with its environment.
This high sensitivity makes Xenon a very useful probe for the characterization of
systems which it can interact with. However, while the central idea of early researchers
was to exploit Xenon’s sensitivity to get detailed structural and chemical information on
the systems studied, it soon appeared clear that the simultaneous presence of different
factors influencing in a variable manner the observed signal often leads to complex
outputs, which are sometimes difficult to be unambiguously interpreted.
Let us concentrate first on Xenon gas. Since the discovery of gaseous Xenon44,45
subsequent studies showed that over a wide range of densities the shift is expressed by
virial expansion of the Xe density46. The most precise values of virial coefficients were
obtained by Jameson et al.47.
Early studies concentrated on studying 129Xe NMR solutions in different solvents,
which clearly demonstrated the sensitivity of Xe chemical shift to physical environments.
For example, it was demonstrated by Jokisaari and coworkers48 although 129Xe gas to
solution shifts are linearly related to 13C gas to solution shifts of methane in the same
solvents, the entity of chemical shift variations proved that 129Xe is 27.1 times more
sensitive to physical interaction with solvent. As the shifts of dissolved gas merely
depend on Van der Waals interaction, they are reasonably expected to have higher values
CHAPTER I - INTRODUCTION
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for heavy atoms. 129Xe NMR of Xenon dissolved in a number of solvents and organic
and bioorganic ligands have been reviewed by Reisse49.
If we assume now that Xe interacts with more complex systems, where more than
one site (target) is available for Xenon atoms, (i.e. many chemically and or structurally
different surfaces Si are present), the observed 129Xe NMR chemical shift (δi) will be an
average between all the possible situations, weighted for the frequency of collisions.
δi = (Term for chemical nature of Si)·(Term for frequency of Xe-Si collisions)
The observed spectrum, therefore, will depend on the lifetime of Xe on each adsorption
site and two limiting conditions can be discussed:
If the lifetime of Xe on each Si is long in the NMR time scale, the spectrum will
be constituted by as many components (each with chemical shift δi) as there are target
types, the intensity being related to the number of targets of each type in the samples.
If instead the lifetime of Xe in each site is very short (fast exchange condition) all the
signals coalesce and the spectrum will therefore consist of only one component whose
chemical shift depends on the values of δi each weighted by the probability αi of the Xe-
Si collision:
δobs = Σ αiδi with Σ αi=1 [1.1]
In general, we can consider the observed 129Xe NMR chemical shift as influenced
simultaneously by the following contributions, as proposed by Fraissard50,51:
δobs = δref + δs + δXe-Xe + δSAS + δE + δM [1.2]
where δref is the chemical shift of Xe gas at zero pressure, which is considered the
reference value and fixed to zero; δs is due to interaction between Xe and the surface of
the sample, thus it provides structural and chemical information on each site where the
Xe atoms interact with (dimensions and shape of cage/channels, ease of Xe diffusion etc);
δXe-Xe arises from the interaction of two or more Xe atoms in cages, channels or pores
CHAPTER I - INTRODUCTION
9
that can contain more than one Xe at the same time. This latter contribution evidently
depends on Xe density. Whenever strong adsorption sites (SAS) are present, Xe atoms
will spend a longer time in contact with them than the cage or channel walls, particularly
at low Xe concentrations.
δE and δM are related to the presence of electric and magnetic fields, that
sometimes arise from the presence of charged ions, paramagnetic metals and/or radical
species.
Longitudinal relaxation time of gaseous Xenon is in principle influenced, in a
homogeneous magnetic field, only by spin-rotation during collisions, according to the
relation
T1 ≈ 56/ρ [1.3]
Where ρ is Xe density in amagat and T1 is in hours. However, experimental results show
that in fact the measured T1 is generally less than the ideal value expressed by the
previous equation. This is basically due to collisions between Xe and the walls of the
sample. This problem was shown to be relevant when hyperpolarized Xenon is used, as
loss of polarization (i.e. longitudinal relaxation) causes considerable loss of the signal
previously enhanced by laser pumping. In this regard, it has been shown that pretreating
the pumping cell’s walls with polymeric coating (Surfasil-Pierce), T1 longer than 20
minutes can be obtained.
While the study of relaxation times has led to interesting achievements in the
characterization of different systems in solution, its employment for solid materials has
not provided the same results. It should be observed that although nuclear relaxation time
T1 of adsorbed Xenon should ideally provide interesting information about local Xenon
structure and dynamics, this study could be carried out reliably only on extremely pure
systems, as paramagnetic impurities are often present in real catalysts and other solid
porous systems. The effect of paramagnetic ions in enhancing nuclear relaxation is well
known, thus in presence of hyperfine coupling between unpaired electrons and nuclear
spins all the other possible sources of relaxation become negligible.
CHAPTER I - INTRODUCTION
10
While in solid materials the presence of paramagnetic sites may generate
unwelcome problems, in solution of paramagnetic proteins it can give very useful
information. Hemoglobin and myoglobin were the first two proteins shown to bind
Xenon. Hemoglobin is currently more difficult to study via NMR because of its large
size, but exploitation of Xe NMR on myoglobins, which is by now relatively well
characterized, has given important insight on the structure of Xe-myoglobin complexes.
An example of 129Xe NMR relaxation studies in myoglobins containing the heme iron ion
in the high spin (S=5/2) form have been discussed by Locci et al. and Corda et al.23,38,
who discussed a method to obtain Xe-Fe distances from the analysis of T1.
We describe here Xenon-binding systems and we correlate results obtained by
NMR and other techniques to describe structural and dynamical characteristics of each
system and to discuss similarities and differences between them.
The systems which will be described throughout this thesis are myoglobins and
peroxidases in solution and solid microporous crystalline dipeptides.
CHAPTER I - INTRODUCTION
11
1.4 Bibliography
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6. P. Sozzani, S. Bracco, A. Comotti, L. Ferretti, R. Simonutti Angew. Chem. Int.
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70(2), 1546-1552
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(1999), 42(6), 1137 – 1145
13. Eckenhoff RG, Johansson JS, Pharmacol. Rev (1997), 49(4), 343-367
14. JW Tanner, JS Johansson, PA Liebman, RG Eckenhoff Biochemistry (2001), 40,
5075-5080
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Acad. Sci. U. S. A. (1999), 96, 3664–3669
17. C.R. Bowers, V. Storhaug, C.E. Webster, J. Bharatam , A. Cottone III, R. Gianna,
K. Betsey, B.J. Gaffney, J. Am. Chem. Soc. (1999), 121, 9370–9377
18. A. Stith, T.K. Hitchens, D.P. Hinton, S.S. Berr, B. Driehuys, J.R. Brokeman, R.G.
Bryant, J. Magn. Reson. (1999), 139, 225– 231;
CHAPTER I - INTRODUCTION
12
19. S.M. Rubin, M.M. Spence, B.M. Goodson, D.E. Wemmer, A. Pines Proc. Natl.
Acad. Sci. (2000), 97, 3472–9475 ;
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Am. Chem. Soc. (2001), 123, 8616–8617;
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3, 812– 814;
24. S.M. Rubin, S.Y. Lee, E.J. Ruiz, A. Pines, D.E. Wemmer, J. Mol. Biol. (2002),
322, 425– 440
25. Schröder L, Lowery TJ, Hilty C, Wemmer DE, Pines A, Science (2006),
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26. Q. Wei, G.K. Seward, P.A. Hill, B. Patton, I. Dimitrov, N.N. Kuzma, I.J.
Dmochowski, J. Am. Chem. Soc., (2006), 128, 13274-13283;
27. V. Roy, T. Brotin, J.P. Dutasta, M.H. Charles, T. Delair, F. Mallet, G. Huber, H.
Desvaux, Y. Boulard, P. Berthault. ChemPhysChem (2007), 8(14), 2082-2085
28. Gröger C, Möglich A, Pons M, Koch B, Hengstenberg W, Kalbitzer HR, Brunner
E, J. Am. Chem. Soc. (2003), 125, 8726-8727;
29. TJ Lowery, SM Rubin, EJ Ruiz, A Pines DE Wemmer, Angew Chem Int Ed
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30. L Dubois , P Da Silva , C Landon , JG Huber , M Ponchet , F Vovelle , P
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13
35. Song, Y.-Q. Concepts Magn. Reson. (2000), 12, 6-20.
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38. Corda M, Era B, Fais A, Casu M, Biochim. Biophys. Acta (2004), 1674,182-192
39. Moglich A, Koch B, Gronwald W, Hengstenberg W, Brunner E & Kalbitzer HR,
Eur J Biochem, (2004), 271, 4815–4824
40. Soldatov DV, Moudrakovsky IL, Grachev EV & Ripmeester JA J Am Chem Soc
(2006), 128, 6737–6744
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Freeman HC & Mitchell Guss J Biochemistry (2003), 42, 15148–15157;
42. Duff AP, Trambaiolo DM, Cohen AE, Ellis PJ, Juda GA, Shepard EM, Langley
DB, Dooley DM, Freeman HC, Mitchell Guss J J Mol Biol (2004), 344, 599–
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Wilmot J Biol Chem (2007), 282(24), 17767–17776
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51. Springuel-Huet MA, Bonardet JL, Fraissard J, Appl Magn Reson (1995), 8, 427
CHAPTER II – DESCRIPTION OF THE SYSTEMS STUDIED
14
Chapter II
The systems studied: void space in biomolecules
CHAPTER II – DESCRIPTION OF THE SYSTEMS STUDIED
15
2.1 Myoglobins: suitable model systems
Myoglobins (Mb) are intracellular hemoproteins that reversibly bind molecular oxygen
(O2). They are expressed in the myocytes of cardiac tissues and in striated muscular
fibers of type I and II vertebrates1.
Earliest studies on myoglobin were carried out by Millikan in late ‘30s and
resulted in a comprehensive review2 in which he assembled a significant body of
knowledge to establish that myoglobin is formed adaptively in tissues in response to the
demand for oxygen. Subsequent important results achieved by many authors further
assessed myoglobin structure and function and this class of proteins is nowadays one of
the most studied systems and commonly believed to be a very useful model compound to
investigate the important issue of structure-function relationship. Additionally, the
extensive experimental results on myoglobin make it a prime example for testing the
applicability of various theoretical techniques to proteins3,4.
2.1.1. - Myoglobin function.
Functionally, myoglobin is well accepted as an O2-storage protein in muscle,
capable of releasing O2 during periods of hypoxia or anoxia. Myoglobin is also thought to
buffer intracellular O2 concentration when muscle activity increases and to facilitate
intracellular O2 diffusion by providing a parallel path that augments simple diffusion of
dissolved O2. It has been extensively demonstrated that the function of myoglobin is
carried out with remarkable variability with genetic origin of the polypeptide chain; the
role of many aminoacids in modulating the process of ligand binding and diffusion has
been deeply investigated by means of computational methods and experimental analysis
on point mutants of myoglobins5,6. Several studies have demonstrated that myoglobin
carries out many other important functions beyond serving as an O2 reservoir and
transporter. In this regard, it was recently proposed7 that oxymyoglobin (MbO2) can also
play the role of intracellular scavenger of nitric oxide (NO), thus protecting respiration in
skeletal muscles and heart. NO, in fact, is known to reversibly inhibit Cytocrome-c
oxidases, the terminal enzyme of the mitochondrial respiratory chain8-12. It was also
pointed out that myoglobin supports oxidative phosphorilation13.
CHAPTER II – DESCRIPTION OF THE SYSTEMS STUDIED
16
Another important ligand with high affinity for myoglobins is carbon monoxide
(CO). Carbon monoxide binds coordinately to heme iron atoms in a manner similar to
that of oxygen, but the binding of carbon monoxide to heme is much stronger than that of
oxygen. The preferential binding of carbon monoxide to heme iron is largely responsible
for the asphyxiation that results from carbon monoxide poisoning. Several amino acids
play important role in regulating the binding of different ligands to myoglobins and in
determining the selectivity of this protein. Among all, distal histidine E7 (His64) in
vertebrate myoglobins has been strongly conserved during evolution and is thought to be
important in fine-tuning the ligand affinities of these proteins14,15.
2.1.2. - Myoglobin structure.
Myoglobin has relatively small size (Mr ∼17 600) and it is formed by a single
polypeptidic chain of 153 aminoacids and a protoporphyrin IX heme prosthetic group, a
tetrapirrole to which is bound an iron atom, identical to that of hemoglobins. The iron
atom (green ball in Figure 2.1) forms five coordination bonds in the deoxy form of
myoglobin, four of which with the nitrogen atoms (in blue) belonging to the tetrapyrrole
and one with the Nε of the imidazolic ring of proximal His93(F8) (in yellow), which has a
particular relevance in stabilizing the heme group.
Figure 2.1: Heme group (Fe-protoporphyrin IX) in deoxy-myoglobin.
CHAPTER II – DESCRIPTION OF THE SYSTEMS STUDIED
17
The sixth coordination position, at the opposite side of the heme plane with respect to
proximal histidine, can be occupied either by oxygen in oxymyoglobin or by other
potential ligands such as CO (Carboxy-Mb), H2O (Meta-myoglobin), N3 (Azide-Mb), CN
(Cyano-Meta-Mb) and NO (Nitroso-Mb).
The polypeptidic chain is arranged in eight separate right handed α-helices,
designated A through H, that are connected by short non helical regions in a highly
conserved globular fold. Amino acid R-groups packed into the interior of the molecule
are predominantly hydrophobic in character while those exposed to the solvent on the
surface of the molecule are generally hydrophilic, thus making the molecule relatively
water soluble. The heme prosthetic group is buried within a hydrophobic cleft of the
globin, sandwiched between the E and F helices. In particular, the heme group is placed
between two Histidine residues that significantly influence the overall function of
myoglobin: the proximal His93(F8) and the distal His64(E7). The helices B, C and E
(purple region in Figure 2.2) form the so called distal side of the active site, while the
proximal side is lined by residues belonging to the helix F (yellow in Figure 2.2).
Figure 2.2: Helices B, C and E (in purple) form the distal region of myoglobins, while helix F (in yellow)
lines the proximal side. In the picture are evidenced proximal histidine (His93) in orange, heme group in
red and iron atom in green.
CHAPTER II – DESCRIPTION OF THE SYSTEMS STUDIED
18
The protoporphirin ring, embedded within the folded globin, is stabilized by Van der
Waals forces or through hydrophobic interactions with the non-polar side chains of the
residues lining the active site16. In particular some residues, Leu89 (F4), His97 (FG3),
Ile99 (FG5) and Leu104 (G5), have been demonstrated to be crucial in stabilizing the
heme group firmly linking it to the globin: Ile99 and Leu104 residues are localized in the
internal region of the heme pocket, close to the prosthetic group and are believed to act as
barriers to the water molecules of the solvent; the residue Leu89 has the same function,
being at the entrance of the hydrophobic active site. Substitutions in these positions have
significant effect on the dissociation of heme17. On the other side of the prosthetic group,
in the distal region, His97 forms a hydrogen bond with 7-propionate, thus dividing the
interior cavity from the solvent. Propionic groups in positions 6 and 7 of the heme ring
are exposed to the solvent and interact with polar residues on the surface of the protein.
Mb can exist in both the reduced iron(II) state as well as oxidized iron(III) state,
and both the diamagnetic and paramagnetic derivatives have been the subject of intense
physicochemical studies to elucidate the mechanism of control of ligand binding18. The
electronic configuration the valence shell of Fe is 3d64s2 while that of Fe2+ ion is 3d6 and
that of Fe3+ ion is d5. When Fe is coordinated with heme within the protein, a splitting of
the energy level characterizes each coordination environment according to the ligand
field theory [Scheme 2.1].
Scheme 2.1: Ligand field theory predicts the splittings of the d orbitals of heme iron.
CHAPTER II – DESCRIPTION OF THE SYSTEMS STUDIED
19
High-spin iron(III) represents the resting state form of many heme proteins. In
this form the heme iron is pentacoordinated with proximal His ligand, or hexacoordinated
with a water molecule in the sixth coordination site. High spin ferric heme proteins have 6A ground state. The electronic configuration of a high-spin d5 system is shown in
Scheme 2.2 reported below:
Scheme 2.2: Electronic configuration of a high-spin d5 system
The low-spin ferric heme cyano-metmyoglobins (CNMMbs) represent an
important subclass of paramagnetic metalloproteins whose 1H NMR spectral parameters
contain a wealth of structural information. Due to the low paramagnetism of these
systems characterized by S = 1/2, they represented the material for the first applications
of new NMR techniques to paramagnetic proteins.
Scheme 2.3: Electronic configuration of a low-spin d5 system
CHAPTER II – DESCRIPTION OF THE SYSTEMS STUDIED
20
NMR assignments for the hyperfine-shifted resonances in this system have relied
primarily on comparisons with model compounds19-21 analysis of paramagnetic
relaxations22,23 and by isotope labeling of heme protons24.
In 1983-1985 some papers appeared which represented the first application of 1D
NOE to a paramagnetic metalloprotein25-28. Moreover, it has been demonstrated that
approximately 95% of the protons within 7.5 Å of the ferric iron of CNMMb can be
assigned on the basis of 2D NOESY29 by exploiting the X-ray crystal coordinates30 to
interpret the cross peaks. It was also demonstrated that these assignments can be used to
determine the orientation of the magnetic axes in solution29.
2.1.3. - Cavities in myoglobin.
Myoglobin has been the first protein to be crystallized and resolved at atomic
resolution31.
The pioneering crystallographic studies of Shoenborn and coworkers evidenced
the presence of cavities within Sperm Whale myoglobin (SW Mb) crystals able to bind
Xenon under moderate pressure32,33. These Xenon complexes were in fact obtained by
subjecting native protein crystals to relatively low gas pressure (2-2.5 bars) and it was
suggested that Xenon atoms are bound to pre-existing atomic-sized cavities in the interior
of these globular proteins through weak Van der Waals forces. Subsequent work carried
out by Tilton and co-workers34 clearly showed that the number and the occupancies of
Xenon binding sites vary with the applied pressure. Thus, at a pressure of 7 bars, only
one fully occupied principal binding site, the proximal cavity (Xe1), was found in Sperm
Whale met-myoglobin crystals, and three additional secondary cavities were
characterized having lower Xenon occupancy. These four sites, referred to as Xe1, Xe2,
Xe3 and Xe4, are shown in Figure 2.3 below.
CHAPTER II – DESCRIPTION OF THE SYSTEMS STUDIED
21
Figure 2.3: Crystal structure of Sperm Whale Myoglobin complexed with Xenon. In red is represented the
prosthetic heme group, in orange, licorice style, is shown proximal His93(F8) and yellow balls are Xenon
atoms bound to the four principal hydrophobic cavities referred to as Xe1, Xe2, Xe3 and Xe4.
Xenon 1 is bound in the proximal pocket and is essentially fully occupied. Xenon
2 binds directly below the proximal cavity in a relatively small cleft near the bottom of
the heme group and it was shown to have very close contacts with protein atoms, in
particular Cε1 and Cδ1 of Phe138(H14). Xenon 3 is located in a cavity lined by residues
belonging to the E-F corner and to the H helix near the surface of the protein. Finally,
Xenon 4 is on the distal side directly below the O2 binding site. It was shown by Tilton
and coworkers that, according to X-ray crystal structures, Xenon binds to myoglobin with
only very little perturbation of the local environment. It was observed that in fact non-
neglibible positional changes correspond to aminoacids such as Leu89 and Ile142, which
have close contacts with Xenon into the main binding site Xe1. One more feature that
characterizes Xenon binding to myoglobin according to Tilton et al. is the observation of
an overall decrease (about 13%) of the temperature factors of both backbone and side
chains atoms, some particular regions, such as helices close to Xe1, being more affected
than others. It should be pointed out in this regard that thermal factors are of interest as
CHAPTER II – DESCRIPTION OF THE SYSTEMS STUDIED
22
they measure dynamic and conformational disorder and this led the authors to the
conclusion that atomic motions in myoglobin are decreased upon Xenon inclusion.
However, although proximal region was shown to be much more influenced by
this restricted motion, the overall flexibility of protein atoms was affected, that can be
explained only by considering a likely ligand-induced restriction of the number of
conformational substates, i.e. a “freezing” of ligand-stabilized substates. The presence of
different conformational states of myoglobin, each having particular activation energy,
was postulated by Austin and coworkers35 and further confirmed by means of FT-IR on
CO-Mb36 and two dimensional infra-red based vibrational echo experiments37.
The role of hydrophobic voids in biomolecules is the source of a very intense
scientific debate. Early researchers referred to those internal cavities using the term
“packing defects” and the uncertainty on their actual relevance in favoring the dynamics
of protein molecules lasted until recently. However, these cavities exist at the expense of
considerable cost in free energy, so that it is unlikely that they are mere packing defects.
Moreover, binding of Xenon to myoglobin has been shown to significantly affect the
functionality of the protein38,39
The hypothesis that cavities are important for the conformational flexibility of
protein molecules is further supported by the observation that conformational states of
myoglobin are restricted by Xenon binding to protein cavities40. At the same time, the
observation of binding of small ligands to internal voids buried within proteins would
contrast with the static representation of cavities as voids closed off by the protein atoms
if concerted movements of the protein backbone and side chains were not taken into
account. It is in fact commonly believed that diffusion of ligands into proteins is made
possible by transient formation of pores, channels and pathways which are not observed
in the average picture usually obtained by crystallographic structures.
Cavities, therefore, are generally hydrophobic, are able to bind exogenous ligands
such as Xenon, which are stabilized by non covalent specific interactions32. Cavities,
moreover, permit proteins to have a stable structure and to perform their function, as they
represent the best compromise between thermodynamic stability and flexibility: it has
been suggested in fact that they are important in catalyzing reactions41,42 and in tracing
pathways for the diffusion of ligands to and from the active site.
CHAPTER II – DESCRIPTION OF THE SYSTEMS STUDIED
23
Both molecular dynamics simulations34,43,44 and laser photolysis studies as well as
time-resolved crystallography45-47 have enlightened the key role of Mb cavities in ligand
dynamics. Within this framework, the competition with Xenon in occupying these
strategic sites has also been exploited for testing possible routes followed by the ligands
inside the protein45,48.
One among the most intriguing features to be understood about Xenon binding to
protein cavities is the diversity of possible binding sites. They can be channel-pores and
pockets transiently exposed to the solvent and they can be buried inaccessible cavities as
well. Moreover Xenon is found to bind to inter- as well as to intra-molecular sites49.
Many studies have exploited Xenon-induced variation of NMR chemical shift of
nuclei which the Xenon comes in contact with when it is included in cavities or
channels50-54. Commonly, 15N, 13C, and 1H NMR chemical shift variations are considered
in order to extract thermodynamical parameters of binding and to detect and characterize
the sites where the complexed Xenon resides51,53-55. Such studies, however, are usually
applied to diamagnetic systems, and, to our knowledge, no attempt has been presented in
literature so far to explain similar results in paramagnetic biomolecules. Deepening the
knowledge of paramagnetic interactions in model proteins as myoglobin is, however,
relevant, as many heme-proteins, along with many other metallo-proteins, exist in their
paramagnetic states.
Analyzing the observed chemical shift and its variations in paramagnetic
biological compound is more challenging than in diamagnetic molecules because many
different contributions, essentially related to the presence of the unpaired electron in the
atomic orbitals of the metal ion, finally influence the experimental result.
The influence of guest molecules on proton NMR spectra of myoglobins was
observed in early studies56,57; however, despite it was clearly demonstrated that the
presence of cyclopropane and Xenon within the internal cavities of myoglobins caused
modifications in the experimental proton NMR spectra no detailed explanation of that
result was attempted. This is probably due to the poorly resolved and not yet assigned
spectra obtained and to the limited knowledge of these systems at that time. Further
studies, mainly NMR and EPR measurements58,59 together with model calculations60,
CHAPTER II – DESCRIPTION OF THE SYSTEMS STUDIED
24
successively expanded the investigation and showed the relevance of parameters such as
Fe-1H dipolar through-space interactions, orientation of unpaired spin density and
electron and spin delocalization from iron to porphyrin orbitals in establishing the
structure of the heme cavity and consequently influencing the observed proton spectra61-
63.
2.2 Copper-containing Amine Oxidases
Copper/quinone–containing amine oxidases [amine:oxygen oxidoreductase
(deaminating)(copper containing); EC 1.4.3.6] (Cu/TPQ AOs) are found in bacteria,
yeasts, fungi, plants and mammals.
2.2.1. - Structure.
Amine oxidases are homodimers: each subunit (molecular mass ∼ 70-90 kDa)
contains an active site composed of a tightly bound Cu(II) and a quinone of 2,4,5-
trihydroxyphenilalanine (TPQ or TOPA). The protein-derived cofactor TPQ is generated
by an endogenous tyrosine residue through a self-catalytic reaction with copper divalent
ions and molecular oxygen64 and has a crucial role in the catalytic process of Copper
amine oxidases, defined for plant amine oxidases.
Figure 2.4: Biogenesis of TPQ
P r o t e i n
O H
O H 2 O
O P r o t e i n
O
+ + 2O 2 + Cu(II)
TPQ
+ H 2 O 2 + Cu(II) + OH -
CHAPTER II – DESCRIPTION OF THE SYSTEMS STUDIED
25
So far, six AOs have been successfully crystallized and their structure has been
resolved by XRD65-70.
Figure 2.5: Crystal structure of a eukaryotic (pea seedling) Copper containing amine oxidase. The picture
shows the structure of dimers where TPQ (in red) cofactors and Copper ions (in orange) are evidenced
The Copper ion is coordinated with the imidazol groups of three conserved
histidine residues and with two water molecules, arranged in a distorted square base
pyramidal geometry (Fig. 2.6).
Figure 2.6: Structure of Copper sites: three histidines (residues His603, His442 and His444) and a water
molecule (the oxygen is a red sphere) are shown. The fifth position is expected to be occupied by another
water molecule to form a distorted square base pyramidal conformation.
CHAPTER II – DESCRIPTION OF THE SYSTEMS STUDIED
26
2.2.2. - Hydrophobic cavities in AOs.
XRD structures of AOs show, similarly to what has been observed in many other
proteins and enzymes, that the active site is buried in a cavity not directly accessible from
the solvent. X-ray crystal structures of Copper-AOs bound to Xenon are available from
bacteria (Arthrobacter globiformis), yeast (Pichia Pastoris), plant (Pisum Sativum), and
mammalian sources (bovine serum albumine oxidases)70,71.
A recent investigation of a Copper-containing Amine Oxidases from Hansenula
polymorpha by means of a combination of XRD analysis on HPAO single-crystals in
presence of Xenon gas, kinetics and computational approaches have given evidences for
the existence of at least four binding sites for Xe inside these AOs .
Figure 2.7: In figure are shown the four Xe sites found in HPAO by means of X-ray Crystallography. In
the picture is shown the structure of a dimer where one of the monomers is whitened. Xenon atoms are
represented as yellow spheres, Copper ions are depicted in orange and TPQ cofactor is evidenced in
licorice style.
Xe1
Xe2
Xe3
Xe4
CHAPTER II – DESCRIPTION OF THE SYSTEMS STUDIED
27
2.2.3. - Biological function: AOs’ catalytic process.
Copper AOs catalyze the conversion of two substrates, primary amines and
molecular oxygen, to aldheydes and hydrogen peroxide, respectively.
The oxidative deamination occurs by transfer of two electrons from amines to molecular
oxygen72.
The ping–pong catalytic mechanism of Cu/TPQ AOs can be basically divided into two
half–reactions: the first, referred to as reductive half–reaction, involves the oxidation of
amine to aldehyde and the formation of a reduced form of the TPQ cofactor:
Eox + R–CH2–NH3+ → Ered + R–CHO [2.1]
The second half–reaction, known as oxidative half–reaction, involves the reoxidation of
the enzyme with the simultaneous release of ammonia and hydrogen peroxide:
Ered + O2 + H2O → Eox + NH4+ + H2O2 [2.2]
A number of biochemical investigations have been carried out in order to shine a
light on the molecular mechanisms implied in both biogenesis of TPQ and catalytic cycle
of Copper AOs, but the debate is still ongoing. In particular, while quite definite results
tend to confirm the reductive half reaction, somewhat unclear appears the mechanism of
activation of the molecular oxygen in the oxidative step of the cycle, which remains
subject of intense study73-76.
Somewhat contentious has appeared the role of Copper ion in the catalytic process
of these amine oxidases and this issue has been the focus of recent controversy. As
Copper AOs contain Cu(II) ion in the active site, it was suggested that Cu(I) ion is likely
responsible of reacting with O2 to give Cu(II)-superoxide77. In anaerobic conditions, in
fact, amine induced reduction of Copper AOs shows an equilibrium between Cu(II)-
aminoquinol and Cu(I)-semiquinone with yelds of Cu(I)-semiquinone depending on the
particular enzyme source78. A catalytic mechanism proposed for plant AOs is reported in
the scheme below79,80.
CHAPTER II – DESCRIPTION OF THE SYSTEMS STUDIED
28
+
CuII
(I)
O
OO
NH3CH2 R
CuI
CuII
+ H2O
-
-
H++
H+
O
NH2
OH
R CHO
OH
HO
NH2
(II)
O2H2O2-
NH4-
+(III)
Figure 2.8: Left: scheme of the proposed catalytic process of Copper amine oxydases defined for plant
AOs. On the right, the active sites where Copper ions (orange) and TPQ cofactors (cyan=carbons;
red=oxygens; blue=nitrogens) are evidenced.
Following the reaction scheme reported in the left side of Figure 2.8, three
principal steps can be described. First (I), the amine substrate reacts with the TPQ
cofactor of the oxidized enzyme to give the Shiff base Cu(II)-quinone ketimine, a short
lived species which is rapidly converted, through the formation of an unstable Cu(II)-
carbanion, another Cu(II)-quinolaldimine Shiff base and release of the aldehyde, into the
reduced Cu(II)-aminoquinol derivative (II), which binds an ammonia molecule ( a more
detailed reaction scheme will be discussed in the Results and discussion, see sections
3.2.2 and 3.2.3) .
It was suggested and demonstrated by EPR measurements that Cu(II)-
aminoquinol (II) forms the yellow-colored intermediate Cu(I)-semiquinolamine (III)
radical in anaerobic conditions78. This latter species, observable only in absence of
oxygen, contains the substrate-derived nitrogen which is covalently bound to the aromatic
ring system and is characterized by a typical UV-vis spectrum having characteristic
absorption bands at 464, 434 and 360 nm,81,82 similar to that shown below:
CHAPTER II – DESCRIPTION OF THE SYSTEMS STUDIED
29
Figure 2.9: UV-vis absorption spectrum of the yellow-colored intermediate Cu(I)-semiquinolamine radical
observed in anaerobic conditions.
Spectroscopic features have been explained by considering both electronic
transitions associated with the quinone and also the possible influence of Copper-cofactor
charge transfer (LMCT transitions) was hypothesized. Ligand field transitions of the
Cu(II) ion in amine oxidases in presence of exogenous ligands that stabilize Cu(I), such
as CN-, have been characterized by means of circular dichroism83.
Both forms of the reduced enzyme (II and III) can further react with molecular
oxygen (if present) to release hydrogen peroxide and ammonia, thereby regenerating the
Cu(II)-quinone species84,85.
Understanding the molecular mechanisms implied in enzymatic activities is of
fundamental relevance. In particular, the role of many aminoacids in the overall catalytic
process of Amine oxidases has been suggested especially on the base of crystal
structures. In this case, a critical issue would be the study and substantiation of possible
pathways involved in the processes of migration and binding of molecular oxygen, as this
species is certainly involved in the enzymatic redox reactions.
Among all the methods that are usually adopted in order to study O2 migration pathways
inside the cavities Xenon atoms represent ideal probes which can be used to investigate
the interior of proteins and enzymes. Because of their analogous properties in size and
CHAPTER II – DESCRIPTION OF THE SYSTEMS STUDIED
30
hydrophobicity, any region that binds Xenon is usually assumed to be favourable for
O2.87
Although protein crystallographers have used Xenon derivatives in order to get
isomorphous form of protein crystals and thus acquire diffraction phase parameters
aiming to elucidate biological activity of proteins and enzyme, it should not be forgotten
that Xenon can sometimes participate in biological reactions.
Although only very few examples in literature give evidence of the relevant role
of Xenon in triggering and/or catalyzing biological reactions, neglecting this possibility a
priori may lead to erroneous conclusion. In this regard, recently the role of Xenon in
increasing electron spin intersystem crossing rates in chemical and enzymatic reactions
with radical pair intermediates has been discussed88-94
CHAPTER II – DESCRIPTION OF THE SYSTEMS STUDIED
31
2.3 Biomaterials: microporous crystalline dipeptides
2.3.1. - Developments of microporous materials.
Microporous materials are currently the subjects of widespread studies because of
the availability of well defined and ordered void space of different sizes and shapes and
due to the variety of possible applications. Current and future possible applications of
these materials include industrial catalysis, gas sensing and storage, isolation and
purification technologies, stabilization of pharmaceuticals, biological molecules and
reactive species, inertization of hazardous waste materials.
Until mid 1990s there were basically two types of microporous materials, namely
inorganic and carbon-based materials.
In the case of microporous inorganic solids the largest two subclasses are the
aluminosilicates and aluminophosphates. Several related crystalline oxides such as
silicoaluminophosphates, metallosilicates, metalloaluminophosphates, but also porous
chalcogenides, halides and nitrides have been discovered.
Carbon-based materials represent another important example of widespread used
microporous materials. A principal negative aspect is however that in this type of
materials microporosity is usually very disordered and a very detailed systematic study of
sorption process is often disadvantaged.
Development of organically-based microporous materials assembled from
building blocks represents a very actual issue in material science, supramolecular
chemistry and crystal engineering, mainly due to the remarkable diversity of possible
modes of assembly and to the multitude of final structural motifs attainable. These
materials offer many important advantages with respect to the more commonly used
inorganic counterparts such as zeolites, clays and various metal oxides. In organic-related
systems, chemical and structural modulations can be introduced in small increments over
a wide range to create a desirable property or function. At the same time, the size and
geometry of the free void space are crucial factors in determining the properties of
microporous materials. In many cases, the species included within the pores are weakly
linked by non-covalent bonds to the host matrix, frequently by only Van der Waals
interactions. Therefore selectivity, total capacity, thermodynamics and kinetics of the
CHAPTER II – DESCRIPTION OF THE SYSTEMS STUDIED
32
inclusion process are often merely determined, and may be therefore predicted and/or
regulated, by knowing the pore geometry.
In the literature, a significant number of examples can be found of the synthesis
and characterization of molecular details of the void space in organic95-97, hybrid metal-
organic97-100 and protein/peptide97,101-104 solid microporous frameworks.
Particularly interesting results have been recently obtained on the characterization of void
space in flexible pore systems such as biozeolites, a new group of microporous materials
based on peptides.
The first peptide-based system whose building blocks form nanotubes in the solid
phase were described in 1975 for cyclic α−β−α−β peptides105. Ghadiri and coworkers
have continued in 1990s to use cyclic peptides with eight to twelve residues106. β-sheet-
like intermolecular hydrogen bonds between the peptidic units formed in both cases
tubular structures.
Other research groups since then have kept working at the synthesis and
characterization of peptide-based nanotubes in crystalline compounds and the variety of
structure modulations is evident107,108.
The first example of much smaller peptide-chains forming nanotubes in the solid
state was reported by Görbitz and Gundersen in 1996 and was represented by dipeptides
L-Val-L-Ala as building units109 which form helical nanochannels resulting from head-to-
tail hydrogen bonds between functional groups of dipeptide molecules and with
hydrophobic inner walls. Other dipeptides were then studied: it seems to be a common
pattern among oligopeptides that, taking into account their combinatorial diversity might
entirely revolutionize the domain of engineering microporous solids.
2.3.2. - Characterization of bioorganic materials
Studies on oligo-peptides formed by long aminoacid chains have shown that these
materials usually show complex behavior and are often difficult to characterize in detail:
consequently, building-blocks formed by smaller peptides can be considered as to
represent simpler model systems to test the fundamental properties of these systems.
Moreover, short-chain peptides are cheaper and it is easier to deal with them for
crystallizations and structural refinement. Sorption studies have already revealed size-
CHAPTER II – DESCRIPTION OF THE SYSTEMS STUDIED
33
matching molecular recognition by the peptidic channels102 and the design of peptide
sorbents that are highly selective to a particular guest will likely be possible taking into
account their diversity.
Gas sorption was tested for some dipeptides nanochannels and it was found that
their frameworks show a high sorption capacity and high selectivity for inert species like
Xenon. This permitted pioneer researchers to refer to these biomaterials with the term
biozeolites. Further advantages of biozeolites are their biocompatibility and
environmental friendliness. Moreover, microporous dipeptides display not only
similarities to inorganic sorbents (uniform pore geometry, ordered porosity,
thermodynamic stable phases), but also some characteristics which are typical of
proteins, such as flexibility of the pores and structural softness. Microporous
oligopeptide-based chains are in fact considered very suitable models of biological ion
channels and may be used for further understanding the mechanisms involved in the
inclusion of ligands in proteins.
Dipeptides represent in fact a rare opportunity to study ordered microporous
biologically-related solid materials that maintain, perhaps to a less extent, the
characteristic flexibility of protein in solution: this could result in a system which
possesses the characteristic stability of crystals and the specificity (selectivity) peculiar of
proteins and enzymes. In fact, the flexibility of the dipeptides channels is well known 97,102,110-112 that “dipeptides structures can be considered as belonging to a class of “soft”
sorbents which tend to adapt their structure relative to the presence, concentration and
chemical nature of the guest species”.
While the development of new synthetic processes enables the design of tailored
materials with pores of known volume and geometry, the continuous improvements of
the characterization techniques make easier the understanding of the correlation between
their structural and functional properties. A successful characterization of nanoporous
materials relies upon the establishment and optimization of suitable techniques that are
able to highlight even subtle details of the structure under investigation. It is reasonable
to think that the selectivity toward different guests that often characterizes nanoporous
channels can be significantly influenced by small differences in the geometry, chemical
composition and flexibility of void space. This is particularly true if a biological
CHAPTER II – DESCRIPTION OF THE SYSTEMS STUDIED
34
functionality is involved: the understanding of the molecular mechanisms underlying the
action of biological ion-channels and receptors is still the subject of vivid interest and
their study gathers the efforts of researchers belonging to different scientific branches. At
the same time, the synthesis and characterization of organically-based soft nanoporous
materials has recently paved the way for a new material science, due to a number of
distinctions that these sorbents show compared to the more diffused inorganic porous
materials such as zeolites, aluminophosphates, activated carbons, silicas, clays, etc113.
Previuos studies on nanochannels of crystalline dipeptides97,112 have shown that
the information elicited about the porosity of such materials appears to be strictly
dependent on the technique employed for their characterization. Although the results
obtained by means of different methods have found to be roughly coincident, it appeared
clear that the dynamics of the host matrix might play a crucial role in determining the
sorption of these soft materials and the reliability of the characterization techniques as
well. In particular, the consistency of single crystal XRD structures with respect to other
characterization techniques has been objected due to the average and static nature of the
information provided97. In this respect, a complete and comprehensive description of soft
materials would require the combination of different and complementary techniques and
the comparison between the results obtained. In order to gain an exhaustive
understanding of the structure and properties of flexible nanoporous materials, therefore,
the improvement and testing of methods that are able to give insights on the dynamic
nature of the sorption mechanism appear to be essential.
The specificity of the dipeptides nanochannels respect to the complexation of
simple chemical species was already observed by Gorbitz et al111. In particular, a fully
retention of I2 into the nanochannels space of LS dipeptides was observed and local
structural adaptations were suggested in order to explain the low degree of channels
filling: however, the efforts, by means of TGA/DSC technique, addressed to the
characterization of the release process of I2 were unsuccessful.
In this regard, is known that most of biological-related nanochannels can be switched
between an open and a closed state and that this transition can be triggered by the binding
of a ligand. In the case of microporous dipeptides complexes the characterization of
CHAPTER II – DESCRIPTION OF THE SYSTEMS STUDIED
35
possible structural rearrangements and the study of their reversibility could be an
interesting challenge.
Other experimental results and calculations based on simple dipeptides systems
suggest the influence of guest Xenon atoms on modification of the internal pore
structure112, however a compelling explanation of these phenomena has never been
attempted.
Moreover, the diffusion through such ultramicropores and the possible trapping of
small organic and inorganic moieties into the nanopore space, along with the capability of
dipeptides to sustain guest solvent exchange and full removal, make the deep
characterization of these phenomena a very interesting challenge.
Despite the fundamental interest on this materials and their numerous possible
applications108, a full characterization of the structure-function correlation and dynamics
of such systems is still not available.
CHAPTER II – DESCRIPTION OF THE SYSTEMS STUDIED
36
2.3.3. - Microporous dipeptides structure.
In this thesis eight different dipeptide structures (all LL-isomers) will be discussed
in particular: Ala-Val (AV), Val-Ala (VA), Leu-Ser (LS), Ala-Ile (AI), Val-Val (VV),
Ile-Ala (IA), Ile-Val (IV), Val-Ile (VI). Formulas of all dipeptides are represented in
Figure 2.10.
Figure 2.10: Molecular structure of the dipeptides units studied
The crystal structures of the eight dipeptides can be divided into two isostructural
groups: seven of them, formed by the hydrophobic residues Alanine (Ala), Valine (Val)
and Isoleucine (Ile), represent in fact an isostructural series, having hexagonal unit cell
with P61 space group. In this structure, the channels are formed by helical H-bonded
assembly of the single dipeptide molecules. As a typical example, H-bonding scheme is
shown for VV in Figure 2.11.
H 3 N +O
N H C O O -
H 3 N+ O
N H C O O -
H 3 N +O
N H C O O
-
H 3 N +O
N H C O O
-
H 3 N +O
N H C O O -
H 3 N +O
N H C O O -
H 3 N+
O NH C O
-
H3 N +O
NH C O O
-
O H
AV
VA
AI
VV
IA
IV
VI
LS
O
CHAPTER II – DESCRIPTION OF THE SYSTEMS STUDIED
37
Figure 2.11: Fragment of the crystal structure of VV (hexagonal, space group P61) showing H-bonding
helical assembly of the dipeptide molecules surrounding the channel (view along the channel). Two
translational periods are shown. N and O atoms are drawn as small black and white balls, respectively. H-
bonds are designated with grey lines.
The channels are one-dimensional and isolated from each other. Single file
diffusion of Xenon atoms inside these nanochannels appears very reasonable from the
analysis of channel diameters by means of XRD crystal structures and other independent
techniques97,114 and it was recently demonstrated by Bowers and coworkers114 by means
of NMR measurements of a continuous flow of hyperpolarized 129Xe. The interior of
channels is lined by hydrophobic parts of the aminoacids and the void space inside the
nanochannels is essentially determined by the size of hydrocarbon fragments of the
dipeptide molecules. The diameter of the nanotubes (which does not equal the inner
diameter of the channels) is identified by the a parameter of the unit cell and varies
from14.2 Å to 10.4 Å and the translation period varies from 10 to 10.4 Å and includes a
screw rotation.
Leu-Ser dipeptides (LS) form a different structure. Crystals have hexagonal unit
cells and P65 space group. Similarly to the other dipeptides, LS units form tubular
channels with approximately 15 Å diameters by means of intermolecular H-bonds.
VV
CHAPTER II – DESCRIPTION OF THE SYSTEMS STUDIED
38
Translation periods are of about 6.5 Å. However, in this case, the inner walls are lined by
hydrophobic fragments of only leucyl residues, as it is shown in Figure 2.12.
Figure 2.12: Fragment of the crystal structure of LS (hexagonal, space group P65). Atoms are represented
as previously described (see Fig 2.11).
Also for LS crystals, channels are isolated from each other. A significant
difference with respect to the other series of dipeptides is that the helicity of LS channels
is left-handed, while in AV, VA, AI, IA, VV, IV and VI the helicity is right-handed. This
characteristic is very important as it has been suggested that crystalline dipeptides can be
used in the separation of organic compounds by chiral recognition. Moreover, while in
LS each isolated nanotube is formed by a single chain of dipeptide molecules, in the other
dipeptides a channel wall is formed of 50% by one nanotube and for the other 50% by six
adiacent nanotubes.
LS
CHAPTER II – DESCRIPTION OF THE SYSTEMS STUDIED
39
2.3.4. - 129Xe NMR of dipeptides microporous crystals. 129Xe NMR has proven to be an extremely sensitive technique for characterization
of void spaces in porous materials and proteins and the suitability of this technique to
provide dynamically averaged information is well known.
Since the earliest 129Xe NMR experiments on characterization of porous
materials, a number of different theories have been proposed, aimed at finding a good
correlation between the NMR parameters of the guest Xenon atom probes (chemical
shifts, line shapes, relaxation times) and the actual structure of the hosts. However,
although high sensitivity of the easily polarizable Xenon electronic cloud to its physical
environment potentially provides plenty of information, the experimental NMR
parameters are often influenced by a number of different concurrent contributions.
Calculations based on Lennard-Jones potentials115,116 demonstrated that, in fact, a single,
simple correlation between 129Xe NMR chemical shift and void space is not expected and
the chemical shift of Xenon adsorbed in microporous materials is a complicated function
of void space geometry, sorption energy and temperature. Considerable progress has been
made in the derivation of useful parameters for sorption from 129Xe NMR spectroscopy
of large pore sytems (d > ~ 1nm). In those cases, the line shape is dominated by
exchange and dynamics, and parameters pertaining to pore size, adsorption energy, etc,
can be derived using a simple model that uses the temperature dependence of the
chemical shift.
Rather consistent 129Xe NMR results concerning the δ-D correlation were in fact
obtained by adopting a semi-empirical approach117 based on the adsorption
thermodynamics to study a number of mesoporous amorphous silica gels with a range of
mean pore diameters from 2 to 40 nm and this approach has been successively extended
to micropores over the 0.5-2 nm range118.
Moreover, a fair number of empirically-derived equations119,120 were proposed in
the early works with the purpose of tentatively explain the correlation between isotropic
Xe chemical shifts (δ) and the pore diameter (D) mainly in zeolites and clathrates. Such
an empirical approach is still largely in use 119,121-125. Although the application of
empirical equations to a number of systems seems to support the proposed models126, this
CHAPTER II – DESCRIPTION OF THE SYSTEMS STUDIED
40
approach is of only little help in understanding the physical nature of the correlation
between the 129Xe chemical shift and the pore size. Besides, their general validity has
been several times objected127-129. The analysis by 129Xe NMR of compounds having pore
sizes comparable or smaller than the Xenon Van der Waals diameter would be very
useful to complement the picture drawn so far and would eventually point out further
possible advantages or drawbacks of the technique
It appears therefore clear from what has been said above, that a more detailed
description of the correlation between the physical nature of the sorption process in
nanochannels and the 129Xe NMR outputs is highly desirable.
The study of model systems with well known chemical composition and ordered
porosity can help to deeper analyze how different effects contribute to the experimental
results, allows extracting general rules on the sorption mechanism and can be useful in
providing hints for the interpretation of more complex systems. Dipeptides nanotubes
represent a very suitable class of compounds for this purpose: as previous studies
confirm97, in fact, they form one-dimensional nanochannels with cylindrical or nearly
cylindrical cross-section and diameter of the same order of magnitude of that of the
Xenon atom (ranging from about 3 Å for VI to about 5.4 Å for AV). The inner walls of
the channels are formed by hydrocarbon fragments of both residues and are essentially
hydrophobic. Moreover, the self-assembling process of different dipeptides allows a fine
modulation of the diameter and the helicity of channels, giving the unique opportunity to
compare 129Xe NMR experimental results obtained from samples having the same
chemical composition but different pore geometry and vice versa.
CHAPTER II – DESCRIPTION OF THE SYSTEMS STUDIED
41
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CHAPTER III – RESULTS AND DISCUSSION
49
3.1 Myoglobins
Clearly, hints on the characterization of cavities and Xe-protein affinity can be
achieved by comparatively studying myoglobins extracted from different species or
mutated myoglobins. For instance, internal structural differences between myoglobins
extracted and purified from different animal species can be highlighted by exploiting the
sensitivity of Xe to its local environment.
Four myoglobins, from horse, rabbit, pig and sheep hearts are described here.
They have been chosen because they differ for several residues, mostly located at the
protein surface and only one residue differentiates their hydrophobic interior. Namely,
the residue in position 142, which lines the proximal cavity (Xe1), is an Isoleucine in
horse and rabbit Mbs, whilst pig and sheep Mbs have a Methionine at the same position.
This substitution introduces changes in both the size and shape of proximal cavity and in
its hydrophobic character: Methionine, indeed, is not as highly hydrophobic as Isoleucine
(hydrophaty indexes: Ile = 4.5, Met = 1.9)1 and additionally it is larger in size.
Figure 3.1: Molecular structure of amino acids methionine and isoleucine
It was shown by means of crystal structures of myoglobins from sperm whale in
presence of 7 atm of Xenon that the proximal cavity is the main binding site for Xenon
inside the protein and that only another site is populated at any time other than Xe1 at 7
CHAPTER III – RESULTS AND DISCUSSION
50
atm of Xe.2 Moreover, the presence of both specific (Xe-cavities) and non-specific
(between Xe and protein surface and/or other environments that are sampled by Xenon
with weak diffusion-mediated interactions) interactions between Xenon and myoglobins
has been previously demonstrated and the effect of non-specific binding with the protein
surface on 129Xe NMR parameters was quantitatively assessed3-6. Equilibrium constants
for Xenon horse myoglobin, metmyoglobin and cyanomyoglobin complexes were also
derived from Xenon adsorption measurements and it was suggested that in metmyoglobin
two suitable binding sites exist for Xenon and that equilibrium constants vary, depending
on temperature, between 85 and 200 M-1 for one cavity and between 1 and 10 M-1 for the
other7. It was suggested afterwards by Locci and coworkers4, by analyzing 129Xe NMR
linewidths in myoglobins of high spin state (Fe3+ S=5/2), that Xe1 is, at first
approximation, the main binding site in horse Mb as it was previously observed in crystal
structures of sperm whale Mb-Xe complexes2. This is very reasonable if one considers
that the amino-acid compositions of the internal cavities in the two proteins are identical.
All these results suggested that proximal cavity in sperm whale and horse
myoglobin combines proper size, shape and chemical composition that make of Xe1 the
cavity for which Xenon atoms have the highest affinity compared to the other (at least
three) internal cavities, and that also surface residues can influence observed 129Xe NMR
parameters.
Additionally, the previous achievements just summarized led to speculate that
structure and amino acid composition of the proximal cavity within myoglobins could
represent crucial factors in determining Xe binding to myoglobin and that they could
have particular relevance in regulating ligand diffusion within the protein, i.e. protein
functionality.
It was successively shown by Corda et al8 that careful analysis of 129Xe NMR
chemical shifts and relaxation rates in aqueous solutions of high-spin Met-myoglobins
from pig can provide useful insight on the distribution of Xe within the internal cavities
of Mbs. In particular it was found that while Xe1 is the main binding site in horse Mb, at
least another cavity was suggested to significantly influence Xe binding to pig Mb8. The
same study also evidenced that the 13 residues that differentiate the protein surfaces of
pig and horse Mbs do not specifically influence observed 129Xe NMR parameters,
CHAPTER III – RESULTS AND DISCUSSION
51
suggesting that the interaction of Xenon with the surfaces of the two proteins is almost
identical.
In order to more clearly highlight structural differences between proximal cavities
of myoglobins from horse and pig, some residues lining the proximal cavity in horse and
rabbit Mbs (a) and in pig and sheep Mbs (b) are shown in Figure 3.2.
(a) (b)
Figure 3.2: Residues lining the Xe1 cavity in (a) horse (Leu89, Ala90, His93, Leu104, Phe138, Ile142,
Heme) and (b) pig (Leu89, Ala90, His93, Leu104, Phe138, Met142, Heme) Mbs. The figures are obtained
with the visualization program VMD9.
CHAPTER III – RESULTS AND DISCUSSION
52
3.1.1 - 129Xe NMR measurements in solutions of low-spin (Fe3+ S=1/2) cyano-
metmyoglobins.
Figure 3.3 collects the 129Xe NMR spectra of solutions of pig, sheep, horse and
rabbit CNMbs. Spectra of ∼1mM solutions of the four myoglobins, pressurized with 3
atm of Xenon overpressure in high-pressure tubes, were acquired at 25°C. Chemical
shifts are referenced to the chemical shift of 129Xe dissolved in buffer solution, in the
same experimental conditions.
-50510129129129129Xe (ppm )Xe (ppm )Xe (ppm )Xe (ppm )
sheepsheepsheepsheep
pigpigpigpig
horsehorsehorsehorse
rabbitrabbitrabbitrabbit
Figure 3.3: 129Xe NMR spectra of Xenon (3 atm of overpressure, 25°C) in ~1 mM solution (phosphate
buffer 0.1 M, 20% D2O) of cyano-metmyoglobins from horse, rabbit, sheep and pig.
Very similar NMR parameters, i.e. isotropic chemical shifts (δiso) and linewidths
(fwhm), characterize 129Xe signals of Xenon in solutions of CNMbs from pig and sheep
(pig : CNMb δiso=3.7 ppm, fwhm=46 Hz; sheep : δiso=3.3 ppm, fwhm=36 Hz).
Analogously, 129Xe spectra of horse and rabbit CNMbs exhibit essentially
identical chemical shifts and line widths: δiso=1.4 ppm and fwhm=55 Hz in horse CNMb
solution and δiso=1.2 ppm and fwhm=50 Hz for rabbit CNMb solution.
CHAPTER III – RESULTS AND DISCUSSION
53
It may be useful at this point to recall again that these four myoglobins differ for
several residues mostly located at the surface of the proteins and that they can be grouped
into two groups according to the only difference concerning the amino acid composition
of internal cavities: in particular, horse and rabbit Mbs have in position 142 an Isoleucine
whereas pig and sheep Mbs have a methionine at the same position. Clearly, 129Xe NMR
is able to evidence this difference, also indicating that 129Xe NMR parameters are not as
much influenced by non-specific interactions with the protein surface as they are affected
by specific interactions with internal cavities.
These preliminary results were in fact not unexpected. It was previously shown by
Locci et al4 that 129Xe NMR chemical shift in horse high-spin metmyoglobins is affected
by two concurrent opposite contributions with respect to 129Xe resonance in water: a
downfield contribution, attributed to non-specific interactions between Xenon and the
protein surface, and an upfield contribution coming from the hyperfine dipolar interaction
between the Xenon bound within the proximal cavity and the high-spin paramagnetic
Fe3+.
Moreover, it was pointed out by Corda et al8 that a large diamagnetic component
characterizes the observed 129Xe signal in pig metmyoglobins (MMbs) compared to what
is observed in horse MMbs; however, it is difficult to separate the single contributions
and assign them to particular local sites, due to the dynamically averaged nature of 129Xe
NMR measurements. This latter work additionally demonstrated that the contributions to
the observed 129Xe chemical shift from non-specific interactions between Xenon and
metmyoglobins from horse and pig are very similar while the contribution due to specific
interactions Xe-cavity are quite different, showing a higher affinity of Xenon towards
horse MMbs.
In order to more deeply investigate the influence of proximal cavity structure and
composition on the affinity of Xenon to myoglobins, we present here the changes in 129Xe NMR chemical shift observed in solutions of myoglobins from horse, rabbit, pig
and sheep as a function of increasing Xe concentration (overpressure).
In Fig. 3.4 the variation of the observed 129Xe chemical shift is reported as a function of
the ratio nXe/Vl between the total number of moles of Xenon in the NMR tube, nXe, and
the volume of the CNMb solution, Vl.
CHAPTER III – RESULTS AND DISCUSSION
54
Figure 3.4 clearly shows the sensitivity of 129Xe chemical shift to the nature of
proximal sites in the different myoglobins analyzed. In particular, ∆δ 129Xe, i.e. chemical
shift changes resulting from increasing Xenon concentration in the four CNMb solutions,
group the four myoglobins into two well distinct classes: noticeably, horse and rabbit
CNMb show different behavior than pig and sheep CNMbs, certainly reflecting different
interactions of these two groups of CNMbs with Xenon. In horse and rabbit CNMb
solutions, the 129Xe chemical shift moves downfield with increasing Xenon
concentration, while a reverse trend is observed in pig and sheep CNMbs. For the highest
concentrations of Xenon investigated the 129Xe ∆δ seem to asymptotically converge to
the same value for all the four CNMb solutions.
1
1.5
2
2.5
3
3.5
4
0 0.5 1 1.5 2
nXe/Vl (M)
∆δ∆δ ∆δ∆δ 12
9 Xe
(ppm
)
Pig
Sheep
Rabbit
Horse
Figure 3.4: Variation (in ppm) of the observed 129Xe chemical shift (∆δ 129Xe) as a function of the total
number of moles of Xenon in the NMR tube divided by the volume of solution (nXe/Vl), in ~1 mM
solutions of cyano-metmyoglobins from pig, sheep, rabbit and horse. Shifts are referenced to the chemical
shift of 129Xe dissolved in buffer.
CHAPTER III – RESULTS AND DISCUSSION
55
In order to characterize the binding of Xenon to the CNMbs in aqueous solution
the observed 129Xe chemical shift variation with Xenon concentration was analysed
adopting a two-site model4,8.
inout XeXeCNMb ↔+ [3.1]
According to this model Xenon exchanges rapidly between specific sites within
the interior of the protein (Xein) and all the remaining possible environments (Xeout),
mainly represented by the solvent and the protein surface. The process is characterized by
an equilibrium binding constant K
][][
][
CNMbXe
XeK
out
in= [3.2]
Under these conditions, the observed chemical shift δobs is given by the following
equation:
l
inoutinoutobs Xe
Xe
][
][)( ⋅−+= δδδδ [3.3]
Here δin is the chemical shift of 129Xe trapped inside the protein, δout the chemical
shift of 129Xe in the other possible environments, [Xe]in the concentration of the
complexed Xenon (Xe bound to Mb), and [Xe]l the total concentration of Xenon
dissolved in solution.
Within the framework of this thermodynamic model [Xe]in can be expressed as a
function of K, and thus the variation of δobs with (nXe/Vl) is a function of δin, δout and K.
For numerical application few reasonable approximations can be introduced without
significantly alter the results: it is in fact assumed that Xenon is an ideal gas, that Henry’s
law holds, and that [Xe]out corresponds to the total concentration of Xenon dissolved in
the buffer solution, i.e. [Xe]out = [Xe]l. Due to the low protein concentration used in the
CHAPTER III – RESULTS AND DISCUSSION
56
experiment and the expected weak binding constant these approximations seem
reasonable.
The just described model can be exploited to fit the experimental variations of 129Xe NMR chemical shifts as a function of nXe/Vl in Figure 3.4 and obtain the values of
δin, δout and K. Table 3.1 collects the results of the fitting procedure.
Table 3.1: Pig, sheep, horse and rabbit CNMb equilibrium constants (K) and 129Xe chemical shifts (δin, δout)
obtained from fitting the thermodynamic model to the experimental data. The errors are fitting errors.
[CNMb] (mM) K (M -1) δδδδin (ppm) δδδδout (ppm)
Horse (1.01)
Rabbit (1.03)
Pig (0.94)
Sheep (0.90)
158±40
131±22
48±8
36±6
-18±4
-22±3
55±10
51±9
2.7±0.2
2.7±0.2
2.1±0.1
2.2±0.1
Within the experimental errors, the binding constants K relative to the complexes
of Xenon with the horse (158 M-1) and rabbit (131 M-1) CNMbs are in good agreement
with that reported in the literature7 for the sperm whale CNMb (145 M-1). The
comparison between binding constants and chemical shifts that characterize bound and
unbound Xenon species in the four myoglobins analyzed seem to group them into two
well defined classes: pig CNMb- sheep CNMb on one side and horse CNMb – rabbit
CNMb on the other, suggesting that differences at the level of the interior structure of
myoglobins are significant in determining 129Xe NMR results. This classification of the
four myoglobins considered into two well distinct groups is further confirmed by the
value of the chemical shift (δin) associated to the 129Xe bound within the cavities of the
proteins, obtained by means of the thermodynamic model applied to fit the experimental
results. δin has in fact very similar negative values for 129Xe bound to horse (δin =-18
ppm) and rabbit (δin = -22 ppm) CNMbs, while positive values characterize Xe-sheep
CNMb (δin = 51 ppm) and Xe-pig CNMb (δin = 55 ppm) complexes. Thus, the two
groups of myoglobins differ in δin of about 70 ppm. Considering, as previously stated,
that the distance-dependent hyperfine dipolar interaction between Xenon and
CHAPTER III – RESULTS AND DISCUSSION
57
paramagnetic heme Iron causes an upfield shift of the observed 129Xe signal we could
suggest that Xenon is much more influenced by the paramagnetic ion in myoglobins from
horse and rabbit than it is in myoglobins from pig and sheep. Clearly, 129Xe NMR
resonances are mirroring differences in the Xenon binding inside the proteins. In
particular, these observations likely originate from a higher affinity of Xenon toward the
proximal site of myoglobins from horse and rabbit. This hypothesis is further confirmed
by the binding constants K extracted for the four myoglobins: the equilibrium constant K
measured in the horse and the rabbit are about three times larger than in the pig and the
sheep Table 3.1. These differences can be ascribed either to changes in the size, shape
and residue composition of the cavities affecting the chemical shift of the bounded 129Xe
and the binding constant or to residues at the surface of the proteins, which differentiate
the four myoglobins analyzed.
The values of δout of Table 3.1 can be essentially attributed to non-specific
interactions of Xenon with the protein surface and Xenon in the bulk4,6. Remarkably, the
∆δout, normalized to 1mM protein, in the CNMbs from pig (2.2 ppm), sheep (2.4 ppm),
horse (2.6 ppm) and rabbit (2.6 ppm) are very alike, suggesting similar interaction
schemes of Xenon with the external surface of the proteins. All these data are in good
agreement with those found for pig (2.41 ppm) and horse (2.35 ppm) MMb4,8, reinforcing
the idea that an interaction of Xenon with the external surface of the protein can be
considered to a good approximation independent from the particular surface composition
of the studied species.
In order to more clearly define the critical factors that characterize the chemical shifts
of 129Xe observed in solutions of the different Mbs considered in this work, we need to
describe in more detail the nature of specific Xenon-protein interactions in paramagnetic
proteins.
The NMR chemical shift of 129Xe bound to the protein, δin, is the sum of three
different contributions: diamagnetic, pseudocontact (or dipolar) and Fermi contact
contributions.
δin = δdia + δdip + δcon [3.4]
CHAPTER III – RESULTS AND DISCUSSION
58
Here, δdia is due to the interactions of Xenon with the residues lining the cavity where
it is trapped in, and depends on cavity structure and shape10-13. δdip is related to the
hyperfine dipolar interactions between the Xenon and the low spin paramagnetic Fe3+ of
the prosthetic heme group and δcon is the contribution of the unpaired electron spin
density of the paramagnetic ion delocalized at the observed nucleus. When non-
covalently bound Xenon is considered, in fast exchange with the bulk solution, δcon is
zero.
Analogously, δdip is zero when the heme iron is in the oxidation state 2+ and has
spin state S=0 (no unpaired electrons). This latter situation arises when diamagnetic
carboxy myoglobin (CO-Mb) species are considered and experiments on this species
allow obtaining estimates of the diamagnetic contribution to the 129Xe NMR chemical
shift.
The high information content of low-spin cyano-myoglobins is due, among other
reasons, to the presence of substantial magnetic anisotropy that imposes significant
dipolar shift to nuclei near the active site. This property is very useful especially because
it allows the exploitation of the iron paramagnetism to probe the geometry of nonbonded
nuclei (residues and/or ligands).
In details, δdip in CNMbs is determined by the anisotropic magnetic moment of
the iron atom and can be written as a function of the polar coordinates (R, ϑ, ϕ) of the
targeted Xenon with respect to a coordinate system in which the magnetic susceptibility χ
is diagonal. The resulting equation is14:
∆+∆−= ),,(2
3),,(
3
1RFRF
N rhrhaxaxdip ϕϑχϕϑχδ [3.5]
with axial and rhombic magnetic anisotropies of the magnetic susceptibility tensor χ,
respectively
∆χax = χzz – ½( χxx+ χyy)
∆χrh = χxx – χyy
CHAPTER III – RESULTS AND DISCUSSION
59
geometric factors
3
2 )cos3(
RFax
ϑ= and 3
2 2cossin
RFrh
ϕϑ=
N is Avogadro’s number and (R, ϑ, ϕ) are the polar coordinates of the targeted
nuclei with respect to a Fe-centred coordinate system.
Equation [3.5] therefore becomes:
3
22rh
3
2ax 2cossin
2
1cos3
3 RNRNdip
ϕϑχϑχδ ∆−−∆−= [3.6]
The magnetic coordinate system adopted following Nguyen et al.14 has the
magnetic z-axis passing through the iron atom and the His93 Cα atom, tilted ~15° from
the heme normal toward the heme δ-meso position. Also the values of ∆χax and ∆χrh, 2.54
x 10-8 m3/mole and -0.62 x 10-8 m3/mole, respectively, are taken from the work on sperm
whale CNMb by Nguyen et al.14. This latter assumption is justified by the similar heme
geometry and stereochemistry of all these CNMbs15.
It is usually adopted, in order to localize nuclei within myoglobins, a coordinate
system in which the tensor χ is diagonal. Thus, the atomic coordinates (x,y,z) extracted
by X-ray crystallography are replaced by new coordinates (x’,y’,z’) of the new coordinate
system. If Γ(α,β,γ) represents the Euler rotation matrix transforming the X-ray derived
coordinates system defined above to one in which the magnetic susceptibility tensor χ is
diagonal (with components χxx, χyy, χzz), equation [3.6] becomes
),,(),,(2
3),,(
3
1 γβαϕϑχϕϑχδ Γ⋅
∆+∆−= RFRFN rhrhaxaxdip [3.7]
The Euler rotation matrix is defined by the angles α,β, and γ, according to the scheme
below.
CHAPTER III – RESULTS AND DISCUSSION
60
Figure 3.5: Schematic representation of the Euler rotation angles α, β and γ which transform the coordinate
system x,y,z in a new system referred to as ‘new x’, ‘new y’, ‘new z’.
Equation [3.7] shows that the chemical shift experienced by each nucleus depends
on its position with respect to the iron, on the magnitude of the anisotropies ∆χax and
∆χrh, which in turn relate to the electronic structure of the heme iron, and on the
orientation of the magnetic susceptibility tensor.
Ideally, if the position of Xenon atoms with respect to the paramagnetic center
Fe3+ were known, we could obtain the dipolar contribution to the observed 129Xe
chemical shift by just applying equation [3.7]. However, due to the fast exchange of
Xenon in the NMR times scales, this calculation is in fact unfeasible. Nevertheless,
knowledge of the geometric factors which characterize the four internal cavities of
myoglobins furnishes useful indication of what cavities could reasonably participate to
the binding process in Xe-myoglobin complexes.
The polar coordinates of the Xenon bound to the cavities Xe1 (R = 5.30 Å; θ =
35.75; φ = 72.20), Xe2 (R = 9.50 Å; θ = 77.14; φ = 70.85), Xe3 (R = 15.09 Å; θ =
62.69; φ = 83.58) and Xe4 (R = 8.46 Å; θ = 44.98; φ = 76.60) can be extracted by
analysing of the crystallographic structure of sperm whale myoglobin (pdb 1J52). In
particular, as it has been already stated8, according to equation [3.7] the 129Xe shift would
shift upfield for Xe1 and Xe4, and downfield for Xe2 and Xe3 relative to the chemical
shift of 129Xe shift in buffer.
CHAPTER III – RESULTS AND DISCUSSION
61
It is worth noting that by using different crystallographic structures (pdb 1MBC,
1YMB and 1MYG) of myoglobins from horse and sperm whale found in literature,
computing the location of the centre of gravity (CG) of the cavity by exploiting the
program VOIDOO (see Materials and Methods’ section) and following the procedure
outlined above we obtained similar results. Moreover, the calculation of paramagnetic
contribution to the observed 129Xe shift from geometric constraints by means of equation
[3.7] suggest that, as it was in fact expected, δdip has decreasing value in the series Xe1
>> Xe2 ≥ Xe4 >> Xe3.
As it is shown in Figure 3.3, in horse CNMb and rabbit CNMb 129Xe signal is
shifted to higher fields than in solutions containing CNMbs from sheep and pig.
However, all myoglobins induce an upfield shift to the 129Xe resonances even if to
slightly different extents.
Moreover, the values estimated for δin in horse and rabbit CNMbs via the two-site
thermodynamic model are upfield shifted with respect to Xenon in the buffer. These
observations, together with the calculations just mentioned would suggest that the
chemical shift of 129Xe is dominated by the dipolar interaction between Xenon and the
unpaired electron of low spin paramagnetic Fe3+ and typify the Xenon binding in the
proximal cavity, in agreement with crystallographic studies on structurally similar sperm
whale Mb2.
In pig and sheep CNMbs δin is downfield shifted, although it results upfield
shifted compared to Xenon binding in the diamagnetic COMbs of the same species. This
latter paramagnetic shift has been explained by the presence of Xenon in the Xe1 cavity
and potentially in the Xe4 cavity of the pig CNMb8. The fast exchange regime of Xenon
in the NMR chemical shift time scale does not allow discriminating certainly between the
paramagnetic contributions from these two sites.
CHAPTER III – RESULTS AND DISCUSSION
62
Table 3.2:* Inaccessible Cavities computed using the program VOIDOO for horse MMb (PDB code =
1YMB) and pig MMb (PDB code = 1MYG)
Cavities V a
Ra θθθθa
GFb Lining
Horse Xe1 43.7 5.3 42.9 40.9 Leu89, Ala90, His93, Leu104, Phe138, Ile142,
Heme Xe2 48.9 9.4 84.5 -11.7 Leu72, Leu104, Ile107, Ser108, Ile111, Leu135,
Phe138, Arg 139, Heme Xe3 59.9 15.6 74.1 -2.6 Trp7, Ile75, Leu76, Lys78, Lys79, Gly80,
His82, Ala134, Leu137, Phe138 Xe4 19.7 8.6 147.4 17.7 Gly25, Val28, Leu29, Gly65, Val68, Leu69
Pig Xe1 30.3 4.9 49.7 21.7 Leu89, His93, Leu104, Phe138, Met142, Heme
Xe2 23.4 9.6 92.8 -11.2 Leu72, Leu104, Ile107, Ser108, Ile111, Leu135, Phe138, Heme
Xe3 46.6 15.7 73.9 -2.0 Trp7, Ile75, Leu76, Lys78, Lys79, Gly80, His82, Ala134, Leu137, Phe138
Xe4 25.8 8.4 147.3 18.9 Gly25, Val28, Leu29, Gly65, Val68, Leu69, Ile107
a) Volume (Å3), distance from centre of gravity and iron (Å) and angles (deg) obtained from VOIDOO. b)
Geometric Factor, (3cos2θ-1)R-3 · 1026 m3. *From ref 8
3.1.2. - 129Xe NMR relaxation measurements of CNMb solutions.
Longitudinal relaxation times T1 have been measured for ~ 1 mM solutions of
CNMbs extracted from pig, sheep, rabbit and horse. For each solution, a single T1 value
fitted the experimental data, indicating that Xenon is in fast exchange, in the 129Xe
relaxation time scale, between all available environments. The 129Xe relaxation rate R1 =
1/T1 measured for buffer solution pressurized with 2 atm of Xenon is R1(buff) ~ 2x10-3 s-
1. This value, as expected, is significantly affected by the presence of low-spin
paramagnetic cyano-metmyoglobins: in particular we measured, in the same experimental
conditions, values of R1(h)=0.95±0.07 s-1 for horse CNMb, R1(r)=0.96±0.10 s-1 for rabbit
CNMb, R1(p)=0.31±0.03 s-1 for pig CNMb and R1(s)=0.36±0.06 s-1 for sheep CNMb.
The increase of 129Xe longitudinal relaxation rates of Xenon in solutions
containing low-spin (Fe3+, S=1/2) paramagnetic myoglobins in the cyano form, however,
is less pronounced than that previously measured for 129Xe in solutions of high-spin
CHAPTER III – RESULTS AND DISCUSSION
63
(Fe3+, S=5/2) Met-myoglobins (horse R1=11.4±0.1 s-1, pig R1=1.4±0.1 s-1),8 thus
substantiating the sensitivity of 129Xe NMR not only to the presence of the paramagnetic
metal ion but even to its spin state.
In order to more clearly understand and explain these experimental data we
should identify and quantitatively assess all the contributions that determine the observed 129Xe relaxation rates and in addition formulate appropriate considerations on the
experimental conditions and on the features that characterize Xe-myoglobin complexes.
It is evident that the presence of the paramagnetic ion (Fe3+, S=1/2 in CNMbs)
plays a fundamental role in determining the value of the observed spin-lattice relaxation
rate R1,obs. The contribute R1,p, merely due to hyperfine interactions between Xenon and
the unpaired electron of the paramagnetic heme iron, is superimposed to that (R1,0) given
by the diamagnetic interactions between Xenon and globins and between Xenon and the
buffer solution in which Xe and myoglobins are dissolved.
In presence of a large excess of free Xenon compared to Xenon bound to protein
cavities, as it is in our case, longitudinal relaxation rate can be expressed by the equation
[3.8]:
0,1,1,1 RRR pobs += [3.8]
Here, the observed spin-lattice 129Xe NMR relaxation rate R1,obs is given by the
sum of paramagnetic spin-lattice relaxation rate R1,p and relaxation rate R1,0, respectively
corresponding to the relaxation rate measured in presence and the absence of Fe3+
species.
Indicating τM the residence time of Xenon bound to Mb cavities, T1M the spin-
lattice relaxation time of the Xenon in the bound state, p the ratio between the total
concentration of metal ion and that of dissolved Xenon, and n the number of moles of
Xenon interacting with each paramagnetic site (i.e. with each myoglobin), equation [3.8]
becomes:
)( 10,1,1,1
MMobsp T
npRRR
τ+=−= [3.9]
CHAPTER III – RESULTS AND DISCUSSION
64
Clearly, np represents the molar fraction of Xenon complexed inside the protein
cavities
l
in
l
in
Xe
Xe
Xe
Fe
Fe
Xenp
][
][
][
][
][
][=⋅= [3.10]
In the absence of contact interactions, as is the case of Xe-myoglobin complex,
the diffusion averaged nuclear relaxation of the weakly bound 129Xe is enhanced by the
motions of both the electron spin and of the entire complex relative to the direction of the
external magnetic field. Therefore, both the longitudinal (T1M-1) and the transverse (T2M
-1)
relaxation rates of the nuclei bound in the proximity of paramagnetic sites are affected by
these motions. When such complexes are considered, where non-bound Xe freely
diffuses through protein cavities, paramagnetic enhancement of nuclear relaxation of 129Xe is merely dominated by distance-dependent dipole-dipole interactions.
The unpaired spin density of Fe3+ electrons is not usually confined to the metal
ion, but it is delocalized over the heme group. It has been stated14,16, however, that the
just mentioned π delocalization is in fact negligible in low-spin Fe(III) complexes, so that
the effects of the paramagnetic ion can be reliably approximated by a point-dipole model
even for nuclei that are very close to the iron.
Equation [3.11] represents the Solomon equation that describes dipolar
longitudinal relaxation of 129Xe bound to myoglobin, in a fast exchange condition
between all the cavities17,18 and is given by:
++
++
=2c
2S
c2c
2Xe
c6
2B
2e
2Xe
2
0M1 τω1
τ7τω1
τ3r
µg)γ1S(Sπ4µ
152
/T1 [3.11]
where r is the distance Fe-Xe, S the electron spin quantum number (S=1/2 for the low
spin Fe3+ in CNMb), γXe the gyromagnetic ratio of the observed 129Xe nuclei, µB the Bohr
magneton, ge the electron g value, µ0 the vacuum permeability, ωS and ωXe the electronic
CHAPTER III – RESULTS AND DISCUSSION
65
and nuclear (129Xe) angular Larmor frequencies in radiants per second at the operating
magnetic field, respectively19.
The correlation time (τc) is the reciprocal of a rate constant which value is
affected by different contributions, according to the following equation [3.12]:
τc-1 = τM
-1 + τr-1 + τs
-1 [3.12]
where τs-1is the electronic relaxation rate , τr
-1 is the contribution from molecular rotation,
and τM-1 takes into account the chemical exchange of the probe nucleus (Xe).
Equation [3.11] has been extensively used for determining the distance between a
paramagnetic metal ion and a probe nucleus in absence of significant trough-bond
interactions 5,20-22.
The rate constants that contribute to define the correlation time have been
previously estimated for several species and their values can be found in literature. The
electronic term τs ranges between 10-11 and 10-12 s for low spin Fe3+,23,24 while the
isotropic rotational correlation time for a molecule of the size of myoglobin ranges
between 10-8 and 10-9 s; τM should not be larger than 10-5 s.25 For low spin Fe3+ CNMb
the correlation time τs (~10-11-10-12 s) is expected to dominate τc. The value τc of 6 x 10-9
s was derived from the 15N T1 and T2 relaxation times measured for 25 α-helical residues
of a diamagnetic COMb sample16.
When 129Xe spin-lattice relaxation times T1 are measured in aqueous solutions
containing the four low-spin CNMbs from horse, rabbit, pig and sheep different
behaviors are observed and again horse CNMb and rabbit CNMb show similar behavior,
but different from that of pig CNMb and sheep CNMb.
In order to clearly define a picture of what is observed let us consider just CNMb
from horse and pig as representative species, and discuss T1 values obtained at variable
pressures of Xenon into tubes containing solutions of these two myoglobins.
Figure 3.6 shows T1 values measured at increasing Xenon pressure in the tubes
containing ~1 mM solution of CNMbs from pig and horse.
CHAPTER III – RESULTS AND DISCUSSION
66
0
1
2
3
4
0 2 4 6 8 10 12
T1 (
sec)
atm Xenon
pig CNMb
horse CNMb
Figure 3.6: T1 values measured at increasing Xenon pressures in tubes containing solutions of CNMbs
from pig and horse
Clearly, an increase in the T1 of 129Xe corresponds to an increase of Xenon
pressure in the tube containing a solution of horse CNMbs, while a reverse trend is
observed for the solution of pig CNMb. It is interesting to point out again that CNMbs
from pig and sheep show similar behavior and the same can be said for the couple rabbit
CNMb and horse CNMb (data not shown). A detailed examination of the data obtained
can be carried out by exploiting the thermodynamic model adopted for the interpretation
of 129Xe chemical shifts. This model, in fact, allows discerning between interactions of
Xenon with the internal region of the proteins and all the other available environments.
CHAPTER III – RESULTS AND DISCUSSION
67
0
0.2
0.4
0.6
0.8
1
1.2
0 0.01 0.02 0.03 0.04 0.05 0.06 0.07
R1o
bs(s
-1)
[Xe]in
/[Xe]tot
pig
horse
rabbit
sheep
Figure 3.7: Variation of the observed 129Xe spin lattice relaxation rate as a function of the ratio of
concentration of Xenon complexed within internal cavities and total Xenon concentration in ~1 mM
solution ([Xe]in/[Xe] tot) of CNMbs from pig, sheep, horse and rabbit.
In Figure 3.7 are plotted the 129Xe R1obs corresponding to Xenon in horse, rabbit,
sheep and pig CNMbs as a function of the ratio [Xe]in/[Xe]tot.
This ratio can be estimated by using7 K = 145 M-1 for horse and rabbit, K = 48 M-1 for
pig and K = 36 M-1 for sheep, and the two-site thermodynamic model previously
described [see page 55].
While in horse and rabbit CNMb the 129Xe relaxation rate increases with
increasing concentration of Xenon bound to the internal cavities, a slight decrease is
observed for the 129Xe relaxation rate as a function of Xenon bound ([Xe]in/[Xe]tot) in pig
CNMb and sheep CNMbs.
An additional very important consideration must be done at this point as adopting
the variable [Xe]in/[Xe]tot may introduce misunderstandings in regard to its correlation
with Xenon pressure in the tube: it is important to note that due to the relatively low
concentration of myoglobins in solution, increasing Xenon pressure results in a decrease
CHAPTER III – RESULTS AND DISCUSSION
68
of [Xe]in/[Xe]tot, as bound Xenon species ([Xe]in) have a very low concentration with
respect to non-bound Xe, while Xenon dissolves in solution in the amount of ∼4 mM/atm.
We can now hypothesize that the residence time of Xenon within the proximal cavity of
the four myoglobins analyzed is the same and, precisely, identical to that previously
measured25 (~ 10-5 s) for sperm whale met-myoglobin. Thus, 1/(T1M+τM) in equation [3.9]
reduces to 1/T1M and this value, which is the longitudinal relaxation rate of 129Xe bound
to the Mb, can be determined for the 129Xe in the four Mbs analyzed.
As expected, the extracted values are grouped in two blocks: horse-rabbit couple
together as well as pig-sheep do. More precisely, 1/(T1M) = 9.18 ± 0.28 s-1 and R1,0 = 0.45
± 0.01 s-1 for the horse CNMb, 1/(T1M) = 9.41 ± 0.71 s-1 and R1,0 = 0.39 ± 0.03 s-1 for the
rabbit CNMb, 1/(T1M) = -0.64 ± 0.24 s-1, R1,0 = 0.35 ± 0.01 s-1 for the pig and 1/(T1M) = -
0.29 ± 0.13 s-1, R1,0 = 0.36 ± 0.01 s-1 for the sheep.
The negative values of 1/(T1M) elicited for the latter group are, of course, non
physical, thus indicating that the starting hypothesis on the residence time of Xenon
within proximal cavity is not reliable.
The increasing 129Xe relaxation rate actually observed as a function of increasing
Xenon pressure in pig and sheep CNMbs (see Figure 3.7) suggests that the Xenon
population in the proximal cavity increases significantly as a function of Xenon pressure.
Because of the 1/r6 dependence in the relaxation rate (equation [3.11]), Xenon in the
proximal cavity, with a CG-Fe distance of 4.9 Å (as determined by the program
VOIDOO) in the pig MMb8, mainly contributes to the average relaxation rate, being the
other cavities 8.4, 9.7 and 15 Å far from the iron centre.
The unphysical values extracted from fitting the relaxation rates measured on pig
and sheep CNMbs might therefore arise from a different distribution of Xenon among
their internal cavities than that in horse and rabbit CNMbs. In the former pair of Mbs this
ratio does not favour the Xe1 as much as in the latter.
The results previously obtained by observing 129Xe NMR parameters4,5,8 together
with those discussed here clearly indicate important differences in the affinity of Xenon
towards internal cavities of myoglobins, they are infected by some problems, mostly
derived from the fast exchange condition of Xenon between all available environments.
CHAPTER III – RESULTS AND DISCUSSION
69
Testing the ability of Xenon as an efficient biomolecular probe of cavities in
globular proteins and, particularly, in myoglobins (Mbs) is crucial to extract important
information on their structure and function, but at the same time challenging due to the
presence of different interaction sites besides the heme iron. 129Xe NMR measurements
so far performed have given therefore exchange-averaged results and lacked of the site-
specific information needed in order to more clearly understand the role played by
structure, shape and composition of proximal cavity in regulating Xenon affinity.
Among the techniques adopted in the characterization of myoglobins, NMR has
proven its suitability in investigating these model systems. The possibility to easily tune
the oxidation state of the metal ion (Fe) in myoglobins gives the unique opportunity to
study interactions of different physical nature in the same protein, which are sensed by
both the guest and the host itself.
In particular, for low-spin cyano myoglobins (CNMbs) most of 1H signals of the
residues in the active site have been unambiguously assigned26. These low-spin systems
have been the subject of extensive study because of the excellent resolution and narrow 1H NMR lines even for the protons close to the iron27-30. It is well known that both
contact and pseudocontact shifts contribute to the observed proton NMR chemical shifts
in paramagnetic species: a number of studies have been carried out so far aiming to
understand and quantitatively separate these two contributions in myoglobins and model
heme compounds22,31-33. Moreover, many efforts have been spent to determine the effect
of the orientation of the magnetic axes of the susceptibility tensor χ and of their
modifications in point mutants of CNMbs on the dipolar contribution to the observed
NMR chemical shifts14,34-39.
The extremely high information content of low-spin (S=1/2) CNMbs can be
exploited with the aim of deepening the role played by internal cavities in these proteins.
The presence of significant magnetic anisotropy imposes large dipolar shift to nonbonded
residues in the active site and allows exploiting iron paramagnetism to probe the
geometry of distal and proximal regions of the heme cavity, thus providing valuable
information on possible ligand-induced modifications of the host matrix. Most
importantly, 1H NMR measurements on myoglobin solutions pressurized with Xenon gas
CHAPTER III – RESULTS AND DISCUSSION
70
allow obtaining site-specific information on the residues close to the iron, i.e. lining the
active site.
3.1.3. - 1H NMR chemical shift in CNMbs from horse and pig.
Figure 3.8 collects 1H NMR spectra of CNMbs from horse and pig and the effect
of 10 atm of Xenon on the chemical shifts of hyperfine-shifted protons. It is evident, even
at a first sight, that the presence of 10 atm Xenon overpressure causes marked changes in
the proton NMR spectra of CNMbs.
Further discussion is needed to point out important distinctions that must be made
to explain the results concerning different protons and myoglobins from different sources.
Tables 3.3 and 3.4 collect the 1H chemical shift of CNMb from horse and pig in
the absence and presence of 10 atm of Xenon, compared to the chemical shift of the
corresponding protons in sperm whale CNMb26.
1216202428 -10-8-6-4-20
Pig
Pig+Xe
Horse
Horse+Xe
5-C
H3
8-C
H3
1-C
H3
His
93 N
δH
His
93 C
εH2-
Hα
Phe
43 C
ζH
His
93 N
pH
Ile99
Cγ1
H
Ile99
CδH
3Ile99
Cγ2
H3
Val
68 C
αHThr
67 C
γH3
Leu1
04 C
δH3
1H (ppm)1H (ppm)
Figure 3.8: 1H NMR spectra in the low-field (from 11 ppm to 29 ppm) and high-field (from -10 ppm to 0
ppm) regions for ~1 mM solutions of CNMbs from horse and pig in the absence and presence of 10 atm of
Xenon overpressure. Labelling of some signals is made on NOESY spectra and based on the assignments
of Emerson and La Mar26.
CHAPTER III – RESULTS AND DISCUSSION
71
Signal assignment is based on the published data on the sperm whale CNMb and
on two-dimensional spectra COSY, TOCSY and NOESY14,26. Some of the assigned
peaks of horse CNMb and pig CNMb are indicated in the mono-dimensional 1H NMR
spectra.
As observed for Sperm Whale CNMb26, 1H NMR spectra of Cyano-
MetMyoglobins from pig and horse show three methyl signals in the region 18-13 ppm,
assigned to 5-CH3, 1-CH3, and 8-CH3 heme groups. In the spectral region from 23 ppm to
9 ppm many one-proton signals are observed and attributed to α-type protons of the heme
substituents and to protons pertaining to the proximal histidine. Some resonances of other
residues present in the active site are also detected in this spectral region, due to the
magnetic anisotropy of the low-spin iron system. In the low-frequency region signals are
resolved in the -1 to -10 ppm range.
CHAPTER III – RESULTS AND DISCUSSION
72
Table 3.3: 1H NMR chemical shifts relative to Heme and His93 protons observed in pig CNMb and horse
CNMb solutions in the absence and the presence of 10 atm of Xenon. The 1H chemical shifts of sperm
whale CNMb are reported for comparison26.
Chemical shift (ppm) Resonances Proton Sperm Whale Horse Horse/Xe Pig Pig/Xe
Heme 1-CH3 18.62 18.37 17.87 18.49 18.41 3-CH3 4.76 4.39 3.82 4.04 3.88 5-CH3 27.03 27.16 26.54 27.52 27.24 8-CH3 12.88 13.49 12.17 13.20 12.75 2-Hα 17.75 17.88 18.43 17.75 17.95 2-Hβc -1.73 -1.49 -1.76 -1.36 -1.52 2-Hβt -2.55 -2.42 -2.64 -2.37 -2.50 4-Hα 5.50 5.53 5.75 5.72 5.62 4-Hβc -1.95 -1.77 -1.24 -1.97 -1.73 4-Hβt -0.77 -0.59 -0.10 -0.81 -0.61 6-Hα 9.18 9.27 9.84 9.23 9.47 6-Hα’ 7.35 7.52 8.10 7.86 8.00 6-Hβ 1.67 1.58 1.78 1.26 1.39 6-Hβ’ -0.48 -0.41 -0.76 7-Hα 1.13 1.43 1.64 1.14 1.20 7-Hα’ -0.45 -0.33 -0.01 -0.35 -0.27 7-Hβ 1.55 1.49 1.32 1.33 1.41 7-Hβ’ 0.78 0.50 0.62 0.71 0.65 Hα 4.40 4.41 2.60 4.32 4.21 Hβ 2.09 2.38 2.94 2.55 2.69 Hγ 5.98 6.08 6.73 6.32 6.05 Hδ 4.40 4.13 3.79 3.59 3.79
His93 CαH 7.51 7.41 7.49 7.49 7.48 CβH 11.68 11.54 11.13 11.61 11.59 CβH’ 6.34 6.46 6.33 6.45 6.45 NpH 13.20 13.78 13.57 13.92 13.88 CδH -4.70 -4.80 -4.58 -5.05 -4.98 NδH 20.11 21.20 21.37 21.15 21.13 CεH 19.20 18.90 19.82 18.65 18.66
CHAPTER III – RESULTS AND DISCUSSION
73
Table 3.4: 1H NMR chemical shifts of residues lining the active site of pig and horse CNMbs in the
absence and presence of 10 atm of Xenon in the NMR tube. The 1H chemical shifts of sperm whale CNMb
are also reported26.
Chemical shift (ppm) Residues Proton
Sperm Whale Horse Horse/Xe Pig Pig/Xe Leu29 Cδ2H 5.53 5.63 5.56
CγH 3.90 3.76 3.90 4.07 4.03 Phe33 CεH 8.32 8.36 8.33 8.31 8.27 Phe43 CεH 12.58 12.42 12.38 12.41 12.40
CζH 17.27 17.16 17.21 16.96 16.91 Phe46 CδH 7.69 8.23 8.19 His64 NpH 8.45 8.68 8.67
CδH 11.61 12.40 12.37 12.43 12.34 NεH 23.70 23.46 23.31 22.57 22.56
Val67 CαH 2.37 2.42 CβH 0.87 0.89 CγH3’ 0.09 0.10 CγH3 -1.93 -1.86
Thr67 CαH 2.47 2.52 2.51 CβH 2.66 2.79 2.78 CγH3 -1.59 -1.63 -1.64
Val68 CαH -2.55 -2.21 -1.78 -2.32 -2.19 CβH 1.42 2.03 2.08 2.00 Cγ2H3 -0.97 -0.61 -0.54 -0.53 -0.60 Cγ1H3 -0.81 -0.61 -0.54 -0.53 -0.60
Ala71 CαH 3.48 3.49 3.55 2.85 2.90 CβH3 -0.12 -0.18 -0.08 -0.20 -0.20
Leu89 Cδ2H3 3.25 3.18 2.87 CαH 8.53 8.64 8.64
Ala90 CαH 6.50 6.46 6.52 6.55 6.54 CβH3 2.63 2.66 2.66 2.79 2.78
Ser92 NH 11.04 11.04 11.03 11.01 11.00 His97 CδH 11.07 11.03 11.01 11.02 11.00
CγH 6.83 6.82 6.83 Ile99 CδH3 -3.83 -3.63 -3.34 -3.74 -3.63
CγH3 -3.46 -3.19 -3.18 -3.30 -3.28 CγH’ -1.91 -1.77 -1.22 CγH -9.60 -9.18 -8.71 -9.44 -9.18
Leu104 Cδ2H3 -1.49 -1.31 -1.45 -1.36 -1.37 CδH3 0.07 0.12 0.04 CγH 0.07 0.06 0.15 0.35 0.34
Ile107 CγH3 -0.25 -0.36 -0.25 -0.26 -0.27 CδH3 0.37 0.34 0.25 0.38 0.37
CHAPTER III – RESULTS AND DISCUSSION
74
Phe138 CδHs 7.05 7.08 7.11 7.12 7.12 CεHs 6.94 7.00 7.11 7.06 7.06 CζH 7.02 7.08 7.11 7.16 7.15
Tyr146 CδHs 7.20 7.41 7.48
Interestingly, most of the hyperfine shifted proton signals in the horse CNMb (see
Fig 3.8 and Tables 3.3 and 3.4) are remarkably shifted upon addition of 10 atm of Xenon
overpressure in the tube. The same behaviour is observed, although less pronounced, in
the 1H NMR signals for the pig CNMb. This evidence is clearer when 1H NMR chemical
shift variation induced by Xenon binding into cyano-metmyoglobins from pig and horse
is plotted as a function of the concentration of Xenon in the NMR tube.
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0 0.5 1 1.5 2
nXe/Vl (M)
∆δ∆δ ∆δ∆δ
1 H (
pp
m)
5CH3 Ho
5CH3 Pig
CαH Val68 Ho
CαH Val68 Pig
CδH
3 Ile99 Ho
CδH
3 Ile99 Pig
Figure 3.9: Total Xenon-induced proton NMR chemical shift for CNMb (~1 mM) from horse and pig at
room temperature as a function of the total number of moles of Xenon in the NMR tube divided by the
volume of solution (nXe/Vl). The open symbols pertain to the pig CNMb while the full symbols to the
horse CNMb. 5-CH3 - Circle; CδH3 Ile99 - rombus; CαH Val68 – square.
CHAPTER III – RESULTS AND DISCUSSION
75
Fig. 3.9 shows NMR chemical shift variation of protons signals assigned to some
residues lining the active site and/or belonging to the heme methyl groups of horse and
pig CNMbs upon addition of Xenon (1 to 10 atm overpressure of Xenon). The data
plotted in Fig. 3.9 are extracted either by a series of mono-dimensional 1H NMR spectra
and/or by a series of two-dimensional NOESY spectra.
Observation of the proton chemical shift variation scheme as a function of the
concentration of Xenon in tubes containing solutions of CNMbs from pig and horse
allows making further interesting considerations. Xenon-induced 1H chemical shift
changes are less significant in absolute value in pig CNMb than in horse CNMb.
Furthermore, this variation is linear for pig-CNMb while in horse-CNMb ∆δ first rapidly
increases at low concentration of Xe and then asymptotically reach a saturation value for
higher concentrations. The overall change of proton NMR chemical shift (∆δ 1H) upon
Xenon addition depends on each particular proton considered and reaches for the heme
methyl 8-CH3 of horse CNMb the significant value of 1,32 ppm, which is the maximum
value observed.
In order to more clearly describe what is observed, it is useful to briefly discuss
the relevance of each different contribution to the experimental chemical shifts in
myoglobins.
NMR shifts in paramagnetic molecules are, as has been explained above, the sum
of diamagnetic, pseudocontact (dipolar) and Fermi contact contributions.
These three contributions, however, have very different significance in
characterizing observed proton chemical shifts in the paramagnetic myoglobins studied
here.
For what concerns the diamagnetic part, 1H NMR experiments on the diamagnetic
horse COMb (spectra not shown) yielded small variations (~ 0.05 ppm) in the δdia of the
His93 and porphyrin protons in the presence of 10 atm of Xenon. This observation is in
agreement with a relatively small effect of Xe on 1H NMR chemical shift ∆δdia (< 0.1
ppm) generally observed in diamagnetic proteins40,41. The only exception to our
knowledge concerns the studies on T4 lysozyme where a 1H chemical shift change of
approximately 0.2 ppm was observed in the protons assigned to some side chain
residues42,43.
CHAPTER III – RESULTS AND DISCUSSION
76
Such values are however considerably smaller than the shift observed in the
present study concerning cyano-metmyoglobins and therefore Xenon-induced variation
of the diamagnetic contribution to the observed proton chemical shift variation are not
sufficient to explain experimental results.
Thus, this suggests that the observed 1H chemical shift changes upon Xenon
addition should be considered to be merely influenced by the hyperfine interaction
between the proton and the unpaired electron of the heme iron and ascribed mostly to
dipolar and contact contributions. On the other hand, hyperfine shifts are determined by
very short-ranged interactions, and it could be reasonably assumed that the part of protein
> 7.5 Å far from the iron center is, for all practical purposes, diamagnetic. Hyperfine
shifted resonances in a region roughly enclosed within a sphere of about 7 Å radius and
centred at the iron ion, are very sensitive probes for the environment of the active site.
Since in this region of the protein reside most of the residues that have been considered
essential for the functionality of the protein44, i.e. those aminoacids that are mainly
implied in the ligand binding process, knowledge of the structure of this region and
characterization of possible ligand-induced conformational distortions would be very
relevant because they would provide new important insights into the processes of ligand
diffusion and binding in hemoproteins.
In order to more clearly understand the origin of Xenon-induced 1H chemical shift
variation in myoglobins, and to further characterize molecular details of Xe-induced
distortions of the protein side-chains at the proximal binding site, it is useful to consider
which structural features could reliably influence the observed 1H chemical shifts.
Equation [3.7] correlates the dipolar chemical shift experienced by each proton
with its polar coordinates with respect to the iron-centred coordinate system in which the
magnetic susceptibility tensor χ is diagonal, i.e. with orientation of the tensor itself. The
pseudocontact shift δdip affects both ligated and nonligated residues according to equation
[3.7] and is strongly characterized by the anisotropic magnetic susceptibility tensor χ.
This latter dependence is very important: the analysis of various CNMb point
mutants38 as well as of model compounds45 has in fact demonstrated that it is in turn
strictly related to structural features of the heme cavity In this regard, some NMR studies
of myoglobin mutants38 aimed to the determination of the orientation of the magnetic
CHAPTER III – RESULTS AND DISCUSSION
77
axes to understand the relationship between the region of the protein where the mutation
is located and the changes in orientation of the tensor χ. It was demonstrated that point
mutations in the distal side can have effect on the orientation of the Fe-ligand axis (e.g.
Fe-CO, Fe-CN) and consequently on the principal axis of the magnetic susceptibility
tensor (described by the angle β), while mutations that interest the proximal side39
influence only the orientation of the rhombic axes of the tensor (described by the angle
κ=α+γ) , either causing a rotation of the heme or of the proximal histidine.
In particular, the orientation of His93 imidazole ring with respect to the heme iron
has been indicated as a critical factor in determining this perturbation in heme proteins:
π-bonding between proximal histidine and Fe-heme porphyrin has been proposed as a
likely reason for this rhombic perturbation observed in myoglobins. Any change in the
observed proton chemical shifts could therefore be traced back to the displacement of the
His93 imidazole plane with respect to the heme. It should also be noted that while a
correlation does exist between axial His plane and rhombic axes, it is still unclear if this
structural modification is the only determinant of the rhombic axes.
Thus, a reliable quantitative assessment of this dependence can potentially
provide valuable structural information of local distortions in CNMbs.
Moreover, any consideration regarding Fe-1H dipolar interactions in paramagnetic
proteins should additionally take into account that observed proton signals experience
sometimes both contact and dipolar shifts simultaneously. Evaluating both contributions,
and quantitatively separating them, appears as an important issue in order to achieve a
correct interpretation of NMR results.
It has been shown in previous studies concerning low-spin
ferrihemoproteins39,46,47 that the heme Hmeso protons exhibit both contact and dipolar
shifts; it was also pointed out that the measured asymmetry in the meso-H hyperfine
patterns, calculated from the equation
[ ]δγβα δδδδδ −+−=∆2
1meso [3.13]
CHAPTER III – RESULTS AND DISCUSSION
78
is quantitatively predicted by the rhombic dipolar shifts, being the contact contributions
to their observed chemical shift very similar to each other. Thus, calculation of the
observed ∆δmeso would lead to better understand possible rhombic (in-plane) magnetic
axes displacements related to local structural rearrangements attributable to the presence
of Xenon.
The experimental ∆δmeso of 1.99 ppm that we can calculate for Horse-CNMb in
absence of Xenon [see Table 3.3] is in relatively good agreement with that previously
found in wild type sperm-whale CNMbs (∆δmeso =1.8 ppm)39. The ∆δmeso decreases to 1.3
ppm in horse CNMb after adding 10atm of Xenon gas, thus suggesting that the rhombic
axes have rotated clockwise relative to the heme, implying that κ is to some extent
increased by the presence of Xenon in the proximal cavity of the protein. These
observations suggest that proximal His93 has rotated counterclockwise with respect to
the stationary heme. This hypotesis can be further substantiated by NOE measurements,
as it is discussed in the following. Additional quantitative information can be easily
obtained: as previously suggested, a plot39 of calculated ∆δmeso versus κ indicates a
gradient of 2.5 ppm per 10 degrees of rotation; this correlation allows achieving a
quantitative rough estimate of the relative rotation of the proximal histidine with respect
to the heme group. The calculation suggests a counterclockwise rotation of His93 of
about 2.8 degrees (viewed from the proximal side).
CHAPTER III – RESULTS AND DISCUSSION
79
3.1.4. - NOE measurements used as a tool to further assess His93 rotation relative to
heme.
A small perturbation of the protein structure such as the rotation of proximal
histidine by less than 10 degrees relative to the heme is considered a local phenomenon
and it was suggested not to influence the remaining structure of the heme cavity. Such
rotation in turn can be easily monitored in low-spin cyano myoglobins by following the
inter-residue dipolar contacts between the rotating residue (His93) and backbone protons
on the E and F helices. Moreover, looking at steady-state NOEs upon saturating either the
heme protons or the His93 protons, allows discriminating between a rotation of the heme
and the rotation of the His93, both leading to a relative displacement of one of them with
respect to the other.
-2,4-2-1,6-1,2-0,8-0,40
1H (ppm)
Val
68 C
αHT
hr67
CγH
7-α'
Ala
71 C
βH
8-CH3
8-CH3 + Xe
(a)
Figure 3.10 (a): NOE spectra of horse CNMb solutions obtained irradiating the 8-CH3 signal. The
spectrum below is relative to the solution of the protein without Xenon and the upper spectrum refers to the
CNMb solution pressurized with 10 atm of Xenon. Arrows indicate correspondent signals in the two
spectra, shifted by the presence of Xenon within the protein cavities.
CHAPTER III – RESULTS AND DISCUSSION
80
891011121314151H (ppm)
+28%
-13% -23%
His
93 N
pH
Leu8
9 C
αH
His
93 C
βHS
er92
NH
NδδδδH93
NδδδδH93 + Xe 8-
CH
3
(b)
Figure 3.10 (b): NOE spectra of horse CNMb solutions obtained irradiating the His93(F8) NδH proton
signal. The spectrum below is relative to the solution of the protein without Xenon and the upper spectrum
refers to the CNMb solution pressurized with 10 atm of Xenon. Percentages represent changes in the signal
intensities. Peak assignments are reported above most relevant signals.
Figure 3.10 (a) shows that steady-state NOEs resulting from saturating 8-CH3 in
samples pressurized with 10 atm of Xenon gas are essentially the same as for degassed
solutions, indicating an unchanged position of the heme with respect to the protein matrix
in the heme cavity. The absence of significant changes has been confirmed by
deconvolution of the signals (not shown). The absence of significant changes has been
confirmed decomposing the signals into individual gaussians by means of the OriginPro
7 program. Figure 3.10 (b) compares steady-state NOEs without and with 10 atm of
Xenon, obtained upon saturating His93(F8) NδH. The spectrum of myoglobin pressurized
with Xenon shows some new signals and various modifications in signal intensities with
CHAPTER III – RESULTS AND DISCUSSION
81
respect to the degassed myoglobin sample. In particular new signals appear at 11.03 ppm
and 12.10 ppm, assigned to Ser92 NH and 8-CH3, respectively. Decreases in the
intensities of the signals pertaining to Leu89 CαH (∼-23%) and to His93 CβH (∼-13%) are
observed in the spectrum of the Mb-Xe complex with respect to that of the degassed
solution. Moreover, the signal assigned to His93 NpH has an increased NOE (∼+28%)
after Xenon addition.
Considering the R-6 dependence of the NOE, simple calculations lead to the
conclusion that His93 NδH is displaced by ∼4% further from Leu89 CαH by the Xenon
residing in the proximal cavity (Xe1). Given the His93 NδH - Leu89 CαH distance in
XRD structure of horse Mb (pdb code 1YMB) is 4.25 Å, a 23% decrease in signal
intensity in NOE spectra is indicative of an increase of this distance to approximately
4.43 Å. In the same way it can be calculated a decrease of the distance His93(F8) NδH –
His93 NpH of ∼4% (analogous calculations based on XRD crystallographyc structures
suggest a distance change from 2.99 Å before Xe pressurization to 2.87 Å after Xe
addition). These observations are in very reasonable agreement with the new signals
visible in the NOE spectrum of pressurized samples and they can be easily understood by
taking a closer look to the structure of the proximal cavity and to the positions of the
residues just mentioned. Fig 3.11(a) and 3.11(b) will certainly help to this end.
CHAPTER III – RESULTS AND DISCUSSION
82
a) b)
Figure 3.11: Proximal side of myoglobins complexed with Xenon. Heme group is shown in licorice style,
Xenon is a yellow ball. (a) Atoms belonging to His93, Ser92 and Leu89 are depicted as balls and sticks and
(b) relative positions of heme group, proximal histidine and Xe1 are reported. Carbon atoms coloured cyan,
Nitrogens in blue and Oxygen in red.
In Figure 3.11, the yellow ball is Xenon in the proximal binding site (Xe1). Figure
on the left [Fig 3.11(a)] shows a particular of the proximal cavity of myoglobins where
residues His93, Leu89 and Ser92 are evidenced by ball and stick representation while
Heme group is represented in licorice style. In particular, atoms Leu89 CαH, Ser92 NpH
and His93NδH are marked in the figure. Figure on the right [Fig 3.11(b)] shows a detail
of the proximal heme cavity: His93 atoms NδH, CβH and NpH are marked. Analysis of
these figures confirms what suggested by NOE measurements obtained by irradiating
His93 NδH, i.e. that the effect of Xenon in the Xe1 cavity on axial histidine - (His93 , F8)
is most likely to displace it toward the δ-meso-H heme side. Whether this displacement is
a translation of the whole F8 residue or a counterclockwise rotation (viewed from the
proximal side) of the imidazole ring about the His93 NεH-Fe bond is uncertain but the
latter hypothesis is the most reasonable, given the strong Fe-His coordination bond.
Further confirmation of the tilt/rotation of His93 ring toward the heme δ-meso-H comes
from the observation of NOE enhancement of the proton signal belonging to 8-CH3
CHAPTER III – RESULTS AND DISCUSSION
83
(12.5ppm) after pressurization with Xenon, which is not present in the absence of Xenon
[see Fig 3.10(b)].
A method that allows to characterize even small differences in the proximal
region of myoglobins from different species and to determine changes induced by
exogenous ligands would represent a very important tool to study the biological function
of the protein. It is in fact recognized that proximal histidine has an important role in
modulating the reactivity of heme proteins: it has been demonstrated by means of a
number of techniques that a displacement of proximal His upon ligand dissociation, also
referred to as doming vibrational mode, plays a fundamental role in characterizing the
reactivity of this type of biological molecules. Proximal histidine acts as a trigger for
structural changes leading to cooperative transition in hemoglobin48. It has also been
shown that CO binding kinetics are modified in proximal mutants of myoglobins with
respect to wild type39,40. It has been also suggested in early studies that complexation
with Xenon and other small molecules such as cyclopropane49 influence the binding
affinity of carbon monoxide in myoglobins. Another important aspect to be underlined is
the relevance of the interaction between His93 and Ser92 residues. It has been shown that
the hydrogen bond linking the hydroxyl group of Ser92 to the NεH of His93 has an
important role in the protein activity. It was observed in this regard that solution studies
yield different results with respect to crystal structures likely due to the existence of some
substates51 in the structure of the proximal region of myoglobin.
CHAPTER III – RESULTS AND DISCUSSION
84
3.1.5. - Thermodynamics of Xenon binding to cyano-metmyoglobins from Xenon-
induced 1H NMR chemical shift variations.
For each assigned peak of the horse and pig CNMb spectra, the 1H chemical shift
variation, ∆δ, as a function of Xenon concentration is fitted by the two-site model4:
[ ]
[ ] max1δδ ∆
+=∆
XeK
XeK [3.14]
Here K is an equilibrium binding constant, [Xe] the concentration of Xenon in the
buffer4, and ∆δmax the chemical shift difference between the complexed and free protein
(degassed solution).
It is interesting to note that equation [3.14] resembles Langmuir adsorption
equation commonly used in gas adsorption on the surface of solid materials.
Only the fitting results having correlation function F larger than 0.99 and standard error
of the calculated K smaller than 10% were considered.
In the horse CNMb, 34 out of 70 1H chemical shift variations associated to assigned
proton signals are well described by the two-site thermodynamic model: 21 proton
signals belong to the porphyrin ring and the other 13 to several residues around the heme
region. The remaining 36 1H signals shift only negligibly or show a correlation function
F lower than 0.90. Table 3.5 contains some of the K values extracted from the fitting
analysis.
CHAPTER III – RESULTS AND DISCUSSION
85
Table 3.5: Values of the binding constant K for the residues of horse CNMb extracted from the fitting of the 1H signals.
Resonances Protons K (M -1) r
1-CH3 126±9 0.9983 3-CH3 96±9 0.9976 5-CH3 127±10 0.9987 8-CH3 90±3 0.9998 2-Hα 80±7 0.9996
2-Hβc 106±10 0.9995
2-Hβt 89±8 0.9989
4-Hβc 85±7 0.9986
4-Hβt 94±9 0.9993
6-Hα 89±3 0.9997
6-Hα’ 93±7 0.9992
6-Hβ 137±13 0.9986
6-Hβ’ 158±11 0.9995
7-Hα 154±15 0.9976
7-Hα’ 87±7 0.9973
7-Hβ 81±8 0.9934
7-Hβ’ 88±8 0.9961
Hα 119±10 0.9986
Hβ 103±10 0.9991
Heme
Hδ 80±10 0.9967 CαH 80±6 0.9988
CβH 110±7 0.9991
CβH’ 112±10 0.9991
NpH 85±4 0.9992
NδH 147±11 0.9995
His93
CεH 94±8 0.9986 CδH3 116±9 0.9978
CγH’ 133±12 0.9938 Ile99
CγH 80±7 0.9910 CγH 159±10 0.9981
Ile104 Cδ2H3 145±9 0.9991
Ile107 CδH3 94±9 0.9938 Val68 CαH 98±9 0.9982
CHAPTER III – RESULTS AND DISCUSSION
86
The 8-CH3 (∆δ=1.32 ppm) and Hα (∆δ=1.81 ppm) protons of the porphyrin ring exhibit
the maximum chemical shift variations.
The binding constant K, as derived from the individual fitting of the 34 signals,
ranges between 80 M-1 and 150 M-1, while fitting all chemical shift variations on the
whole (software package Origin from Microcal) leads to K=109 ±7 M-1. ∆δmax lies
between 2.1 and -0.69 ppm, revealing non-negligible proton chemical shift variations
when Xenon is bound to the protein.
Negligible shifts or linear trends characterize the 56 assigned proton signals in the
pig CNMb when Xenon is added up to 10 atm overpressure in the tube. 24 signals have a
shift larger than 0.05 ppm, 19 belonging to the porphyrin ring and the others to the CδH3
and CγH group of Ile99, to CαH and Cγ2H3 of Val68 and to CδH of Val64. The maximum
∆δ is associated with the 8-CH3 (∆δ=0.45 ppm) and Hα (∆δ=0.37 ppm) protons of the
porphyrin ring [see Table 3.3]. The chemical shift variation of these signals changes
linearly upon Xenon addition. Using equation [3.14] to fit these chemical shift variations
we estimate a binding constant K lower than 5 M-1 for pig CNMb samples, while K=109
M-1 is obtained for the horse CNMb.
The use of a single K value in a linear regime can be justified by the 129Xe spin
lattice relaxation rate, which clearly indicates the presence of Xe in the proximal cavity.
CHAPTER III – RESULTS AND DISCUSSION
87
3.1.6. - Myoglobins: CONCLUSIONS
The present work summarizes the results of our efforts addressed to the
challenging issue of characterizing the structural properties of the cavities in different
paramagnetic myoglobins in solution. The application of 129Xe NMR to the investigation
of biomolecules has proven to be very useful in providing essential information on their
structure and dynamics involved in the binding process.
The analysis of paramagnetic proteins in solution is time consuming and requires extreme
care in the preparation of the sample. Moreover, the presence of a paramagnetic ion
hinders the application of laser-polarized Xenon and the hyperfine couplings between the
unpaired electron of the metal and the neighboring atoms significantly influence the
NMR results. We have shown here that these strong interactions, far from representing a
drawback of the technique, can be exploited to study molecular details the Xe-protein
interactions in solution. The study combines the NMR analysis of the proton nuclei lining
the cavities within the host protein and the 129Xe that probes all the available
environments in a fast exchange condition.
The analysis of the 129Xe chemical shift and the spin lattice relaxation time
monitors features of Xe-Mb interactions. The Xe binding characteristics are found to be
strongly influenced by the interior structure and by the hydrophobicity of the proximal
(Xe1) cavity as evidenced by the comparison of the data obtained for pig (K=48 M-1;
δin=55 ppm; rFe-Xe=6.8 Å) and sheep (K=36 M-1; δin=51 ppm) CNMbs with those of
horse (K=158 M-1; δin=-18 ppm; rFe-Xe=5.3 Å ) and rabbit (K=131 M-1; δin=-22 ppm )
CNMbs. The agreement between 129Xe and 1H NMR findings indicates that the residue
substitution in the proximal cavity of Mbs causes a different distribution of Xenon inside
the protein. In myoglobins from horse and rabbit, Xe1 is the main binding site, while in
pig and sheep Mbs there is evidence of the presence of Xe in the Xe1 cavity and most
likely in Xe3, as competing binding site of Xe1. This suggested assignment is based on a
previous study where a second xenon binding site was detected in an NMR study25 in
solution and it was believed to be located in a cavity close to the surface of the protein
In particular, the major novelty brought by the present work concerns the detailed
discussion of of the 129Xe/1H - NMR method used to describe the cavities close to the
CHAPTER III – RESULTS AND DISCUSSION
88
active site of paramagnetic myoglobins, which are supposed to play a fundamental role in
the protein functionality.
It appears clear from this study that the Xenon population of the proximal cavity,
i.e. Xenon affinity for this region of the protein, is strongly influenced by the structure
and the hydrophobicity of the cavity itself.
It is demonstrated that proton chemical shift variations in these systems are
understandable considering changes in the orientation of the principal magnetic axes of
the CNMb-Xe complexes. Besides complementing 129Xe NMR results with site-specific
information, 1H NMR measurements help to achieve a picture of Xenon-induced local
distortions of the protein. Xenon-induced local distorsions of the protein structure are
spotlighted and associated to a residue located right at the active site. According to the 1H
NMR data, Xenon induces the tilt of the residue His93 relative to the heme plane and
consequently causes an alteration of the magnetic axes. These findings have, in our
opinion, very general involvements, since structural modifications of the cavities and
structural perturbations of the ligand tilt could not only affect the kinetics of ligand
binding, but also determine relative affinities, and consequently the physiological
function of the myoglobin
CHAPTER III – RESULTS AND DISCUSSION
89
3.2. Copper-containing amine oxidases enzymes: Xenon-induced
reactions
3.2.1. - Lens Esculenta Amine oxidases (LSAO) in solution: 129Xe NMR chemical
shifts
Several Amine Oxidases have been investigated in this work in order to clarify
the interaction of this class of enzymes with Xenon in solution. Our studies first
concentrated on the characterization of one of them, Lens esculenta Amine Oxidase
(LSAO), with the attempt to characterize the main features of Xe-enzyme complexes. In
particular the work has focused on the spectroscopic characterization of Xe-enzyme
interaction and on the challenging issue of understanding the relationship structure-
function in these complex biological systems.
Figure 3.12 shows 129Xe NMR spectra of solutions of LSAO and buffer, both
pressurized with 10 atm of Xenon gas. The spectra were collected 48 hours after Xenon
addition.
-15-10-505101520
ppm
Buffer
LSAO 0.33 mM
Figure 3.12: 129Xe NMR spectra of a solution (Na+-phosphate buffer 1 mM, pH 7.0, 20% D2O) of 0.33 mM
LSAO pressurized with 10 atm of Xenon gas. Shifts refer to the chemical shift of 129Xe dissolved in buffer.
CHAPTER III – RESULTS AND DISCUSSION
90
The 129Xe NMR signal in the solution containing the LSAO enzyme is shifted
downfield with respect to the resonance of Xenon in buffer, which has been fixed to 0
ppm and used as a reference value of chemical shifts.
The presence of a single resonance in protein solution indicates that Xenon is in
fast exchange, in the NMR time scale, between all the available environments, i.e.
enzyme cavities, enzyme surface and solvent.
Further characterization of the interaction between Xenon and LSAO can be performed
by monitoring the 129Xe chemical shift, spin lattice relaxation time T1 and NMR signal
linewidth. These parameters were measured as a function of both Xenon and protein
concentration.
By increasing LSAO concentration from 0.26 mM to 3.4 mM, while keeping
constant at 8 atm the Xe pressure, the 129Xe NMR resonance is shifted downfield from
0.33 ppm to 2.8 ppm.
Xenon NMR chemical shift are also influenced by Xenon pressure: 129Xe NMR
signal is shifted at 2.5 ppm and 2.9 ppm at 5 atm and 10 atm of Xenon respectively, at
constant protein concentration (0.25 mM LSAO). 129Xe line width, taken as the full width at half maximum (fwhm), increased with
increasing protein concentration (104 Hz and 63 Hz at 0.37 mM and 0.26 mM of LSAO
concentration, respectively) and showed for all the studied concentrations of enzyme
larger fwhm values than the signal of Xenon in buffer solution.
The 129Xe spin lattice relaxation times (T1) in the LSAO and Copper–free LSAO were
measured at 25°C and at the Xenon gas overpressure of 10 atm. The T1 value (3.2 ± 0.5 s)
measured in the LSAO solution (0.3 mM) is longer than the T1 measured in Copper–free
LSAO (18 ± 2 s-1). However, both the T1 values were much smaller than the spin-lattice
relaxation time of Xenon in buffer (T1 ~ 500 s).
All these observations point out that an interaction between Xenon and the LSAO
actually exists which modulates the observed 129Xe NMR parameters: these features were
in fact observed in other protein solutions and resulted from the fast exchange of Xenon
between both specific and non-specific sites of the protein and the buffer 4,8,25,52-54.
The measurement of shorter longitudinal (spin-lattice) relaxation times in LSAO
compared to the T1 observed in Copper-free enzymes suggests that most likely additional
CHAPTER III – RESULTS AND DISCUSSION
91
relaxation mechanisms associated with the presence of the metal ions characterize Xe-
LSAO interaction. This observation could be further evaluated by explaining the
molecular mechanism of Xe-protein interaction and describing the intermediate species
involved in the enzymatic activity of LSAO.
3.2.2. - Spectral changes in the UV-vis region of LSAO solutions induced by
substrates and Xenon.
600500400300
0.2
0.15
0.1
0.05
0
Wavelength (nm)
Abs
orba
nce
Figure 3.13: UV-vis spectra of native LSAO solution (32 µM of active sites in 1 mM sodium phosphate
buffer, pH 7.0) in anaerobic conditions before () and after (---) addition of 100 µM benzylamine.
Electronic absorption spectra of LSAO show characteristic features of oxidized
Cu-TPQ (resting enzyme) cofactor and of its reduced form, depending on the
experimental conditions. In anaerobiosis, the oxidized form is observed in absence of the
substrate and its typical spetrum is reported in Figure 3.13. A broad absorption band in
the UV region with maximum at 278 nm is ascribed to aromatic amino acids. In addition,
LSAO has a characteristic pink color due to the presence of the oxidized TPQ cofactor,
which has a broad absorption band around 498 nm in the visible spectrum. The extinction
CHAPTER III – RESULTS AND DISCUSSION
92
coefficients ε at 498 and 278 nm are determined to be 4.5 mM-1 cm-1 and 245 mM-1 cm-1,
respectively, for LSAO55.
The addition of 1 mM benzylamine to a solution containing 16 µM LSAO (32 µM
active sites), in the absence of air, caused the immediate changes in the spectra, which
can be explained considering the reaction path reported Scheme 3.1 below.
The bleaching of the 498 nm absorption band indicates the rapid formation of a
reduced TPQ intermediate and it is in particular associated with the formation of the CuII-
quinolaldimine (IV) (see Scheme 3.1). Oxidation of the bound substrate (followed by
hydrolysis) releases the aldehydic product, leaving the CuII-aminoresorcinol derivative
(V), which has a bound ammonia molecule. This species is still colorless. The CuII-
aminoresorcinol is in equilibrium with the yellow, EPR-detectable CuI-semiquinolamine
radical (VI) , containing the substrate-derived nitrogen covalently bound to the aromatic
ring system56-58, and characterized by absorption bands at 464, 434 and 360 nm.59,60 The
extinction coefficients of reduced LSAO at pH 7 and 298 K are: ε464 = 7.1 mM-1 cm-1,
ε434 = 4.6 mM-1 cm-1.61
CHAPTER III – RESULTS AND DISCUSSION
93
Scheme 3.1: Molecular reaction pathway so far described for plant Amine Oxidases
CHAPTER III – RESULTS AND DISCUSSION
94
In our experiments, approximately 30% of the active LSAO enzyme was
converted into the radical, which was determined by ESR measurements of the CuII to
CuI ratio (not shown). The amount of benzaldehyde produced was measured to be 1.0 ±
0.1 mol mol–1 LSAO subunit [see Fig 3.14].
40200
40
20
0
LSAO (nmol active sites)
Pro
duct
(nm
ol)
Figure 3.14: Anaerobic product release. Formation of aldeyde () and ammonia () at different LSAO
concentrations. Slope of the benzaldehyde line is 1 nmol benzaldehyde of LSAO subunits.
It is clear from Figure 3.14 that while aldeyde is a product of the catalytic process
of LSAO in presence of benzylamine in anaerobiosis, ammonia is not formed at this
stage.
Readmission of oxygen rapidly regenerated the oxidized TPQ cofactor with
release of hydrogen peroxide and ammonia.
In order to characterize the effect of Xenon on the spectral features of the enzyme
solution, i.e. on the catalytic process of LSAO, a LSAO solution (1 ml solution; 26 µM
LSAO active sites) has been pressurized with 10 atm of Xenon gas in absence of oxygen
and without any amine substrate. After approximately six hours spectral changes in the
CHAPTER III – RESULTS AND DISCUSSION
95
UV-vis region appear [see Fig 3.15]: the broad absorption at 498 nm starts to disappear
and simultaneously characteristic absorption bands become detectable suggesting that the
radical CuI-semiquinolamine radical is formed. The reaction is over after 48 h and the
quantitative analysis of absorbances at 464 and 434 nm shows that more than 60% of the
native enzyme was converted into the radical form.
600500400
0.12
0.08
0.04
Wavelength (nm)
Abs
orba
nce
600500400300
0.2
0.1
0
W avelength (nm )
Abs
orba
nce
Figure 3.15: Uv-vis spectra of native LSAO acquired as a function of time after pressurization with 10 atm
of Xenon gas. Arrows indicate formation of the radical species and disappearance of the oxidized TPQ
form. The spectrum of LSAO-Xe complex after 48 hrs is reported in the small panel.
Ammonia production was not detected under 10 atm Xenon gas overpressure in
anaerobic condition. After readmission of Oxygen the absorption spectrum of oxidized
TPQ was rapidly recovered and the formation of approximately 2 mol of ammonia and 2
mol of hydrogen peroxide were detected per mol of LSAO.
As a comparison, two more LSAO samples, prepared with the same procedure as
those treated with Xenon or the substrate, have been respectively kept in presence and
absence of Oxygen and analyzed after 48 hours: their UV-vis spectra do not show
CHAPTER III – RESULTS AND DISCUSSION
96
modifications of the characteristic absorption at 498 nm suggesting that the observation
of spectral absorptions typical of the reduced Copper-TPQ form can be only ascribed to
the presence of either the amine substrate or Xenon.
Xenon does not cause significant modifications in the secondary or tertiary
structure of LSAO as suggested by measurements on Xenon-treated enzymes by means
of CD spectroscopy, tryptophan fluorescence and fluorescence of the hydrophobic probe
1-anilinonaphtalene 8-sulphonate, which is sensitive to microenvironmental changes of
proteins (spectra not shown). Also X-ray crystallographic studies so far performed on
Xe*AOs complexes have pointed out that only minor structural arrangements of the
protein structure are observable upon Xenon binding.
When a second cycle is performed, i.e. when Xe-treated LSAO is exhaustively
dialyzed and pressurized again with 10 atm of Xenon, neither the radical is observed nor
the production of ammonia and hydrogen peroxide.
On the other hand, Xe-treated LSAO, after being exhaustively dyalized, is able to
react again with an amine substrate, thus forming the radical species (spectra not shown)
in anaerobic conditions and producing after readmission of Oxigen hydrogen peroxide
and ammonia, thus confirming the similarity of native and Xe-treated LSAO enzymes
regarding their activity toward amine substrates.
In order to more clearly describe the catalytic process of AOs and aiming to get
further insight on the role of Copper ion on the redox process, Copper-free enzymes were
also studied. Copper can be removed from native LSAO by treatment with
diethyldithiocarbamate62. In our experiments, the residual copper, measured by atomic
absorption spectroscopy, was measured to be 0.2 ± 0.02% of the original content.
A band at 480 nm chacterizes the visible spectrum of Copper-free LSAO as it is shown in
Figure 3.16.
After treatment with 10 atm Xenon the absorbance relative to the band at 480 nm
of the apoprotein progressively decreased in intensity, clearly indicating the formation of
the colorless aminoquinol, without formation of the characteristic bands of the radical at
464 nm and 434 nm. A new band at around 360 nm instead appeared reaching its
maximum after 24 h [see Fig. 3.16]. Quantitatively analyzing the absorbance at 480 nm,
CHAPTER III – RESULTS AND DISCUSSION
97
we calculated that about 50% of the apoenzyme remained in its oxidized form after Xe
treatment.
600400
0.3
0.2
0.1
0
Wavelength (nm)
Abs
orba
nce
Figure 3.16: Absorption spectrum of a solution of Copper–free LSAO (40 µM active sites) pressurized
with 8 atm of Xe. It is shown with arrows the bleaching of the absorption band at 480 nm and the appearing
of a new band at 360 nm, which indicates the formation of aminoquinol.
Thus, Copper-free enzyme under 10 atm Xenon, clearly unable to form the radical
species due to the absence of the metal ion necessary to allow the electron transfer, to
some extent forms the aminoquinol species (≈ 50%). Reliably, the absorption band
around 360 nm [see Fig. 3.16] is due to the formation of a neutral form of the product
Schiff base quinolaldimine, similarly to what has been already observed in model
compounds and in bacterial Copper amine oxidases63. This observation, moreover, would
suggest that there is a back reaction between aminoquinol and the allysine residue,
according to the Scheme 3.2 below.
CHAPTER III – RESULTS AND DISCUSSION
98
-O
OH
N
CH
H
CH2
CH2
CH2
CHCO NH
HO
OH
NH2
HO
OH
N
CH
CH2
CH2
CH2
CHCO NH
(a)(b) (c)
CH O
CH2
CH2
CH2
CHCO NH
Scheme 3.2: Proposed Xe-induced reaction in Copper–free LSAO enzyme. (a) aminoquinol; (b)
quinolaldimine, protonated form; (c) quinolaldimine, neutral form. It is worthnoting that in Copper–free
enzyme the radical is not formed and the quinolaldimine accumulates (λ = 360 nm) over the time of
treatment.
Therefore, quinolaldimine species, well characterized in bacterial amine oxidases
so far, is for the first time isolated and evidenced also in plant enzymes.
In conclusion, while it has been proposed that in some AOs, such as BSAO,
Copper would have just a structural role64,65, mainly interfering in the electrostatic
stabilization of oxygen, the results obtained here rule out this possibility in plant amine
oxidases and in contrast they show that Copper is certainly involved in the amine
oxidation process.
CHAPTER III – RESULTS AND DISCUSSION
99
3.2.3. - Involvement of a lysine residue in the intra-molecular catalytic mechanism of
LSAO
The observation just described of the activation of LSAO enzyme in the absence
of its substrate led to hypothesize the role of intramolecular reactions induced by the
presence of Xenon atoms inside the hydrophobic cavities of the protein. The side chains
near the active site appear to be likely responsible for the reaction. The candidate residue,
in order to act as intramolecular enzymatic substrate, must clearly mimic the amine
substrate, thus the choice should be restricted to glutamines, asparagines and lysines,
which contain an amine group at the end of their side chain. Between these three amino
acids, lysine appears to be the most eligible because of its relatively long hydrophobic
chain which makes it the the less sterically hindered among the three of them.
LSAO contains 38 lysines in each subunit66. Attempts to identify the lysine
residue converted into allysine under Xenon pressure were carried out by proteolytic
digestion with trypsin and lysyl endopeptidase, followed by HPLC analysis. This
approach, however, revealed to be unsuccessful at a first attempt, as it resulted in very
complicated elution profiles making not easy the unequivocal identification of the
modified lysine.
Moreover, 129Xe NMR experiments cannot provide a more detailed
characterization of the interaction between Xenon and the protein, and the actual location
of a possible cavity or cavities involved remains unknown and would require further
studies that should involve NMR structural determination and signal assignments. These
achievements are however still far from what NMR technique is able to accomplish
nowadays. The dimer of LSAO is in fact formed by more than 1860 residues, which are
way too many to allow a complete structural characterization in solution by NMR, even
with the most advanced instrumentations. The primary limitation in determining protein
structure by NMR is in fact the size of the protein. The size limitation for complete
atomic-resolution structure determination by NMR is currently ~30 kDa, though
backbone assignments and general folds have been described for proteins up to 100 kDa.
Few considerations may help in formulating reliable hypothesis on molecular
mechanisms involved in the catalytic process of AOs.
CHAPTER III – RESULTS AND DISCUSSION
100
A lysine residue, Lys296, located at the active site of Pea Seedling Amine
Oxidase has been established to be structurally important from X-ray crystallographic
studies of the enzyme.
a) b)
Figure 3.17: Relative positions of Copper, TPQ cofactor and Lysine 296. (a) Top view and (b) side view.
The Cu is represented as an orange sphere, TPQ in red licorice style and lysine is in licorice with Carbon
atoms coloured cyan, Nitrogens in blue and Oxygen in red. Pictures are derived from crystallographic
structure of PSAO (PDB code 1KSI)72
The X–ray crystallography of PSAO has demonstrated67 that Lys296 makes a
hydrogen bond with the phenolic group at C–4 of TPQ [see Figure 3.17]. This residue is
located into domain D4, between β-sheet C5 and α helix H8, close to the entrance to a
channel which has been found to be suitable for the movement of substrate and products
to and from copper/TPQ active site buried in the protein interior. The hydrogen bond
between TPQ and a lysine residue is typical of plant amine oxidase while it has never
been observed in crystals of other AOs.68-71. The role of Lys296 is not yet precisely known
but it has been suggested that it might favor the deprotonation of the TPQ contributing to
the observed differences among the catalytic efficiencies of several AOs72.
The internal surface of the channel above mentioned is lined by residues which become
more hydrophobic as the active site is approached. This would make the channel suitable
for Xenon, which is known to prefer hydrophobic regions73. A direct experimental
evidence that Xenon binds to several sites has recently been obtained in the crystal of
various AOs67. Only one of Xe–binding site is closest to Cu/TPQ center. In pea seedling
AO the Xe atom is bound in a pocket at 7.7 Å from the copper atom and 9.3 Å from TPQ;
CHAPTER III – RESULTS AND DISCUSSION
101
the nearest neighbor amino acid residues are Ile405, Leu407, Tyr446, Ile601, Leu616 and
Thr618.
Since the identity in amino acid sequences of LSAO and PSAO72,74,75 is about
92% it should be safe to accept that both enzymes have an almost identical structure.
Moreover, PSAO and LSAO are similar in functional properties,60 and this would be
compatible with their structural similarity. As observed in PSAO72 the Copper ion and
TPQ are in close proximity (6 Å) but they are not coordinated. Moreover a small
displacement of the TPQ would be required to facilitate the extremely rapid electron
transfer CuII–aminoquinol/CuI–semiquinolamine, and the TPQ side chain appears to be
sufficiently flexible to accommodate this change. A considerable conformational
flexibility of TPQ is also proposed when an amine substrate attacks at C–5 of TPQ and
H+ abstraction of the active site base Asp300 requires that TPQ rotates by 180 degrees.
Figure 3.18: XRD crystallographyc structure of PSAO Copper in the so called ‘on copper’ conformation67, suggested to be the only productive conformation relative to the catalytic activity of AOs.
In considering the results obtained, we propose a possible reaction pathway that
would explain the results observed. The suggested catalytic process is reported in Scheme
3.3. In native LSAO, under 10 atm Xenon, a movement of either the TPQ cofactor or an
α helix containing a lysine residue may occur making the C-5 of TPQ closer to ε-amino
group of this residue [see Scheme 3.3], and it is well known that TPQ side chain appears
to be sufficiently flexible. The quinoneketimine intermediate (2) occurs between TPQ
CHAPTER III – RESULTS AND DISCUSSION
102
and ε-amino group of lysine and accumulates during the lag phase. Then, the
quinoneketimine (through intermediate species like the CuII-carbanion and the CuII-
quinolaldimine (3) forms the colorless reduced CuII-aminoquinol derivative (4) which is
in equilibrium to the yellow radical intermediate (5). Quantitative assessment of the
radical species from UV-visible spectra indicates that more than 60% of the Xe-treated
enzyme is in the reduced radical form, suggesting that both monomers in a dimer can
generate the radical. Readmission of oxygen causes the release of ammonia from the
reduced TPQ that is converted to the oxidized form (1’), together with the simoultaneous
formation of hydrogen peroxide. As a consequence, the lysine residue must be converted
into allysine by oxidative deamination.
In order to confirm the proposed Xe-induced molecular mechanism the oxidative
deamination of a lysine residue was monitored by the detection of α-amino-adipic-δ-
semialdehyde-fluoresceinamine derivative (AASF) by HPLC in native Xe-treated LSAO
and Copper-free Xe-treated LSAO.
In Xe-treated LSAO a peak was detected with retention time about 9.7 min and
was identified as AASF by comparison with a Lys homopolymer-fluoresceinamine
derivative as standard. One mol of allysine residue per mol of monomeric enzyme was
detected (not shown). The allysine containing LSAO is unable to regenerate the radical
species under 10 atm Xenon in the absence of the substrate in anaerobiosis. This clearly
demonstrates the relevance of a lysine residue at the active site in the observed Xe-
induced chemical reaction in the plant enzyme oxidases analyzed. It is however very
tough to exactly localize the residue, due to the complexity of the systems.
CHAPTER III – RESULTS AND DISCUSSION
103
Scheme 3.3: Proposed intramolecular reaction pathway that would explain our experimental results.
Finally, in addressing the usefulness of 129Xe NMR spectroscopy in the
characterization of biological compounds in solution, it must be pointed out that these
systems are generally characterized by complex structures and often by the presence of
more than one specific site for ligands and/or substrates. The nearest neighbor residues of
the bound Xenon atoms in the cavities are predominantly nonpolar side chains, but they
include polar side chains and backbone peptide groups. This, together with the fact that
the observed 129Xe chemical shift is dynamically averaged among different binding sites
and at the same time interacts with the protein surface, makes it difficult to separate the
individual contributions so as to show whether a particular Xenon-binding site is
responsible for the different components observed in the AOs studied in solution.
CHAPTER III – RESULTS AND DISCUSSION
104
Hyperfine interactions with unpaired electrons in radical species and/or paramagnetic
metal ions could be a further source of information, as long as their effect on 129Xe NMR
observables can be distinguished from other structural or dynamic factors affecting NMR
parameters. These 129Xe NMR outputs cannot provide local information on the host–
guest interaction involved. Experimental evidence of the fast diffusion of Xenon within
AOs clearly opposes the static and average pictures given by single-crystal XRD
structures, which seem to show that Xenon atoms are localized at specific sites.
Moreover, it is worth noting that, as the single crystal XRD results utterly ignore the
fundamental dynamic features involved in the functionality of the biomolecules in
solution, hypotheses on biological activities based on crystal structures should be
considered critically.
X-ray crystallography, on the other hand, does not suffer from the size restrictions
of NMR, with protein size having no direct bearing on the solvability of the protein or
protein complex. This is at least partly why most protein structures have been determined
by X-ray rather than NMR. The limitation of X-ray crystallography is its static nature.
This means that only a single structure can be determined and any protein movement
during data collection results in decreased resolution. Indeed, in many structures there are
segments of the protein that are so disordered they are not contained in the structure.
Aiming to confirm the role of a lysine residue in the enzymatic reaction process of
Amine Oxidases we have investigated the binding of Xenon to AOs purified from various
sources, and our results strongly support the hypothesis that a lysine residue is implicated
in the catalytic mechanism of plant enzymes. In particular, three AOs [pea seedling AO
(PSAO), Euphorbia Characias AO (ELAO) and pig kidney AO (PKAO)] were tested by 129Xe NMR spectroscopy and optical spectroscopy.
CHAPTER III – RESULTS AND DISCUSSION
105
3.2.4. - 129Xe NMR of PKAO, ELAO and LSAO solutions.
Chemical shifts.
Figure 3.19 shows 129Xe NMR spectra of PKAO, ELAO LSAO solutions
compared to the 129Xe NMR spectrum of Xenon dissolved in buffer solution. A single 129Xe NMR resonance in these solutions indicates that Xenon experiences fast exchange
between all the available environments, i.e. protein cavities, protein surface and solvent:
in this condition the observed NMR parameters are a weighted average of the values of
the same parameters that would be observed in each of all the possible sites in absence of
exchange.
When protein solutions are pressurized in NMR tubes with 10 atm of Xenon gas,
the 129Xe NMR signal in AO samples is shifted downfield as compared to the resonance
of the same amount of Xenon in the buffer, which is used as a reference and set to 0
p.p.m.
Due to intrinsic difficulties of the purification process, only limited and variable
quantities of these enzymes could be succesfully purified. Observed chemical shifts must
be analyzed therefore also taking into account the concentration of each solution
analyzed. Measured chemical shifts in the different protein solutions analyzed are as
follows: ELAO 3.96 p.p.m. per 0.35 mM, corresponding to 11.3 p.p.m.·mM-1; and PKAO
1.4 p.p.m. per 0.15 mM, corresponding to 9.4 p.p.m.·mM-1.
CHAPTER III – RESULTS AND DISCUSSION
106
-12-8-40481216
ppm
ELAO
LSAO
PKAO
Buffer
Figure 3.19: 129Xe NMR spectra of Xenon. 129Xe (10 atm) spectra in a solution (Na+–phosphate buffer 1
mM, pH 7.0, 20% D2O) containing 0.35 mM ELAO, 0.28 mM LSAO and 0.15 mM PKAO. Shifts refer to
the 129Xe chemical shift dissolved in buffer. Spectra were collected approximately 48 hours after Xe
addition.
Relaxation time T1.
A single value of longitudinal relaxation times (T1) has been sufficient to fit
experimental results, confirming that Xenon atoms are in a fast exchange condition in the
time scale of NMR longitudinal relaxation. The T1 measured for all native enzymes was
found to be much shorter than the T1 value of Xenon in the buffer (ELAO T1 = 4.3 ± 0.5
s; PKAO T1 = 5.5 ± 0.8 s; buffer T1 = ∼500 s). These features, which were also observed
in LSAO and other protein solutions, confirm that there is an interaction between the
dissolved Xenon and the interior of the protein. However, the actual location of the
possible cavity or cavities involved in the binding of Xenon remains unknown.
The relatively high enzyme concentrations (0.25– 0.35 mM) used and the low
ionic strength (1 mM) of the phosphate buffer in the experiments on solutions of Xenon-
enzyme complexes have led to the formation of inactive precipitates. We were therefore
unable to obtain reliable results from bovine serum AO (BSAO).
CHAPTER III – RESULTS AND DISCUSSION
107
3.2.5. - Spectroscopic features induced by amine substrates and Xenon in several
AOs.
Due to the slightly different amino acid composition and structural conformation
of the three AOs considered, slightly different absorption bands characterize the UV-
visible spectra of BSAO, PKAO and ELAO. BSAO shows an electronic absorption band
at 476 nm (ε476 = 3800 M-1·cm-1)76, PKAO at 490 nm (ε490 = 4000 M-1·cm-1)77, PSAO and
LSAO at 498 nm (ε498 = 4100 M-1·cm-1)78,79, and ELAO at 490 nm (ε490 = 6000 M-1·cm-
1)80.
Spectra of evacuated solutions of native highly purified enzymes and spectral
changes in the UV-visible region observed after the addition of the amine substrate are
reported in Figure 3.20 and Figure 3.21. Immediately after the addition of the substrate,
in the absence of air, the visible absorption band that characterizes spectra of native AOs
disappears, indicating the formation of a reduced TPQ intermediate. However, while in
plant AOs (LSAO, ELAO and PSAO) this reduced TPQ form equilibrates with the
yellow-coloured semiquinolamine radical by transferring one electron to Copper, in
mammalian AOs a different behaviour is observed. In PKAO the transformation of the
radical species can be observed only in the presence of cyanide ions81 [Fig. 3.21(b)]. On
the other hand, BSAO, that does not form the radical species during the normal catalytic
cycle65, stayed in the reduced aminoquinol form.
600500400300
0.2
0.15
0.1
0.05
0
Wavelength (nm)
Abs
orba
nce
A
600500400300
0.2
0.1
0
Wavelength (nm)
Abs
orba
nce
B
Figure 3.20: UV-visible absorption spectra of LSAO and PKAO and spectral changes induced by amine
substrates. (A) 16 µM native LSAO in 1 mM Na+–phosphate buffer, pH 7.0, in anaerobic conditions before
(---) and after () addition of 10 mM putrescine. (B) 19 µM PKAO in 100 mM Tris/HCl buffer, pH 7.2,
before (---) and after () addition of 10 mM cadaverine in anaerobic conditions and in the presence of 100
µM CN–.
CHAPTER III – RESULTS AND DISCUSSION
108
600500400300
0.3
0.2
0.1
0
Wavelength (nm)
Abs
orba
nce
A
600500400300
0.15
0.1
0.05
0
Wavelength (nm)
Abs
orba
nce
B
Figure 3.21: Absorption spectra of native ELAO and PKAO and spectral changes of their solutions
pressurized with 10 atm Xenon gas. Conditions: (A) 11 µM ELAO and (B) 19 µM PKAO in 1 mM Na+–
phosphate buffer, pH 7.0, in 10 atm Xenon. Spectra of the reduced forms () were recorded after 48 h.
As previously discussed, when a solution containing LSAO (10 mM) is
equilibrated with 10 atm of Xenon gas without a substrate and in absence of air, the
semiquinolamine radical form can be observed after a lag period. Similar behavior was
observed with AOs from pea seedlings and E. characias latex ELAO and from pea
seedling PSAO.
Interestingly, the results we obtained with mammalian proteins were different. For
PKAO81, where the semiquinolamine radical appears in the presence of the substrate and
CN–, bleaching of the 490 nm band started with a marked time lag (∼ 6 h) after addition
of 10 atm of Xenon gas [Fig. 3.21(b)] but that the radical species formed neither in the
presence nor in the absence of Cyanides. As observed in plant enzymes, the absorption
spectrum of oxidized TPQ was recovered after readmission of oxygen, and 1 mol of
ammonia and 1 mol of hydrogen peroxide per mole of active site were detected.
In BSAO, no changes in the spectral features were observed in presence of 10 atm of
Xenon gas, indicating that the TPQ cofactor remained in its oxidized form.
CHAPTER III – RESULTS AND DISCUSSION
109
Interesting experimental observations come from following reactions of Xe-
treated enzymes with amine substrates, in particular in regard with the reduced catalytic
activity of the enzymes toward specific substrates. Although the general behaviour of Xe-
treated enzymes very closely resembles that of native species, catalytic activity
(described by the kinetic constant kc [sec-1]) of Xenon-treated LSAO towards diamine
putrescine was shown to be about 40% of that of the native LSAO, whereas the kc for
monoamine aromatic benzylamine does not change.
Decreased activity was similarly detected in Xe-treated PSAO and in ELAO. The
catalytic activity of Xenon-treated PKAO towards cadaverine and benzylamine was
shown to be about 20% of that of the native enzyme. Xenon-treated BSAO, which is
unmodified by the effect of Xenon and retains its oxidized form, showed the same
activity as the corresponding native enzyme.
These results are most likely related to two distinct molecular mechanisms of
catalytic activity in enzymes from mammalian and plant sources. We suggest that in the
plant enzyme the ε-amino group of Lys296 may interact with the positive charge of the
amino group of putrescine. This lysine residue could have an important role in conferring
substrate specificity to the enzyme; therefore, the transformation of this lysine into
allysine would have important implications in the catalytic efficiency.
CHAPTER III – RESULTS AND DISCUSSION
110
TPQ
Asp300 Lys296
H3N+ CH2 CH2 CH2 CH2 NH3
+
Asp300
TPQ
COO-NH3
+
COO- CHOAllys296
H3N+ CH2 CH2 CH2 CH2 NH3
+
Allys296 CHOCOO-
TPQ
Asp300
H3N+ CH2
Figure 3.22: Active site of plant AOs. The model of the active site shows the possible interaction of native
and Xe-treated AOs with two substrates: benzylamine, which represents a substrate with an apolar chain,
and putrescine, with a positively charged amino group. The positively charged ε-amino group of lysine
exerts a repulsive force towards substrates characterized by the presence of a positively charged amino
group, such as putrescine, leading to a lower catalytic efficiency of the enzyme when lysine is transformed
into allysine. Neither lysine nor allysine can interact with the apolar chain of benzylamine, suggesting that
this amino acid residue is responsible for the different substrate specificities.
As stated above, X-ray crystallography of PSAO has demonstrated that a lysine
residue, the Lys296 located in domain D4, forms a hydrogen bond with the phenolic
group of TPQ when in an unproductive on-copper conformation72 while its role in the
off-copper conformation is still unknown.
Currently, new forms of PSAO native protein crystal are available67 in the so-
called ‘off-copper conformation’. In this structure, the O-4 of TPQ is hydrogen bonded to
the hydroxyl group of conserved tyrosinyl residue Tyr286, and the TPQ orientation is in
the active form, with the aspartic active site base residue (Asp300) in an excellent
CHAPTER III – RESULTS AND DISCUSSION
111
position for abstraction of the Cα proton from the substrate, so that TPQ does not rotate
during the catalytic mechanism.
Figure 3.23: Relative positions of TPQ cofactor, Asp300, Tyr286, Copper atom (orange), and Xenon atom
in crystallographyc structure of PSAO*Xe complex. TPQ cofactor is in the so called ‘off Copper’
conformation.67
Lys296 is conserved in LSAO as well as in ELAO (Lys302), while in BSAO, the
residue corresponding to Lys296 of LSAO is Thr381. However, an arginine is present at
position 382 [Fig. 3.24].
Figure 3.24: Relative position of TPQ cofactor, Copper atom and Arg382 residue in BSAO enzyme from
crystallographic studies
CHAPTER III – RESULTS AND DISCUSSION
112
The amino acid sequence of PKAO is unknown. However, considering its
significant homology with known reported sequences82 there may be a threonine residue,
as in human kidney AO (Thr369). In this case, the lysine residue that flanks the threonine
(Lys370) could react with TPQ. This would further substantiate the importance of a
lysine residue in the active site for the formation of the radical semiquinolamine species
in plant enzymes and of the aminoquinol in PKAO due to Xenon inclusion. For what
concerns BSAO, no lysine residue can be found close to TPQ and only arginine Arg382
would be able to react with TPQ cofactor such as to mimic the amine substrate. However,
as the arginine residue in BSAO possesses a highly basic guanidine group, it is expected
to be unreactive with TPQ under Xenon pressure.
A nucleophilic residue has been shown with certainty to be involved in the
inhibition mechanism of AOs during the oxidation of 1,4-diamino-2-butyne (DABY)83-87
and other selective AO inhibitors have been tested. It has been demonstrated that the
product of the reaction between plant AOs and DABY is a very reactive aminoallene
given by DABY oxidation. This species reacts with an essential nucleophilic group at the
enzyme active site, forming a covalently bound pyrrole and producing an inactive
enzyme
The possible relevance of a lysine residue has been suggested84,87, but no
experimental evidence has been presented so far.
The structure–function of an enzyme can be successfully studied finding specific
inhibitors and following their effects on the catalytic process. Our interest in the present
study has been the mechanism-based inhibitor 2-butyne-1,4-diamine (DABY), for
basically two reasons: Firstly, the inhibitor has been found to be a suicide substrate for
plant copper AO (Cu-AO) from pea seedlings84 and grass pea85, for mammalian AOs
from pig kidney81 and from beef serum87; secondly, it has been postulated that the
irreversible inhibition of all the enzymes mentioned involves an intermediate
aminoallenic compound that forms covalently bound pyrrole in the reaction with a
nucleophile at the active site.
CHAPTER III – RESULTS AND DISCUSSION
113
The exact mechanism of inhibition is unclear, and it was only in grass pea AO
that the involved nucleophile was identified as Glu113, a residue corresponding to a
Lys113 in PSAO84. DABY was also shown to be a mechanism-based inactivator for
native LSAO and ELAO. All the Xenon-treated AOs were inactivated by the reaction
with DABY, clearly indicating that the lysine residue involved in the reduction of TPQ
under Xenon pressure is not the nucleophilic residue involved in the DABY inhibition
mechanism; i.e. the reactive turnover product of DABY binds an amino acid residue
without interfering with the TPQ function.
3.2.6. - Copper containing Amine Oxidases: CONCLUSION
As it clearly appears from our results, Xenon is capable of inducing a structural
modification in a number of Amine Oxidases, such that most of them react with one of
their own lysine residues.
As previously reported, changes in active site architecture and charge distribution
seem to be critical during catalysis in AOs. Even relatively subtle conformational
changes at the active site may significantly alterate the biological function of proteins and
enzymes. These experimental observations represent a rare demonstration of Xe-induced
chemical reactions and suggest that using Xenon as a biomolecular probe must be
carefully evaluated for each particular system.
In particular, we have demonstrated, for the first time, the formation of two important
enzyme intermediates:
• the radical species in absence of a substrate in native LSAO, PSAO, ELAO and
PKAO enzymes, and
• the quinolaldimine in the Copper-free LSAO enzyme.
Moreover, hints on the role of Copper ions in the catalytic mechanism of several AOs
have been proposed and substantiated: while in BSAO Copper has a structural role
interfering in the electrostatic stabilization of oxygen, and the CuI-semiquinone state is
off the reaction pathway. The results obtained rule out the possibility that in plant amine
CHAPTER III – RESULTS AND DISCUSSION
114
oxidase Copper has just a structural role but it is certainly involved in amine oxidation
process and the CuI/semiquinolamine radical represents the highly reactive species with
Oxygen. This confirms the possibility that more than one oxidative reaction pathway is
available in AOs from different sources.
Finally we propose the role of a lysine residue that seems to play an important
role in assisting enzyme catalysis.
The TPQ/semiquinolamine radical represents the highly reactive species with the
Oxygen molecule in the catalytic cycle of plant AOs. Thus, the radical species observed
under 10 atm of Xenon without a substrate in plant AOs, and the fact that in these
systems always a lysine residue was identified at the active site, revealed key aspects of
the structure–function relationship among the various AOs studied. The transformation of
a lysine residue, most likely Lys296, into allysine, together with a residue identified in a
conserved aspartate residue (Asp300), could play an important role in the selectivity of
AOs towards substrates with a positively charged amino group.
In conclusion, although the data reported in the present article may well be valid
generally, the exact location and nature of the observed interactions between Xenon and
the enzymes studied remain somewhat hypothetical due to the size and complexity of
these biological macromolecules which hamper the obtainment of site-specific
information.
Further comparative investigation of the active site in AOs from plants, mammals
and bacteria would be helpful in understanding whether these enzymes, which differ in
structure and action mechanism, follow a similar metabolic pathway.
CHAPTER III – RESULTS AND DISCUSSION
115
3.3 – Microporous Crystalline Dipeptides
3.3.1 - Variable Temperature continuous flow HP 129Xe NMR: General spectral
features.
In Figure 3.25 are shown, as a typical example of the experimental results, CF HP 129Xe NMR full spectra of VA dipeptides acquired at different temperatures.
Three signals, corresponding to Xenon in three different environments, can be
observed in the high temperature region: the narrow signal at 0 ppm represents the most
shielded Xenon atoms of the gas-stream, which are not adsorbed on the dipeptides
powder; the broader band, partially overlapping the gas-phase signal and slightly shifted
towards lower fields (centered at about 5 ppm at 343 K in VA), can be ascribed to Xenon
adsorbed on the crystal surface and in intercrystallite regions; finally, the signal resulting
from highly deshielded Xenon absorbed inside the nanochannels is shifted to about 120
ppm at 343 K from the gas-phase signal and shows a pronounced axially symmetric
chemical shift anisotropy (CSA) line shape.
Figure 3.25: The experimental continuous-flow hyperpolarized 129Xe NMR spectra of VA dipeptides at
variable temperature.
CHAPTER III – RESULTS AND DISCUSSION
116
When the temperature is decreased, together with complex changes in the relative
intensities of the signals, modifications of the lineshapes and chemical shifts are observed
for the NMR signals pertaining to the Xenon atoms inside the nanochannels and adsorbed
in intercrystallite regions. More precisely, a broadening, together with its slight shift to
lower fields and a progressive decrease in intensity which leads to its disappearance is
observed for the intercrystallite signal. A global downfield shift and an inversion of the
anisotropy is generally detected for the powder pattern relative to the Xenon inside the
channels; the span of this signal decreases with decreasing temperature and the sign of
the anisotropy is finally inverted at the lowest temperature analyzed (173 K), while an
isotropic line shape can be observed at intermediate temperature.
These general spectral features are common to all the dipeptides studied in this
work and they are in good qualitative agreement with the literature concerning 129Xe
NMR of nanochannels 13,88-96. However, a more detailed analysis of the spectra reveals
subtle but fundamental differences concerning Xenon NMR results in nanochannels
having different structure.
It is worth noting that only the dipeptides AV and VA show the intercrystallite (or
surface) adsorption signal, which in AV is still observable even at 173 K and it overlaps
the anisotropic signal in the temperature range between 243 K and 263 K. AI also shows
intercrystallyte adsorption upon aging of the sample. 129Xe NMR full spectra concerning the other dipeptides, obtained at variable
temperature and continuous flow of hyperpolarized Xenon, are collected in Figure 3.26.
They are in qualitative agreement with other NMR studies on systems where Xenon is
absorbed in nanochannels. At the same time, each dipeptide reveals its own characteristic
spectra, primarily because of the sensitivity of the 129Xe NMR parameters to the
distinctive size and unique geometry of its nanochannels.
CHAPTER III – RESULTS AND DISCUSSION
118
Figure 3.26: Continuous-flow HP 129Xe NMR spectra of AI, AV, LS, VV, IA, IV and VI dipeptides in the
173-343 K temperature range
CHAPTER III – RESULTS AND DISCUSSION
119
3.3.2. - Temperature dependence of the 129Xe NMR Chemical Shift Anisotropy
(CSA) tensor.
When an atom is placed in a strong magnetic field B0, its nucleus is subjected to
two magnetic fields, B0 and B1.
B1 is a local magnetic field generated by the circulation of the electrons that
surround the nucleus with respect to the direction of B0. The effective field at the nucleus
(Beff) is usually expressed as
Beff = B0 – B1 = B0(1-σ) [3.15]
The shielding σ experienced by a nucleus is the result of how the electron
distribution of the atom to which it belongs is influenced by the environment. The name
shielding basically derives from the fact that generally the magnetic field generated by
electrons is such as to oppose the external principal magnetic field B0.
Depending on the particular electronic and magnetic environment, a particular
shielding value characterizes the observed nuclei and the difference in shielding with
respect to a reference molecule or system is known as chemical shift. Thus, in NMR, the
observable directly related to the shielding is the chemical shift δ.
Although rapid molecular reorientation (especially in solution) often leads to the
observation of only one averaged chemical shift value for a given nucleus in a molecule,
the shielding is anisotropic. Thus, σ is well described by a second rank tensor (3×3
matrix), which expresses the dependency of the shielding on the orientation of the
nucleus with respect to the direction of B0. Three principal components of the tensor,
referred to as σ11, σ22 and σ33, describe the shielding experienced by the detected nuclei
along three different directions with respect to the external magnetic field B0.
In static single crystals97, where all the nuclei have a fixed orientation with respect
to the principal magnetic field, each orientation of the observed nucleus with respect to
B0 can be observed as a narrow line in a NMR spectrum. In polycrystalline samples,
however, all the orientations are possible and the NMR signal broadens, giving rise
sometimes to featureless bands and sometimes to the observation of a so called powder
pattern, where the values of σ11, σ22 and σ33 can be precisely correlated to the
CHAPTER III – RESULTS AND DISCUSSION
120
singularities of the signal line shape. In a powder pattern, the signals having chemical
shifts relative to each molecular orientation overlap with each other, the intensities being
proportional to the number of atoms/molecules oriented in each particular direction with
respect to B0.
An anisotropic line shape characterizes the NMR signal of the Xenon atoms
trapped within some confined spaces, such as clathrates and a number of one-dimensional
channels 11,13,94,95,98-104.
This feature bears within itself useful information on the dynamic averaging
experienced by the Xenon atoms and on the size and geometry of the environments in
which they are encapsulated, number of other sorbate molecules and/or atoms within the
same cavity as Xe atom. In this respect, the extensive experimental work done on 129Xe
NMR of clathrates11,105,106, studies on a variety of one-dimensional channels 13,88-96, as
well as theoretical calculations 89,107-110, have pointed out the sensitivity of the 129Xe
chemical shift tensor to the shape, symmetry, size and chemical composition of the cages
and channels in which the Xenon is enclosed.
The number of singularities of the line shapes observed in 129Xe NMR spectra for
Xenon adsorbed in polycrystalline nanoporous materials has been found to be strictly
correlated to the aspect ratio (symmetry) of the cross section of the one-dimensional
channel and the changes of the line shapes, i.e. the behavior of the principal components
σ33 σ11 σ22
CHAPTER III – RESULTS AND DISCUSSION
121
of the chemical shift tensor as a function of loading provide further information on the
arrangement of the Xenon atoms within the channels and on the existence of energy-
favorable sites where the Xenon fit in registery94,103,108,109. A number of theories have
been suggested to account for the anisotropic line shapes in 129Xe spectra of confined Xe,
some of which are briefly analyzed in ref. 107 [see bibliography, Section 3.4].
One of the major contribution to the interpretation of observed anisotropic NMR
line shapes of Xenon in nanochannels has been provided in the recent years by the
combination of ab initio calculations of the 129Xe shielding surfaces107 and Grand
Canonical Monte Carlo simulations in a process based on the so called additive dimer
tensor model110. Such method has been by now successfully applied to different
systems89,108,109, and it has been demonstrated that it is able to provide results reasonably
close to the experimental observations.
The availability of new experimental results concerning similar porous structures,
possibly varying in channel shapes and diameters, would be desirable in order to confirm
the picture drawn by theoretical calculations and clarify the basic rules governing the
observed anisotropy. Even though the relationship between Xe shielding tensors and
pore size and shape is well understood for small pore systems, a general approach to the
actual derivation of useful parameters from NMR spectroscopy has not been
demonstrated. The establishment of a consistent relationship between 129Xe NMR
parameters and properties of sorbent-Xenon systems, both physical (macroscopic) and
structural (molecular level), relies on detailed studies on a series of similar sorbents,
where a property changes in small increments over a wide range111-113.
In the presence of an axially symmetric CSA, only two principal values of the
shielding tensor are generally reported, being two of them coincident. In particular, in the
ideal case of a Xenon atom adsorbed in nanochannels with cylindrical cross-section in the
zero-loading limit, the shielding tensor is axially symmetric, with components σ11 ≤ σ22 =
σ33. Generally, in this condition, it is indicated σ11 = σ and σ22 = σ33 = σ⊥; in this case,
the component of the 129Xe NMR chemical shift tensor parallel to the axis of the channel
(σ) is more deshielded with respect to the other two components perpendicular to the
channel walls (σ⊥), which in turn show the same shielding value107.
CHAPTER III – RESULTS AND DISCUSSION
122
As the chemical shift δ and the effective field Beff are directly proportional, the
directly observed NMR chemical shifts are often discussed instead of the shieldings. In
particular, the NMR anisotropic signals are completely described by three parameters: the
isotropic chemical shift δiso = (δ11 + δ22 + δ33)/3, the span Ω =δ11 - δ33 and the skew κ =
3(δ22 + δiso)/Ω. In the following, the components δ⊥ and δ relative to the directions
perpendicular and parallel with respect to both B0 and the channels axis are described.
In this thesis we will discuss the experimental chemical shifts values δ and δ⊥
correspondent to the shielding just mentioned.
Different behaviors of δ and δ⊥ as a function of the temperature correspond to
different dipeptides, the dissimilarities obviously being related to each particular
electronic environment sensed by the Xenon atoms inside each nanochannel.
In order to more clearly describe and understand the results just mentioned, it
would be useful to very briefly review at least some of the most important concepts so far
drawn by theoretical studies, in particular those obtained by ab-initio calculations
performed by C.J. Jameson and coworkers regarding how Xe shielding tensor
components are influenced by interatomic and intermolecular interactions with other
atoms/molecules. These results revealed the following general behavior: as the
intermolecular partner gets closer to Xe, the component of the Xe shielding tensor along
σ
σ σiso
CHAPTER III – RESULTS AND DISCUSSION
123
the direction of approach changes slightly, increasing shielding. At the same time, the
tensor components in the plane perpendicular to the direction of approach become
uniformly less shielded.
In all cases of Xe interacting with an atom or approaching collinearly to a linear
molecule, the components of the Xe shielding tensor in the plane perpendicular to the
direction of approach are uniformly deshielded, whereas the tensor component along the
direction of approach is changed only slightly.
For Xe interacting with a wall of atoms, all components are deshielded relative to
the free isolated Xe atom, with the component parallel to the wall being the most
deshielded. The least deshielding occurs for the component along the line of approach of
the Xe to the wall.
Aiming at simulating the case of two or more Xenon atoms sorbed into a
nanochannel at high loading, ab initio calculation of the Xe shielding tensor in the
presence of of two more Xenon atoms have been performed by Jameson and coworkers,
and results showed to be comparable to experimental studies107,108. The shielding tensor
of the central Xe in a Xe3 trimer is very representative of what is actually observed when
the tensor is dominated by Xe-Xe interactions and Xe-wall interactions are negligible in
highly loaded channels. This work demonstrated that the angle formed by the three Xe
atoms strongly influences the shielding tensor and therefore the resulting observed line
shape of 129Xe NMR signals.
We will make use of these results in understanding the Xe shielding in
nanochannels under full loading.
It is now clear that important qualitative information concerning the interaction
between both the Xenon atoms and the channel walls (Xe-wall) and between neighbors
sorbed Xenon atoms with respect to each other (Xe-Xe) can be gained by analyzing the
chemical shift tensor. In particular details will be given here on how hints on the role of
each particular channel structure in differentiating the adsorption of Xenon and
consequently the observed 129Xe NMR spectra can be found and discussed.
The observed line shapes, moreover, are influenced by the dynamics of the Xenon
atoms sorbed in the pores, which are evident in the zero-loading limit and are correlated
CHAPTER III – RESULTS AND DISCUSSION
124
with the channel size. These evidences are discussed in some more detail in the
following, and divided in four short paragraphs.
3.3.2.1. Effect of channels loading on the CSA
The behavior of the components of chemical shift anisotropy tensor as a function
of channel loading has been observed and described for different nanochannels so far89-
91,93,94,96,107,111 .
According to the calculations performed by Jameson et al., in a diamagnetic
narrow-bore pipe, the chemical shift anisotropy should change as a function of channel
loading from positive values at zero loading, with the component δ⊥ going from being the
most shielded, to negative values at high loadings, the δ⊥ being in this case the most
deshielded component and the component δ remaining unaffected by the filling of the
channels.
These trends are generally observed for all the dipeptides analyzed and prove the
sensitivity of the line shapes to the loading of a number of different channels.
It is interesting to observe, however, that in situations where Xenon is in channels
with a diameter narrower than its Van der Waals diameter (VI, IV, IA and VV, which
have diameters that range from 4.0 Å for VV to 3.0 Å for VI compared to the Xenon’s
Van der Waals diameter of about 4.3 Å), a significantly large positive anisotropy (~100
ppm for VI at room temperature) characterize the signals at high temperatures (low
loading) and a positive anisotropy is still observed also at the lowest temperature (high
loading), although with reduced span compared to the high-temperature signal (an
isotropic line shape is observed for VV at 173K). This observation is consistent with the
presence of very strong Xe-wall interactions that most likely characterize the Xenon
atoms tightly wrapped by these very narrow, although flexible, channels and with Xe-Xe
interactions being unable to balance these effects. It is also reasonable to believe that in
the case of the narrowest channels, the Xe-Xe interactions could be somewhat hampered
due to the constitution of the pores, that can be thought as formed by an alternation of
wide regions and constrictions in a corrugated channel. This hypothesis is furthermore
discussed and substantiated in the next paragraph.
CHAPTER III – RESULTS AND DISCUSSION
125
3.3.2.2. Presence of specific sites (niches)
Distinctive features characterize the observed CSA in LS, VI and IV with respect
to the other channels. In the high temperature region, all three show a linear behavior of
each tensor component with decreasing temperature. The narrowest channels VI and IV
show similar behavior to LS in that the line shapes do not change in the high-temperature
range (which is up to about 220 K in VI and IV and about 260 K for LS) showing, in fact,
a constant span: nevertheless, LS differs from VI and IV because, decreasing the
temperature, the whole signal is shifted toward high frequencies in the latter two while it
shifts to lower frequencies in the former. Such a linear dependence of the 129Xe chemical
shift tensor components with average occupancy has been observed94 already in
molecular sieves ALPO-11 and SAPO-11 and ascribed to the presence of an ordered
arrangement of Xenon atoms within the channels driven by the existence of energetically
favorable sites along the channel walls where the adsorbed atoms fit. It was recently
pointed out that when these niches are too close to each other no linear behavior of the
chemical shift tensor components should not be observed109. It is interesting to note,
however, that the linear behavior of the tensor components observed in VI and IV is
interrupted at some intermediate temperature and a non-linear behavior is observed at
lower temperatures. This suggests that also the structure of the channel is most likely
changed by the increase of the Xenon loading inside the channels, going from a
corrugated channel to a more smooth one, where the Xe atoms have chance to get closer
to each other, similarly to what observed in the larger channels.
3.3.2.3. Effect of helicity and diameter of the channels on CSA
In order to explain the influence of the helicity of the channels on the observed
spectra, it is useful to consider again the ideal model of a single Xenon atom in a straight
nanochannel. In this case, the presence Xe-wall interactions are merely reflected in the
deshielding of the parallel component of the Xe chemical shift tensor with respect to its
perpendicular component, this latter being in fact influenced only by interactions along
the axis of the channel. This model suggests that varying the loading, which can be
achieved by increasing the Xenon pressure or decreasing the temperature as well, would
CHAPTER III – RESULTS AND DISCUSSION
126
cause a modification of only the perpendicular component of the tensor leaving
unchanged the parallel one. As we expected, real systems are more complicated than
ideal models. According to our experiments on dipeptides indeed, δ generally increases
with decreasing temperature up to the lowest temperature (173 K) although it shows
almost a constant value for AI. It is worth noting at this point that, according to previous
helium pycnometry measurements and to XRD single-crystal structures88, AI channels
are characterized by having a diameter very close to the Van der Waals diameter of
Xenon atoms and by a low helicity.
Figure 3.27: HP CF 129Xe NMR spectra of AI dipeptides showing the constancy of the parallel component
of the chemical shift tensor at variable temperatures.
The observation of a constant value for δ as a function of experimental
temperature in AI dipeptides is a further confirmation of the results previously obtained
and moreover this finding complements the previous results suggesting that the Xenon
atoms adsorbed inside the AI channels are aligned along the channel axis in a fairly
straight line. Calculations suggested108 that when angular arrangements of Xenon atoms
in highly loaded channels is characterized by angles in the range 180º-150º (i.e., to a good
approximation, in a straight line) the component of the chemical shift tensor along the
axis of the channel (δ) will hardly change with Xenon occupancy.
This, however, is not expected to strictly apply to helical channels, where the
effects of Xe-wall and Xe-Xe interactions are spread out also along the direction
perpendicular to the axis of the channel, thus influencing the observed δ⊥ and to some
extent the δ as well. It is not straightforward, however, to directly correlate the helicity
of the channels to the shift of the parallel component of the 129Xe NMR signal. It is
CHAPTER III – RESULTS AND DISCUSSION
127
reasonable to think that the deviation from the constancy of δ observed in all the
dipeptides except for AI can be attributed both to the helicity and to the diameter of the
channels. Larger channels than the Xenon Van der Waals diameter should allow zig-zag
conformations of Xenon atoms even in straight highly loaded channels.
3.3.2.4. Dynamics of Xe in the cross section of the pores and CSA of 129Xe NMR signal
Previous calculation based on Lennard-Jones potentials114,115 and ab initio
calculations107, suggested that when Xe atoms are sorbed in narrow pores of the same
size of Xenon atoms, in the limit of zero loading (high temperature region), the atoms
remain at the center of the channels and that 129Xe NMR signal shifts toward lower fields
as a function of increasing temperature. This expected result has been demonstrated by ab
initio calculations by considering the effect that the temperature-dependent dynamics of
the Xenon atoms inside the nanopores would have on the shielding of Xenon. In
particular, an increase in the span of the anisotropic 129Xe NMR signal of adsorbed
Xenons with increasing temperature due to an increasing deshielding of the parallel
component is expected and actually observed in our experiments on almost all dipeptides.
In other words, it is assumed that at higher temperatures the Xenon atoms are supposed to
get closer to the channel walls107. It can be seen that this effect is more or less marked
depending on the diameter of the channel where the Xe are trapped and it will be
considered again later.
The aforementioned theoretical predictions, however, do not apply to the
experimental results for the channels LS, IV and VI which show, at high temperature, a
linear increase of the isotropic chemical shift when the temperature is decreased. The
effects that characterize the observed CSA of these compounds have been discussed
above.
CHAPTER III – RESULTS AND DISCUSSION
128
3.3.3. - 129Xe NMR isotropic chemical shifts as a function of temperature
Figure 3.28: Variation of the experimental 129Xe NMR isotropic chemical shift (δiso) as a function of
temperature for the eight dipeptides AV (), VA(), AI(), VV(), LS (), IA (), IV(×) and VI (+).
Solid lines represent the fitting of the experimental points according to the thermodynamic model discussed
in the text. The dotted line is the fit to a straight line relative to the linear behavior of δiso observed in IV in
the high temperature region
CHAPTER III – RESULTS AND DISCUSSION
129
In Figure 3.28 are reported the variations of the 129Xe NMR isotropic chemical
shifts (δiso) pertaining to the anisotropic signal observed for the eight dipeptides studied
as a function of the experimental temperature. The resulting curves clearly resemble
sigmoid functions and each of them can be roughly divided into regions according to the
scheme reported below [see Fig. 3.29]:
Figure 3.29: Schematic representation of the ideal change of isotropic chemical shift for intrachannel Xe
with temperature at isobaric conditions (assuming that Xe-wall interactions are not temperature dependent,
xenon atoms freely enter 1D cylindrical pores and the pore structure remains rigid upon guest inclusion).
The drawings show the cross section of ideal nanochannels at different loadings corresponding to different
temperature regions
In the high-temperature region (A), the concentration of Xenon atoms into the
channels is expected to be very low, thus 129Xe chemical shifts are expected to be merely
characterized by Xe-wall interactions and to correspond to that of a single Xenon atom in
the empty channels.
In the lowest-temperature region (C), the concentration of Xenon atoms in the
nanochannels reaches a maximal, saturated value; the chemical shift δf is defined by both
CHAPTER III – RESULTS AND DISCUSSION
130
Xe-wall and Xe-Xe interactions and corresponds to 1D close packing of Xenon atoms
inside the channel, with Xe-Xe interactions dominating the observed shielding.
The low-temperature region (B) represents an intermediate situation, where the
concentration of Xenon atoms in the nanochannel and average chemical shift of the
atoms smoothly grow from their minimal to their maximal values.
The experimentally observed behavior of 129Xe isotropic chemical shifts as a
function of decreasing temperature follow different trends for different dipeptides: four of
them (AV, VA, AI and LS) show in the high temperature region a decreasing value, a
minimum being reached at some temperatures between 260 K and 280 K depending on
the particular dipeptide; two (VV and IA) show almost constant chemical shift in this
region and in the last two (IV and VI) the δiso linearly increase.
The behaviors just described are generally in good agreement with what said
above about the relationship existing between the temperature dependence of the
shielding components of Xe in a narrow-bore pipe at zero loading and the dynamics of
the adsorbed Xenon in the cross-sectional plane of the channels. The guidelines provided
by the theoretical works suggest that in the larger channels of AV, VA, AI and LS, the
effect of thermal averaging among different position of Xenon with respect to the center
of the channels should be more pronounced, since the Xe atoms have more chance to get
closer to the channel walls when the temperature is increased rather than what it is
expected in the narrower channels VV and IA, where instead the Xe are most likely more
constricted in the same position corresponding to both the center of the channel cross-
section and to the closest distance to the channel walls. In this sense, the restricted motion
of Xenon inside the narrow channels of VV and IA can explain the observed invariability
of the isotropic chemical shift with respect to the temperature. As a matter of fact, the
observed trends can be classified according to the channel diameters. As it resulted from
He pycnometry88 the sequence from the largest to the narrowest channel is in fact
AV>VA>LS>AI>VV>IA>IV>VI.
Although we should expect that also in this temperature region the loading
slightly increases when lowering the temperature, the chemical shifts are not significantly
influenced by the loading at this stage, because Xenon atoms are still too far to each other
for the Xe-Xe interaction to be sensed, and we can consider to a good approximation that
CHAPTER III – RESULTS AND DISCUSSION
131
the observed spectra are dominated by only Xe-wall interactions, i.e., the zero-loading
condition is a good approximation for the analysis of the isotropic chemical shift.
In the low-temperature region (B), the experimental chemical shift steeply
increases with decreasing T, showing a distinctive curvature for each particular dipeptide.
An inflection point is observed at some intermediate temperature in all the plots except
those concerning VI, IV and IA.
By lowering the temperature the Xe concentration inside the channel increases
and the average Xe-Xe distance inside the channels decreases. The dependence of the
observed chemical shift upon loading is usually explained in terms of the contribution
due to collisions between Xe atoms in the micropores. At the highest temperatures
analyzed the Xenon atoms are mainly affected by strong Van der Waals interactions with
the channels walls. This zero-loading condition lasts until the loading inside the channels
is such that the absorbed Xenon atoms are able to sense the presence of their neighbors,
that is, when sorbed Xenon atoms get close enough to each other in the nanopore for
allow Xe-Xe interactions to compete with the Xe-wall interactions to dominate the
shielding tensor. Further decrease of the temperature leads to a change in the curvature,
which is most apparent at the lowest temperatures analyzed. It is reasonable to assume
that Xe-Xe interactions, to which has been attributed the steep increase of the chemical
shift would reach a maximum constant value in the completely filled channels, where the
trapped Xenon atoms have reached the closest positions with respect to each other.
At the lowest temperature, the narrow channels IA, VI and IV show different
behavior: no inflection point or changes in curvature are in fact observed in the plots of
isotropic chemical shifts vs temperature. This further suggests that in such very narrow
channels the Xenon atoms are so tightly wrapped by the channel walls that the average
interatomic distance between Xe atoms remains much larger than that in the larger
nanochannels. Similar behavior has been already observed in other one-dimensional
channels91. The variation of chemical shifts in the low-temperature region can be better
understood if we consider that the isotropic chemical shift is the expression of the
probability of collisions between neighboring Xenon atoms, which is of course directly
related to the channel loading. More precisely, as suggested by ab initio calculations of
the intermolecular shielding tensors for pairs of noble gases107,110, the variation of
CHAPTER III – RESULTS AND DISCUSSION
132
shielding as a function of loading should be considered as a continuous process, being
this dependent upon the variation of the interatomic distances between the observed
Xenon and its neighbors more than on the population of specific neighboring adsorption
sites. In the particular case of Xenon adsorbed in nanochannels, we expect the 129Xe
chemical shift to be affected at each temperature by the average Xe-Xe distance
experienced by each Xenon atom in the chemical shifts NMR time scale, assuming that
the range of temperature considered is such that the influence of Xe-wall interactions on
the observed signal can be neglected. These considerations imply that a smooth variation
should be observed between the lowest chemical shift value related to Xenon in almost
empty channels and the highest one corresponding to Xenon atoms in almost completely
filled channels. This smooth variation is, in fact, what is actually observed.
Nevertheless, the two limiting chemical shift values of Xenon in empty and
completely filled channels cannot be directly related to observed chemical shifts: the
temperature dependence of Xe-wall interactions, in fact, significantly affects the observed
chemical shift of Xe in the empty channels at high temperatures and the possible
condensation of Xenon below 170 K could most likely hinder the continuous flow
measurements and does not allow to study the highest loadings.
3.3.4. - Thermodynamics of adsorption: the Langmuir model
In order to extract quantitative information on the adsorption of Xenon in these
systems, a thermodynamic model based on the observations discussed above can be
suggested.
The solid lines in Figure 3.28 represent the fitting of the experimental points
according to the model explained in the following.
The Xenon adsorption isotherms previously acquired116 for AV and VA were well
approximated by a Langmuir equation, which has the well known general form
PTK
PTKPT
L
Lr ⋅+
⋅=Θ)(1
)(),( [3.16]
CHAPTER III – RESULTS AND DISCUSSION
133
where satr ΘΘ=Θ is the relative coverage (a fraction of the total sorption sites occupied
by Xenon atoms, 10 <Θ< rel ), KL(T) the temperature dependent Langmuir constant and
P the experimental Xe pressure at equilibrium. The equation [3.16] applies therefore to
isotherms as well as to isobars.
In order to show the dependence of the relative coverage on temperature, equation
[3.16] should be written in a more general form112,117,118:
RTq
RTq
relst
st
ekpT
ekppT
⋅⋅+
⋅⋅=Θ'
'),(
[3.17]
where k' is the temperature-independent portion of the Langmuir constant and qst is the
isosteric heat of sorption already mentioned previously. It should be noted that the square
root of temperature is always a part of the Langmuir117 constant in equation [3.16] but it
is not evident as long as the equation is applied to sorption isotherms (T=const). Equation
[3.17] is quite general as it may be applied to both sorption isotherms and sorption
isobars. In the experiments of this work the partial pressure of Xenon is maintained
constant (0.01 bar), while temperature is varied. Therefore, the coverage in the
experiment changes isobarically.
The Langmuir theory has proven to be a good model to provide useful
information both on the kinetics of adsorption and desorption of gases on crystal
surfaces118 or on the behavior of thermodynamic functions in microporous adsorbents119.
For constant pressure and variable temperature, the equation [3.16] represents an isobar
and its shape is a sigmoid.
In order to relate the observed 129Xe chemical shift to the relative coverage, we
recall the equation proposed by Fraissard and coworkers111:
MXeXesobs δδδδδ +++= −0 [3.18]
CHAPTER III – RESULTS AND DISCUSSION
134
where δ0 is the reference (gaseous Xenon at zero pressure), δs arises from interactions
with the inner surface of the pores (Xe-wall), δXe-Xe corresponds to interatomic
interactions between adsorbed Xenon atoms and δM accounts for magnetic fields from
paramagnetic ions if they are present in the porous material. The last term δM is zero in
our case and δ0 is zero by convention. As discussed in the previous section, Xe-wall
interactions expressed by δobs define the observed chemical shift in the high-temperature
region. In this region, the Xe-wall interactions depend on temperature due to the
dynamics of the absorbed species over the cross-section of the nanochannel. Therefore,
the variation of δobs in this region is dependent on temperature and is likely to be
independent of loading.
In the low-temperature region, Xe-Xe interactions start to dominate and the only
coverage-dependent term of equation [3.18], δXe-Xe, becomes a significant contributor to
the chemical shift. The chemical shift observed at any particular temperature can be
considered as a sum of the chemical shift defined by the Xe-walls interactions in the
empty nanochannels (δs) and a fraction of the total chemical shift variation ∆δ = δf - δs
[see Figure 3.29] between the chemical shifts corresponding to the completely filled (δf)
and empty (δs) nanochannels:
relsobs Θ⋅∆+= δδδ [3.19]
Previously, such an approach proved to be valid in fitting experimentally
determined Xenon sorption isotherms for zeolites120.
Combining equations [3.17] and [3.19], we obtain the dependence of the observed
chemical shift on pressure and temperature:
CHAPTER III – RESULTS AND DISCUSSION
135
RTq
RTq
sobsst
st
ekpT
ekp
⋅⋅+
⋅⋅∆+='
'δδδ [3.20]
Under the experimental conditions of this work, p = 0.01 (relative pressure,
dimensionless) and the dependence δobs(T) becomes an isobar, a sigmoid-shaped curve
defined by four parameters: δs, ∆δ, k' and qst. In order to simplify the task of fitting the
experimental data with equation [3.20], δs was found from the extrapolation of the
chemical shifts in the high-temperature region. The value of δs was assumed to be
constant in the lower-temperature region considered (region (B) in Figure 3.29) as it is
relatively narrow and the dynamics of Xenon species responsible for the deviation of the
observed chemical shifts from δs is expected to be suppressed at higher loadings. Fitting
of experimental data with [3.20] is shown in Figure 3.28 (solid lines) and the derived
values of k', qst, δs and ∆δ= δf - δs are reported in Tables 3.6 and 3.7.
It should be mentioned that fitting was impossible for the narrowest channel of
VI. For IA and IV, it was necessary to fix qst to the values obtained from the signal
intensities in high-temperature region (see section 3.2.1) in order to obtain the other
parameters.
CHAPTER III – RESULTS AND DISCUSSION
136
Table 3.6: Thermodynamic parameters of Xenon sorption in the studied dipeptides derived from the experimental data for the indicated temperature intervals (K): isosteric heats of sorption qst [kJ/(mol of Xe)], k and k' [dimensionless]
Sample Analysis of signal intensities
(high temperatures, low loadings)
Analysis of chemical shifts
(low temperatures, high loadings)
T-rangea k · 103 qst T-rangea k' · 103 qst
343 – 293 (5) 30.8 ± 0.9 8.21 ± 0.08 AV
293 – 273 (3) 1.1 ± 0.2 16.51 ± 0.59 273 – 173 (11) 4.8 ± 1.4 20.80 ± 0.54
333 – 293 (4) 22.4 ± 1.8 12.16 ± 0.23 VA
293 – 253 (5) 8.4 ± 1.0 14.42 ± 0.28 253 – 173 (9) 2.4 ± 0.7 20.96 ± 0.50
LS 333 – 293 (5) 5.9 ± 1.4 20.07 ± 0.72 253 – 173 (9) 6.5 ± 2.5 20.54 ± 0.71
343 – 323 (3) 1.1 ± 0.4 19.00 ± 1.11 AI
323 – 283 (5) 7.4 ± 1.0 14.01 ± 0.37 273 – 183 (10) 8.0 ± 4.3 20.84 ± 1.02
VV 333 – 283 (6) 9.1± 1.2 20.69 ± 0.36 293 – 173 (10) 8.9 ± 2.8 19.81 ± 0.57
IA 333 – 293 (3) 1.8 ± 0.4 17.67 ± 0.65 273 – 173 (6) 5.5 ± 0.5 17.8b
IV 343 – 253 (5) 4.8 ± 1.0 17.76 ± 0.56 213 – 173 (5) 7.5 ± 1.3 17.8b
VI 293 – 193 (6) 2.6 ± 0.4 17.01 ± 0.37 - - - a The number of experimental points used in the fit is given in brackets. b Fixed values
CHAPTER III – RESULTS AND DISCUSSION
137
Table 3.7: Isotropic chemical shifts (ppm) of absorbed 129Xe at zero loading (δs) and their total variations
(δ) calculated from the experimental data for dipeptides studied
Calculated chemical shift
variation with temperature Sample
δs δ = δf - δs
AV 93.0 63.6
VA 124.1 50.3
LS 166.0 42.0
AI 134.6 53.8
VV 155.0 53.2
IA 196.3 43.8
IV 231.4 46.0
VI - -
CHAPTER III – RESULTS AND DISCUSSION
138
3.3.5. - Signal intensities
In Figure 3.30 (a) is reported the temperature dependence of the integrated signal
intensities (I=Ichannel/Igas) obtained for VV, as a typical example of what observed in all
dipeptides. In Fig. 3.30 (b) is shown the logarithmic plot of Ichannel/Igas in CF HP 129Xe
NMR for Xenon inside all the dipeptides nanochannels as a function of the inverse
temperature (1/T).
(a)
(b)
Figure 3.30: a) Signal intensities I = Ich/Igas for VV dipeptide as a function of temperature. b) The same
data presented in the ln(I) – 1000/T coordinates and fitting of the data with a straight line (high-temperature
region)
CHAPTER III – RESULTS AND DISCUSSION
139
As it clearly appears from the observation of Fig. 3.30 (a), a first exponential
increase of Ichannel/Igas at high temperatures is followed by its decrease at low T, a
maximum being observed at some intermediate temperature, which has a different value
for each dipeptide.
This dependence is a result of two opposite effects. On the one hand, the
decreasing temperature results in higher channel loading because of a favorable sorption
enthalpy. On the other hand, the decreasing temperature has a negative kinetic effect,
slowing down the diffusion of Xenon species inside the nanochannels. The condition of
fast diffusion is a critical factor in hyperpolarized 129Xe NMR studies as it guarantees a
continuous replacement of the Xenon nuclei that relax to a thermally polarized state (and
therefore become undetectable) with new hyperpolarized Xenon.
In the high-temperature region, the intensity of the NMR signal is merely
controlled by the thermodynamics of Xenon adsorption in the dipeptide channel and the
ratio Ichannel/Igas is indeed representative of the relative amount of Xenon absorbed.
In the low-temperature region, the kinetic factors become dominant: reduced
diffusion, which is well described by the temperature-dependent diffusion constant D0,
slows down the process of the replacement of relaxed Xenon with hyperpolarized nuclei,
causing a non negligible loss of signal121. Similar behavior has been observed for sorption
of Xenon in tris(o-phenylenedioxy)cyclotriphosphazene (TPP) nanochannels90 and this
might be expected for other sorbents having their micropore space organized in isolated
narrow channels. In order to extract quantitative information on the sorption process from
the intensities of the HP 129Xe NMR signal, therefore, only the high-temperature region,
where the diffusion of Xenon in the nanochannels is fast, should be considered.
Assuming that Xenon can fill a limited number of discrete sorption sites in the
nanochannels, the sorption process can be described by the following equation:
[ ]solid + Xegas = [Xe]solid, [3.21]
where [ ] represents a single sorption site and [Xe] denotes an atom of the sorbate on the
site. The equilibrium (sorption) constant for the process (K) is the ratio of activities of
adsorbed and gas phase Xenon, the ratio being proportional to experimentally measurable
CHAPTER III – RESULTS AND DISCUSSION
140
ratio of intensities of 129Xe NMR signals from Xenon atoms absorbed in the
nanochannels and Xenon atoms in the gas phase:
IkIIkK probe
gas
channelprobe ⋅=
⋅= [3.22]
where kprobe is a proportional coefficient, the ratio of volumes of the gas phase and
sorbent solid phase in the coil region of the probe. The coefficient depends on how the
sample was loaded in the probe and its precise value is unknown (the estimated value for
highly packed fine powders was kprobe ~ 1). Sets of experiments conducted on the same
sample show that kprobe remains almost constant as long as the same probe in the same
instrumental configuration is used.
In the high temperature region, the exponential growth of intensities is directly
related to the standard free energy of sorption and can be expressed as follows
RT
GeK
∆−= [3.23]
Considering that the adsorption is always driven by the two opposing effects of
the energetic and the entropic terms, the previous equation [3.23] becomes
RTq
RTq
RS
RTG stst
ekeee ⋅=⋅=°∆∆−
0 [3.24]
where ∆So is the standard entropy change in the process [3.21], k0 is a "pre-exponential
coefficient" (entropy term which does not depend on temperature), and qst is the isosteric
heat of sorption122-124 which refers to a particular loading and equals, with opposite sign,
the isosteric sorption enthalpy of process [3.21].
CHAPTER III – RESULTS AND DISCUSSION
141
Taking logarithms
TR
q
R
S
RT
G st 1⋅+∆=∆− o
[3.25]
which is a straight line with slope Rqst and intercept R
S°∆− .
Combining [3.22], [3.23] and [3.24] produces the dependence of the integral
intensity of the intrachannel Xenon on temperature,
RTq
RTq
probe
stst
ekek
kTI ⋅=⋅= 0)(
[3.26]
where k = k0/kprobe is a temperature-independent term. In coordinates ln(I) – 1/T the
experimental data may be fitted with a straight line according to the linear equation
TR
qkI st 1)ln()ln( ⋅+= [3.27]
with the intercept ln(k) and slope qst/R.
Plots of experimentally determined ln(Ichannel/Igas) vs 1/T, in fact, show for all
dipeptides a linear region at high temperatures, while a decisive deviation from linearity,
due to the contribution of reduced diffusion, can be observed in the low-temperature
region, indicating that even though thermodynamic equilibrium was established, the
method used is not suitable because only a small percentage of the adsorbed Xenon atoms
can be detected .
The fitting of experimental points in the high-temperature region provides values
for isosteric heats of sorption and k (as an estimate of k0) in the observed temperature
intervals, the values being valid for low loadings. Values obtained by fitting are collected
CHAPTER III – RESULTS AND DISCUSSION
142
in Table 3.6. The correlation factors for the fitted regions are higher than 0.99 indicating
an excellent agreement of the experimental data with the model applied.
Six dipeptides (VV, LS, IA, AI, VI, IV) show only one linear region before they
deviate from the linear behavior, while two of them have more (AV, VA, AI). Solid lines
in the plots reported below correspond to the fitting of the experimental points with
straight lines, which provide standard entropies and heats of adsorption.
Figure 3.31: Experimentally observed ln(I) – 1000/T dependences for AV, VA, LS, AI, IV and VI
dipeptides and fitting of the data in the high-temperature regions with straight lines
CHAPTER III – RESULTS AND DISCUSSION
143
At this point, some considerations should be made. The presence of more than
one linear region in ln(Ichannel/Igas) vs 1/T plots is observed only in AI, AV and VA, which
are also the only dipeptides between those analyzed that show intercrystallite adsorption.
The exact nature of intercrystallite regions and its actual effect on the adsorption of
Xenon in the channels was quite undefined at this point but it appeared clear from the
comparison of the spectra that the presence of such signal is always associated to a loss of
signal arising from Xenon atoms inside the channels.
Further attempts to characterize this phenomenon suggested that a continuous
aging process takes place in dipeptides crystals, which will be further described in a
separate paragraph in the following.
It must be pointed out here, however, that while signal intensities can be
influenced by the presence of the intercrystallite adsorption due to aging phenomena, the
chemical shifts do not seem to be influenced by this effect to any extent.
3.3.6. - Aging of dipeptide samples
The experiments conducted in this work were complicated by the observation of
irreversible changes in the samples that resulted in an apparent decrease in microporosity.
In particular, the decrease in intensity of the intrachannel Xenon signal was observed
down to its complete disappearance in some cases. Here the changes are referred to as
aging to emphasize their irreversible nature and the fact that the changes progressed
steadily in time. At least three different sources of aging were identified, as discussed
below.
The first type of aging was emblematic particularly in AV, VA and AI. These
three dipeptides, unlike the others, also showed intercrystallite adsorption, with the
appearance of the intercrystallite signal (especially for AI) occurring after new samples
were subjected to a sorption-desorption cycle. The presence or appearance of the
intercrystallite signal was always associated with some loss in intensity of signal from
Xenon residing inside the nanochannels.
These observations are consistent with a stepwise phase transition occurring at a
certain loading and temperature for each of the dipeptides. Indeed, the AV, VA and AI
CHAPTER III – RESULTS AND DISCUSSION
144
dipeptides show two linear regions on the ln(I) – 1/T plot [Figure 3.31] that indicates the
probable presence of a phase transition in the dipeptide-Xenon inclusion compounds.
Additional experiments revealed a visually evident disintegration of large single crystals
(VA) that had been subjected to Xenon pressure above a certain value.
Further attempts to the characterization of Xe-dipeptides complexes by means of
X-ray crystallography have been so far unsuccessful due to the low degree of crystal
order caused by these phase transitions. Different experimental approaches to the
preparation of Xe derivatives of dipeptides crystals are currently underway.
Although the transitions are reversible, mechanical stresses in different parts of
the crystal, in the vicinity of the transition temperature, cause extensive cracking and
break the crystals. This explains the growth of the intercrystallite Xe signal, to some
extent at the expense of the signal from the intrachannel Xenon as relatively large crystals
are ground into fine crystals or a partially amorphous phase with a highly developed
surface. Such mechanical changes occur every time a sample passes through the phase
transition point and this way the sample "memorizes" how many sorption-desorption
cycles it has experienced.
The second type of aging was observed particularly for VA but might, at some
point, become a curse for any of the dipeptides under study. As reported earlier88,116, VA
in its microporous hexagonal form has a packing coefficient of 0.537(4) which is too low
for a stable molecular crystal, even taking into account stabilization by hydrogen bonds.
Therefore, the existence of a more stable, dense polymorphic form of VA could not be
excluded (note that it is impossible to prove the non-existence of such polymorph). The
problem with the dense form is of kinetic origin: the microporous form exists as a
metastable polymorph for kinetic reasons but starts to disappear upon contact with the
seeds of a stable dense polymorph once they appear in the surroundings. Extensive
experiments with VA samples eventually generated the stable form of the dipeptide and
an almost complete disappearance of microporosity was observed in some samples after
less than two months of work. Some results concerning 129Xe NMR characterization of
sorption in aged VA crystals are shown in Figure 3.32 below.
CHAPTER III – RESULTS AND DISCUSSION
145
Figure 3.32: 129Xe NMR spectra of as received VA dipeptides crystals and of the same materials after
approximately 3 months aging of the sample. Experiments were done using CF HP 129Xe at a temperature
of 203K and several (∼10) sorption-desorption cicles.
The polymorphic transition appears to be irreversible and occurs for kinetic
reasons; a guest component (Xenon in our case) does not necessarily participate but, as in
other previously described systems125,126, may act, especially in combination with
temperature changes, as a catalyst. The study of the new polymorph is currently
underway.
The third type of aging is an intrinsic property of all the dipeptides studied. The
organization of micropore space in narrow, mutually isolated (non-interconnected)
nanochannels makes such systems subject to easy "poisoning" by the presence of defects
in their crystal structure or low-volatility impurities. The defects may appear as stacking
faults between crystallite domains127 or as molecular-size irregularities. The impurities
may completely block the nanochannels even in very low concentrations. Noteworthy,
CHAPTER III – RESULTS AND DISCUSSION
146
"poisoning" of zeolite catalysts due to blockage of micropores is one of the major
problems in industry; chabazite128, mordenite128,129, faujasite130 and ferrierite131 were
reported as being subject to this phenomenon. In peptide microporous materials the
problem is rather worse: the impurities cannot be removed by thermal or chemical
treatment because of the limited stability of these materials and the dipeptides can even
produce such impurities themselves as a result of their gradual degradation.
3.3.7. - Thermodynamics of adsorption in nanochannels. General remarks
(summary).
The thermodynamic description of sorption is a primary fundamental account of a
sorbent material which defines its applicability to a desired separation, purification,
storing or catalytic process. On the other hand, the investigation of the relation between
the thermodynamic parameters of sorption and structural parameters of the sorbent-
sorbate components reveals issues defining the energetics and selectivity of sorption and
contribute to the design of new porous materials. The development of numerous synthetic
zeolites and their industrial applications were accompanied by extensive and detailed
sorption studies documented in the literature132.
The thermodynamic parameters of sorption experimentally derived in this study
are summarized in Table 3.6. In general, the sorption of Xenon in all eight dipeptides is
very favorable energetically, as judged from the qst values of 8-21 kJ/(mol of Xe).
Characteristically, at the high loadings, the heats of sorption are similar, while at low
loadings they vary, with the variation being obviously related to the variation in the
sorption entropy. This result likely indicates that at high loadings and low temperatures in
all the channels there is similar dense packing of Xenon atoms having like environments
and low degrees of freedom. This similarity is observed in spite of substantially different
sizes of the channels and may signify the change in channel structure induced by the
Xenon species absorbed.
In contrast, at low loadings and high temperatures different situations may arise,
with various degrees of freedom of the Xenon species in the channel. This possibility is
CHAPTER III – RESULTS AND DISCUSSION
147
especially well illustrated by stepwise changes in AV, VA and AI dipeptides, implying
possible phase transitions in these dipeptides. From the comparison of thermodynamic
parameters in the two temperature intervals [see Table 3.6], the enthalpies and
approximate temperatures of the phase transitions are 8.3(6), 2.3(4) and -5(1) kJ/(mol of
Xe) and 298, 298 and 323 K for AV, VA and AI, respectively. The entropy terms change
in order to compensate for the contribution of the enthalpy changes to the free energy.
This observation is known as a compensation effect, it having been observed for sorption
in zeolites133. The physical sense of this phenomenon is that a higher ordering of
adsorbed sorbate species in the channel ensures a better interaction with the surroundings
(favorable enthalpy term) but it also implies some loss of freedom (unfavorable entropy
term), while higher mobility creates the opposite effects.
The phase transitions in the dipeptide-Xenon inclusion compounds are likely to
arise from a not very dramatic but stepwise structural adjustment in the dipeptide matrix,
or packing of Xenon atoms inside the nanochannels. As can be seen from the enthalpy
and entropy changes, there are less efficient interactions but more degrees of freedom in
the higher-temperature modifications of AV and VA. Therefore, cooling might induce a
better fit between a Xenon atom and a sorption site and higher degree of guest ordering in
the nanochannels. Phase transitions of this kind have been observed and studied in other
inclusion compounds and may be induced by either temperature134-139. In case of AI, the
increase in entropy and decrease in qst might be a result of a stepwise increase in the
channel diameter that takes place at a certain loading. Such phase transitions are triggered
at a certain "gate pressure" of guest141-145. Possible structural motifs in the lower-
temperature phases may be seen in recent studies showing that the hexagonal structure of
AV transforms into a superstructure with four symmetrically nonequivalent channels
upon inclusion of 2-propanol/water (120 K)146 and the hexagonal structure of VA distorts
to monoclinic in its inclusion compound with acetonitrile/water (105 K)147. In general,
the observed phase changes in the dipeptides studied characterize them as stimuli-
responsive host materials148,149.
CHAPTER III – RESULTS AND DISCUSSION
148
The heats of sorption [see Table 3.6] either increase or remain almost unchanged
(within experimental error) at low temperatures and high loadings. While the temperature
dependence of isosteric heats of sorption is generally considered to be weak and may be
neglected, the increase of qst with loading is usually ascribed to the presence of strong
lateral interactions between sorbate molecules within the micropores. In general, the
isosteric heat of sorption is defined by a number of factors and previous studies on
sorption in zeolites suggest that it may be a complex function of loading150-155. Other
studies156 signify that deformations of the sorbent may contribute significantly to the
variation of qst, the factor being important for the flexible sorbent materials of this study.
The thermodynamic values derived in this work may be compared with those of
zeolites and other sorbents. Zeolites present host frameworks with much stronger local
charges distributed over the crystal framework that may imply stronger inductive host-
guest interactions. Another major distinction is the rigidity of the zeolite framework. At
the same time, zeolites may reveal similarity to the materials of this work in the topology
of their micropores. Sorption of Xenon in 1D channels of ferrierite having 4.2 x 5.4 Å
cross-section dimensions158 is characterized by an initial (lowest loading) qst of 31.4
kJ/mol.159,160 Sorption of Xenon in the 1D channels of mordenite (6.5 x 7.0 Å) is
characterized by an initial qst of 35.1 kJ/mol.161 Sorption of Xenon in the 3D system of
channels in Linde 5A zeolite (diameter ~5Å) is accompanied by an initial qst of 22.5
kJ/mol which increases to ~25 kJ/mol at higher loading. Sorption of Xenon in 3D system
of channels in silicalite158 (structural type ZSM-5, 5.3 x 5.6 Å and 5.1 x 5.5 Å) is
accompanied by an initial qst of 26.6 kJ/mol.162 From another study163, the sorption of
argon, methane and sulfur hexafluoride in silicalite is characterized with k0 of 50 10-3, 27
10-3 and 3.7 10-3, respectively. The sorption of Xenon in the 3D system of channels in
faujasite158 (NaX, diameter ~7.4 Å) is accompanied by an initial qst of 19.2 kJ/mol and k0
of 7.4 10-3 (zero filling, 193 K)164. In one study, sorption of Xenon in an organo-clay with
~6 Å micropores was reported with a qst of 14 kJ/mol.165 As can be seen from these
comparisons, the materials used in the present study have thermodynamic characteristics
with respect to the sorption of Xenon that are almost in the same range as for zeolites, in
spite of the differences mentioned above. This may well imply that the sorption process is
CHAPTER III – RESULTS AND DISCUSSION
149
controlled by van der Waals forces to a good approximation and is defined to the greatest
extent by the fit between the Xenon atom and the host cavity. Similar conclusions for
silicalite were made from molecular dynamics simulations166. It also can be speculated
that the advantage of zeolites as sorbents ascribed to the presence of local charges may be
partially compensated by flexibility of dipeptide matrices that can provide better Xenon-
cavity fits by adjusting the pore structure during the course of sorption.
It is interesting to follow the changes in the isosteric heat of sorption with the
diameter of nanochannels in the series studied. As the driving force for the absorption is
the affinity between the guest and the pore walls, pores with dimensions complementary
to the diameter of Xenon might be expected to result in a higher heat of sorption due to
the optimal guest-host contacts106. Figure 3.33 shows qst versus channel diameter (from
He pycnometry88 for the series studied. As becomes apparent from the Figure, the highest
heat of sorption occurs for a channel with a diameter of ~4.4 Å, which is virtually equal
to the diameter of the Xenon atom. It is also interesting that the decrease of the channel
diameter to values lower than 4.4 Å causes only a slight decrease in the sorption energy.
Again, this result must be a consequence of the high flexibility of the dipeptide matrices
where the close contact of the channel with Xe requires some expansion.
In Figure 3.33 are summarized the values derived for enthalpies and entropies of
adsorption as obtained by the analysis of 129Xe NMR isotropic chemical shift (a,c) and
signal intensities (b,d) for all the eight dipeptides studied in this work, plotted as a
function of the channel diameters as previously obtained from He pycnometry analysis88.
It is worth noting that the analysis of the 129Xe NMR signal intensities can provide
reliable results only about the thermodynamic of adsorption in the high temperature
region, due to the effects of reduced diffusivity at temperatures below about 300K while
the fitting of chemical shifts is mostly influenced by the low temperature points, the Xe-
Xe interaction dominating the observed chemical shift only below about 260K.
The knowledge of the physical constants which describe the adsorption
characteristics of an adsorbate-adsorbent system is of fundamental significance in the
design and development of nanoporous materials. Future improvements of the adsorption
CHAPTER III – RESULTS AND DISCUSSION
150
technology rely on understanding the issues that control the selectivity of the adsorption
process and on elucidating the correlation between energetic and structural parameters.
The analysis of the values reported in Table 3.6 points out the variation of the enthalpies
of adsorption with temperature found in some of the dipeptides studied. VA and AV
show an increase of enthalpy of adsorption with increasing adsorbate loading and/or
decreasing temperature. AI on the other hand shows a first decrease and then an increase
of isosteric heats of adsorption and the results were found to be strongly dependent on the
aging of the sample. While the temperature dependence of isosteric heats of adsorption is
generally considered weak and neglecting this effect is a commonly adopted
approximation124, the increase of qst with increasing loading is sometimes ascribed to the
presence of strong lateral interactions between adsorbate molecules within the micropores
while the decrease of this parameter with decreasing temperature can be ascribed to
heterogeneous guest-host interactions155,167. The results concerning AI dipeptides suggest
that the heterogeneity of adsorption characterize this process at high temperature, while
the effect of Xe-Xe interactions is dominant at low T. A single value in the limits of the
experimental and fitting errors characterizes instead the heats of adsorption of Xenon in
all the other dipeptides studied as a function of the temperature, indicating an almost
perfect balance between the strength of the gas-gas interactions and the heterogeneity of
guest-host interactions. The agreement between the values extracted by the analysis of
the chemical shift and those derived from the fitting of signal intensities confirms the
validity of the thermodynamic model proposed and substantiates the characterization
method. A further comment should be done about the interesting relationship existing
between the enthalpic and entropic terms of adsorption and the diameters of the channels.
As the driving force for the adsorption is the affinity between the guest and the pore
walls, pores with the same size of the guest will have the more favorable energy of
adsorption due to the optimization of guest-host contacts. This is evident from Figure
3.33 a and b, where the qst clearly increase up to the diameter of the Xenon atom (about
4.4 angstrom) and then decrease again for larger diameters.
CHAPTER III – RESULTS AND DISCUSSION
151
a) b)
Figure 3.33: Heats of sorption qst [kJ/(mol of Xe)] plotted as a function of the pore diameter [Å]
(experimental data from helium pycnometry88. The values for qst correspond to a) high loadings, low
temperatures (from isotropic chemical shifts) and b) low loadings, high temperatures (from signal
intensities). Dashed lines are linear fittings to guide the eye.
CHAPTER III – RESULTS AND DISCUSSION
152
3.3.8. - Dipeptides: CONCLUSIONS
This study exploits the continuous-flow HP 129Xe NMR technique to extract
comprehensive knowledge on the thermodynamics and molecular-level structural features
of Xenon sorption in a series of microporous dipeptides. In particular, quantitative
thermodynamic parameters, such as isosteric heat of sorption have been determined for
each material with a good accuracy for two compositions corresponding to low and high
loading of the microporous solid with Xenon atoms.
The approach introduced is based on the derivation of sorption isobars under the
dynamic conditions imposed by the continuous-flow HP 129Xe NMR experiments. The
interaction of sorbent with sorbate is ananlyzed with two independent methods based on
the temperature dependence of
1) the intensities
2) isotropic chemical shifts
of the Xenon NMR signal.
The first method is applicable in the high-temperature region, where the loadings
are close to zero. The second method applies to the low-temperature region, where the
loadings are close to maximum. Reasonable agreements between the isosteric heats of
sorption derived by the two methods (the values are expected to be different but not more
than 20-30%) as well as reasonable trends in the values in the series studied confirm the
validity of the thermodynamic model proposed and substantiates the characterization
approach introduced. So far, isobaric determinations have been applied to surface
adsorbents112,118, while the approach illustrated in this study may become a practically
important tool for the express evaluation of a wide range of porous sorbent materials.
As compared to traditional studies based on the determination of a series of
sorption isotherms, the sorption isobar approach introduced is much faster and may be
used, as illustrated well in this study, for characterization of less stable materials or
materials subjected to deterioration progressing in time. Also, the method is much more
applicable to materials with very narrow pores when the equilibrium with the total bulk
of the sorbent is not easy attainable. The possibility to cross-check the data obtained from
signal intensities and from chemical shifts makes it possible to control the adequacy of
CHAPTER III – RESULTS AND DISCUSSION
153
measurements: the data extracted from the chemical shifts do not depend on the total
amount of the sorbent, while the underestimation of this amount in a sorption isotherm
measurement (for example, due to a non-porous impurity) could essentially ruin the
results. The quantity of the material used was ~70 mg; that was sufficient to collect all
necessary spectra and the material was recovered after the measurements.
Another advantage is the simultaneous availability of information on the
structural properties of the micropore space and sorption events occurring on a
molecular-scale from the spectra. In particular, an estimate of the pore diameter is
instantly available and reasonable assumptions about the pore geometry may be made.
On the contrary to these advantages, one drawback of the approach used is the
lack of information on the absolute value of maximal loading, that is, the capacity of a
sorbent studied with respect to Xenon sorbate. This information should be obtained from
an independent experiment; for example, the value may be calculated from a single
sorption isotherm or estimated from the crystal structure of the sorbent material. For the
dipeptides in the present study, the capacity of AV and VA was calculated as 0.5 mol of
Xe per mol of dipeptide116, while the values for the other studied materials, as estimated
from their crystal structures88 should be in the range 0.33-0.5 mol of Xe per mol of
dipeptide.
Previous work has illustrated the use of the continuous-flow HP 129Xe NMR
technique mostly for qualitative characterization of porous solids88,,102,113,116. Recently
continuous-flow NMR techniques on various nuclei were also used to study catalyzed
chemical reactions in zeolites168-170. This study provides a precedent for the use of
continuous-flow NMR measurements to obtain the comprehensive description of a
sorption process rapidly, providing both quantitative determination of its fundamental
thermodynamic parameters and qualitative understanding of the structural changes and
dynamics accompanying the process on the micro-level.
CHAPTER III – RESULTS AND DISCUSSION
154
3.4 Bibliography
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2. RF Tilton, ID Kuntz, GA Petsko Biochemistry (1984), 23, 2849-2857
3. Rubin SM, Spence MM, Pines A, Wemmer DE, J Magn Reson (2001), 152, 79-
86
4. Locci, E., Dehouck Y., Casu M., Saba G., Lai A., Luhmer M., Reisse J., Bartik
K. J.Magn. Reson. (2001), 150, 167-174
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CHAPTER VI – MATERIALS AND METHODS
165
4.1 - Hyperpolarized Xenon: solving sensitivity problems
Early studies, dated about 1980, first anticipated the usefulness of combining
Xenon as an ideal probe and nuclear magnetic resonance as the spectroscopic technique
of choice for the characterization of void space in porous materials and biomolecules1-4.
Since then, Xenon NMR has been applied to a number of different systems thus
confirming the actual sensitivity of NMR parameters of Xenon to its local physical and
chemical environment.
However, very likely Xenon NMR wouldn’t have been so widespread diffused as
it actually became if it wasn’t for the discovery and development of hyperpolarization
processes that allow enhancing Xenon NMR signal usually by factors of 105-106. The
notorious lack of sensitivity, embedded in the nature of NMR measurements, was in fact
one of the most important drawbacks of this technique and somewhat limited its power of
applicability until when, in 1991, a group of researchers at Berkeley University, led by
Prof. Pines, first applied5 NMR measurements to the characterization of materials
surfaces with hyperpolarized 129Xe.
The possibility of inducing non-equilibrium distribution in electronic spins of
gaseous metal vapors was previously demonstrated6-8 by Prof. Alfred Kastler, who won
the Nobel Prize in Physics in 1966 "for the discovery and development of optical
methods for studying Hertzian resonances in atoms". Kastler’s studies basically showed
that circularly polarized light could be used to pump electronic spin polarization to non-
equilibrium population distributions in metal gases.
Only few decades later it was shown that also nuclear spin polarization of the
noble gases present as buffer gases within the pumping cell could be enhanced by means
of spin exchange induced by collisions with electronically spin-polarized alkali metal
atoms9,10. Happer and coworkers successively further studied and developed the
hyperpolarization process in detail11.
In order to achieve hyperpolarization of Xenon nuclei angular momentum must be
transferred first to electronic and then to nuclear spins thus requiring the simultaneous
presence of different species in the polarizing batch apparatus. In particular, the most
used method for producing optically polarized Xenon is the so-called alkali metal spin
exchange.
CHAPTER VI – MATERIALS AND METHODS
166
This method basically consists of two steps: in the first step circularly polarized
laser light polarizes electrons of gaseous alkali metal atoms and in the second step
collisions between unpolarized Xenon atoms and electronically polarized alkali metal
atoms permit spin exchange between electron spins and noble gas nuclear spins. Often
nitrogen is added to the gaseous mixtures in order to quench the fluorescence of the
electronically excited metal atoms that would otherwise depolarize the electron spins.
In more detail the hyperpolarization process can be described as follows:
In the first step, circularly polarized laser light tuned to the D1 transition of
Rubidium atoms (D1 = 794.7 nm) drives their electronic population from mJ = -1/2
sublevel of the ground state into the mJ = +1/2 excited state. Thus electronic spin state of
Rb, 52S1/2, is excited to 52P1/2.
Figure 4.1: Schematic representation of the alkali-metal spin exchange processes.
The population Pel of Rb electrons is determined by the relation
SDOP
OPelP
ρρρ+
= [4.1]
where ρOP is the rate at which Rb is electronically hyperpolarized by means of laser light;
ρSD is the so-called “spin destruction” rate, which basically identifies the spin-relaxation
rate of Rubidium atoms. PRb drops to zero near the cell walls but it permits significant
CHAPTER VI – MATERIALS AND METHODS
167
polarization of Xe gas by spin exchange far from the cell walls. Over time (seconds to
hours, depending upon experimental conditions), the nuclear polarization of the noble gas
accumulates, yielding values as high as several tens percent.
In the second step, collision-induced spin exchange is made possible by Fermi-
contact hyperfine interactions between spin polarized electron spins of Rb atoms and
unpolarized Xenon nuclear spins.
The final nuclear polarization Pnu reached in nuclear spins of Xenon after a time t of
optical pumping is given by the equation:
( )[ ]tRb
SE
SE SEePP 010
ρρ
ρρρ +−−⋅⋅
+= [4.2]
where PRb is the electron spin polarization of Rb atoms, ρSE is the rate of spin exchange
between Rb electrons and Xenon nuclei, ρ0 is a factor which takes into account
longitudinal relaxation of noble gas nuclei.
4.1.1. - Continuous-flow measurements.
An apparatus for the production of large quantities of hyperpolarized Xenon was
first proposed by Happer and coworkers12. In the original design, a high power diode
array (130W) coupled to a polarizer produces a circularly polarized light at the proper
wavelength to electronically polarize Rubidium atoms; the gas used was composed by
few hundred torr (∼0.3-0.5atm) of the mixture of Xenon and Nitrogen, while Helium (8-
10 atm) was used as buffer gas. Moreover, in the original experimental setup the
hyperpolarized Xenon was collected into a Dewar of liquid nitrogen and stored. With
careful preparation of storage vessels, hyperpolarized Xe can be maintained for long
periods of time (hours to days). It has been in fact demonstrated, for example, that T1 of 129Xe is ∼3 hours at 77 K (liquid nitrogen) and >100 hours at 4.2 K (liquid helium) when
it is kept under a high magnetic field (> 500 G).
CHAPTER VI – MATERIALS AND METHODS
168
The experimental setup used at National Research Council (NRC-SIMS) in
Ottawa, Canada, is based on the just described apparatus and uses a continuous flow of a
gas mixture of Xenon (∼1%), Nitrogen (3%) and Helium (98%) and is therefore
commonly referred to as “continuous flow” device.
Here, two cells are connected by a tube to the gas cylinder where the gas mixture
is kept under pressure: the first cell contains Rb (Rb reservoir) vapors and in the second,
the so called “pumping cell”, spin exchange between Rb and flowing Xe atoms takes
place. Both cells need to be heated to about 150 °C during the polarization process. The
pumping cell is placed in the fringe field (8-20mT) that is present outside the
superconducting magnet, which is exploited in order to split electronic levels of Rb atoms
with different mJ. Two traps are also connected to the gas line in order to avoid presence
of oxygen and water in the pumping cell. At the end of the line, usually just after the coil
region of the NMR probe, the gas mixture is blown off into the atmosphere [see Figure
4.2].
A gas flow of about 0.3-0.5 liters per hour is typically used with the setup just
described. Laser power does not usually need to be too high especially because it could
result in cell overheating and possibly explosions. Additionally, a very high laser power
would be almost unnecessary as reduction in laser power often causes only small
reductions in spin polarization.
CHAPTER VI – MATERIALS AND METHODS
169
Figure 4.2: Experimental setup Continuous flow-type for the production of hyperpolarized Xenon installed
at NRC-Canada, SIMS (Ripmeester’s group), Ottawa. Detailed description is given in the text.]
4.1.2. Advantages (and drawbacks) implied in the use of hyperpolarized and
thermally polarized Xenon.
Several important advantages are connected to the use of hyperpolarized gases
beyond sensitivity enhancement.
A very important involvement of developing and improving optical pumping
processes is that when hyperpolarized species are used the polarization of the spins is no
longer dependent on the magnetic field strength they experience, because the
magnetization of the sample is no longer given by the Boltzmann distribution. Therefore
the technique of hyperpolarization allows also measurements of small samples in very
small magnetic fields, which is possibly the major breakthrough of NMR measurements,
CHAPTER VI – MATERIALS AND METHODS
170
because of the intrinsic difficulty in the construction of ultra-high field magnets. This, for
instance, has provided the opportunity to develop mobile devices that are able to perform
high resolution Nuclear Magnetic Resonance measurements in the Earth’s magnetic
field13. Mobile, zero-field measurements and squid detectors is therefore greatly favoured
by the use of hyperpolarized species. Moreover, by avoiding the use of large and strong
magnets, the safety, cost, comfort flexibility and portability are improved. The ease of
management is also much enhanced. The use of a low partial pressure of Xenon and high polarizations simultaneously
allows obtaining well resolved spectra in relatively short time and neglecting Xe-Xe
interactions which usually influence spectra obtained by using higher Xe pressures. Thus,
spectra obtained by utilizing this setup are characteristic of the framework or surface of
the systems analyzed14. Polarization transfer by SPINOE is another important tool which
is available only if HP Xenon is used.
It is worthnoting, however, that only thermally polarized Xenon samples the bulk
of materials while fastly relaxing HP atoms can give information of just the surface of a
material. Also, use of hyperpolarized Xenon in systems containing paramagnetic centers
is usually unfruitful because hyperfine couplings between hyperpolarized atoms and
unpaired electrons act as an additional significant relaxation mechanism.
CHAPTER VI – MATERIALS AND METHODS
171
4.2 Myoglobins.
Myoglobins (wild-type) were isolated and purified according to the Wittenberg
and Wittenberg method15 with some modifications. Fresh or frozen animal heart tissue
was homogenized in 10 mM Tris–HCl pH 8.0 and differentially precipitated with
ammonium sulfate (65%, 75% and 90%). The precipitated myoglobin was dissolved in
0.02 M Tris–HCl pH 8.2 plus 1 mM EDTA and passed in a column (5±50 cm) of
Sephadex G-100 equilibrated with the same buffer. The solution of myoglobin was
completely purified by passing it on a column (2.5_25 cm) of DEAE-cellulose
equilibrated with 0.02 M Tris–HCl pH 8.4 containing 1 mM EDTA. Pig MMb was
obtained with a very small excess of potassium ferricyanide followed by exhaustive
dialysis against water to remove the excess of salt. The purity of both pig and horse MMb
was checked by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)
under reducing conditions according to the classical procedure16.
The CNMb solutions were prepared from the ferric derivatives (MMb) by adding
the sodium cyanide (Sigma) in large excess over the heme. After the reaction, the excess
cyanide sodium was removed by mild dialysis with H2O/NaOH pH 8.0. The UV-visible
spectra of aqueous CNMb samples, in the absence of Xenon, show the typical features of
a low-spin hemoprotein17 with absorption maxima at 422 and 540 nm.
Xenon gas at natural isotope abundance (purity of 99.99%) was purchased from
SIAD (Italy), Xenon (90.9% 129Xe, 99.95% purity) from Chemical Research 2000, Italy.
Wilmad high-pressure NMR tubes (OD 10 mm and ID 7.1 mm, OD 5 mm and ID 4.2
mm) were used for all measurements.
Before NMR analysis, the Mb solutions were freshly prepared at room
temperature by dissolving the protein in a phosphate buffer 0.01 M, 20% D2O. The exact
protein concentration was determined spectrophotometrically at λmax=280 nm (ε=31000
M-1cm-1). Samples were degassed on a vacuum line without freezing to avoid protein
denaturation. Up to 10 atm of Xenon gas were pressurized into the samples at room
temperature. Before acquiring the NMR spectra, the samples were equilibrated for one
hour.
Carbonmonoxy myoglobins (COMb) were prepared by extensively purging the
solution with CO, followed by reduction with a minimum amount of freshly prepared 1
CHAPTER VI – MATERIALS AND METHODS
172
M sodium dithionite solution. Then the solution was eluted by ECONOPAC 10DG
column in a resin to get rid of the sodium dithionite and then again the protein solution
was extensively purged with CO. This solution was lyophilized and then anaerobically
transferred to a dry box filled with argon atmosphere. COMb was diluted in a phosphate
buffer, which was previously degassed using three freeze–pump–thaw cycles. A small
aliquot was then removed to search for protein concentration and oxidation status. The
sample absorbance at 540 and 580 nm for COMb and at 505 and 640 nm for MMb were
used to test the oxidation status. The solution of COMb was then prepared only in the
case of total absence of MMb bands. The COMb solution was prepared in a dry box and
the sample was then transferred to Wilmad high-pressure NMR tubes. Sample was
degassed of argon on a vacuum line without freezing. At the end of each experiment
session, the sample was checked to verify the oxidation status of our protein by UV-vis
absorption spectrum. In the samples, a content of MMb lower than 4% is accepted. 129Xe NMR spectra were performed on a Varian VXR-300 spectrometer at a
resonance frequency of 82.968 MHz. The chemical shift measurements were carried out
at 25.0 ± 0.1°C, using 21 µs pulse (90°), 0.5 s repetition time, and spectral width of 20
kHz. The 129Xe chemical shift of Xenon (1 atm Xenon overpressure) dissolved in
phosphate buffer 0.1 mM, 20% D2O represented our reference. Each experiment run,
consisting of 10 and 12 points, lasted 4 days. Before and after each experimental session,
a buffer solution containing 1 atm of Xenon was run and the 129Xe chemical shift checked
in order to ensure that the measured values were not caused by artefacts.
Titration data were obtained by a non-linear fitting procedure by means of the
Kaleidagraph program; reported errors in binding constants and 129Xe chemical shifts
must be intended as fitting errors. 129Xe NMR spin lattice relaxation times were measured using the inversion
recovery method with an acquisition time of 0.5 s and a recycling delay of 5 s. The
average T1 values were obtained by three-parameters non-linear least square fitting
procedure. The reported errors were estimated from fitting errors. The number of scans
recorded varied from spectrum to spectrum to achieve a good signal to noise ratio (6<
S/N < 10 in the spectra with 1 atm of Xenon overpressure, S/N >10 in all the others).
CHAPTER VI – MATERIALS AND METHODS
173
We collected 1H NMR spectra on a Varian Unity-Inova spectrometer at a
resonance frequency of 399.948 MHz and at T = 28 + 0.1 °C. The experiments were
carried out on a 5 mm high-pressure tube using 7 µs pulse (90°), 1 s repetition time, and
spectral width of 100 kHz. Chemical shifts in all spectra are referenced to DSS (2,2-
dimethyl-2-silapentane-5-sulfonate) through the residual solvent signal. CNMb solutions
were ~1 mM in 100 mM sodium phosphate, pH 7.5. Each experiment session,
corresponding to 11 points, took 6 days.
The magnitude COSY spectra18 were acquired in D2O over a spectral window of
20000 Hz using 4096 t2 complex points. We performed 128 scans for each block with a
total of 1024 t1 blocks. The acquisition time was 0.102 ms, and the total acquisition time
~20 h. The residual H2O was suppressed by pre-irradiating the water signal for 0.5 s.
Phase-sensitive NOESY spectra19 were collected in H2O. 10% D2O was added for the
lock, over a spectra window of 20000 Hz using 4096 t2 complex points. Each of the 512
t1 increments was sampled by 128 scans. The mixing time was 50 ms with a repetition
delay of 0.102 ms resulting in a total acquisition time of 12 h. The pre-irradiating
treatment of the water signal was carried out for 0.5 s. Phase-sensitive TOCSY spectra20
were acquired in D2O over a spectra window of 20000 Hz using 4096 t2 complex points;
64 scans were collected for each block with a total of 916 t1 blocks. The spin lock time
used was 50 ms with a recycle time of 0.6 s using the MLEV-17 mixing scheme.
Suppression of the residual H2O signal was obtained by pre-irradiating the water signal
for 0.5 s.
Volumes of the cavities and centres of gravity (CGs) were calculated with
VOIDOO21, which is a program for computing molecular volumes and studying cavities
in macromolecules such as proteins. This program uses an approach where the cavity
volume is defined as the volume swept out by a probe sphere rolling over the surfaces.
The contacts between the probe-sphere and the van der Waals’ protein surface delimit the
probe-occupied cavity, which is similar to the Connolly-type surface22. In addition, all
atoms/residues lining the cavities are identified.
The calculated volumes were sensitive to the inputted parameter values chosen for
the grid size and the probe radius parameters. Different probe sizes, from 1.0 to 1.4 Å,
and grids, from 0.3 to 0.5 Å, were tried. At least five cavity calculations were carried out
CHAPTER VI – MATERIALS AND METHODS
174
for each molecule with randomly generated orientations in order to minimize
measurement artefacts. The use of a probe radius of 1.2 Å and a grid of 0.35 Å gave the
most reliable results since the volumes were found to be consistent with those reported23
in the analysis of cavities in sperm whale MMb.
4.3 Microporous Dipeptides
Eight dipeptides (AV (Ala-Val), VA (Val-Ala), AI (Ala-Ile), LS (Leu-Ser), VV
(Val-Val), IA (Ile-Ala), IV (Ile-Val) and VI (Val-Ile)) were purchased from Bachem.
Powdered samples for 129Xe NMR analysis were prepared by grinding as-received
materials followed by drying them overnight at 60ºC to remove moisture from the pores.
LS had to be recrystallized and desolvated prior to preparation, as described previously24.
In addition, the samples were purged with a continuous flow of the Xe-N2-He gas mixture
(BOC, Canada, volume composition Xe:N2:He = 1%:3%:96%) at room temperature for
10-15 minutes in the NMR probe before each NMR experiment. About 70 mg of each
dipeptide was sufficient to obtain a good S/N ratio for all spectra.
All 129Xe NMR spectra were obtained on a Bruker AMX300 spectrometer
operating at 83.013 MHz (magnetic field 7.05 T) using a customized probe from Morris
Instruments. The majority of the experiments were performed using a continuous flow of
hyperpolarized Xe gas, as described previously14. The flow rate was monitored using a
Fathom Technologies flow controller (model GR-116 3-A-PV). The gas flow was set to
0.3 L/hr in order to achieve a good signal to noise ratio and kept constant for each
experiment. A Solid-State Spin-Echo sequence was used to acquire all of the data25 with
90º pulse length of 3 µs and 180º pulse of 6 µs τ1 and τ2 delays were chosen as short as
100 µs and a recycle time of 1 s was used.
The continuous flow of hyperpolarized (CF HP) Xe was delivered to the NMR
coil through a 2 mm plastic tubing. The temperature in the probe was controlled using a
Bruker BT1000 temperature controller with an accuracy of 0.1 K. The variable
temperature experiments were performed with decreasing temperature stepwise from 343
K to 173 K. The sample was allowed to equilibrate at each temperature for 10 minutes
before the corresponding spectrum was collected.
CHAPTER VI – MATERIALS AND METHODS
175
The observed intensities, chemical shifts and signal anisotropies were constant for
the same dipeptide at a given temperature and they showed reproducible values over a
series of repeated measurements. Also, the temperature dependent changes upon heating
and cooling were reversible.
The reported 129Xe NMR chemical shifts were referenced to Xenon gas, set to 0
ppm. Analysis of the anisotropic lineshapes was performed using a Bruker simulation
module, Topspin 1.3. Integration of the signals was done using the Bruker processing
program XWinNMR. The relative integral intensity of the signal (I) was calculated as the
ratio of intensity from the signal of Xenon residing in the nanochannels (Ich) and the
combined intensity of Xenon in the gas phase and Xenon adsorbed on the external
particle surface and intercrystallite regions (Igas).
4.4 Copper containing Amine Oxidases
Materials - All reagents were of the highest purity degree available. 1,4-
Diaminobutane dihydrochloride (putrescine), glutamate dehydrogenase, NADH,
phenylhydrazine, α–oxoglutarate acid and 3–aminopropionic acid, 1,5-diaminopentane
dihydrochloride (cadaverine), benzylamine hydrochloride and N,N’-bis(3-aminopropyl)-
1,4-butane diamine tetrahydrochloride (spermine) were purchased from Sigma Aldrich
(St. Louis, MO). Xenon chemical shift measurements were made using 92% enriched 129Xe (Chemical Research 2000; Rome, Italy). Wilmad high–pressure NMR tubes
(Buena, NJ; OD 5 mm and ID 7.1 mm, OD 5 mm and ID 2.2 mm) were used for all
measurements.1,4-Diamino-2-butyne (DABY) was synthesized as previously reported26.
.
Enzymes - Only protein of the highest quality was utilized on the basis of the ratio
TPQ/dimer in the range 1.9-2.1. The concentration of the quinone content was
determined by titration with the carbonyl reagent PHY which gives a hydrazone with a
very high extinction coefficient27 at 445 nm (ε445 = 6.4 × 104 M–1 cm–1). An ε498 of 4.1 ×
103 M-1 cm-1 or an ε278 of 2.45 × 105 M-1 cm-1 for the purified enzyme (2 copper ions and
a Mr = 150000) was used to estimate the enzyme concentration27. Catalytic activity, (kc)
defined as mol of substrate used per mol of active sites in 1 s. Amine oxidases from
CHAPTER VI – MATERIALS AND METHODS
176
bovine plasma (BSAO; kc = 0.35 s–1 using benzylamine as substrate)28, pig kidney
(PKAO; kc = 4.5 s–1 using cadaverine as substrate)29, pea seedlings (PSAO; kc = 140 s–1
using putrescine as substrate)30, lentil seedlings (LSAO; kc = 155 s–1 using putrescine as
substrate)31 and Euphorbia characias latex (ELAO; kc = 23 s–1 using putrescine as
substrate)32, were each extracted and purified according to previously described
procedures. Copper–free lentil amine oxidase was prepared as previously described33.
Residual copper, measured by atomic absorption spectroscopy, was 0.2 ± 0.02% of the
original content.
Activities of the tested enzymes were performed according to the procedures
reported in the related references. Oxygen uptake was determined with a Clark–type
electrode coupled to a OXYG1 Hansatech oxygraph (Hansatech Instruments Ltd. King’s
Lynn, Norfolk, England). The temperature of reaction chamber was controlled at 37 °C
using a circulating water bath. The solution (1 ml) containing the enzyme in 1 mM Na+–
phosphate buffer, pH 7.0, was maintained for 20 min at constant level of oxygen as
previously reported34,35 and the reaction was started by addition of the related substrate.
The value of KM for AOs using different substrate concentrations at saturating
concentration of oxygen (219 µM), or varying concentrations of oxygen at a saturating
concentration of substrate, was calculated from initial velocity data fitted to the
Michaelis–Menten equation by nonlinear regression and by double reciprocal plots by
Michaelis–Menten analysis in 1 mM Na+–phosphate buffer, pH 7.0. Benzylamine oxidase
activity was measured in 1 mM Na+–phosphate buffer, pH 7.0, by monitoring the
increase in absorbance of UV-light at 250 nm using an ε250 = 12.8 mM-1 cm-1 for
benzaldehyde36. Catalytic centre activity, (kc) is defined as mol of substrate consumed per
mol of active sites × s–1.
Spectroscopic Methods - UV/Vis Experiments. Absorption spectra of LSAO in 1
mM sodium phosphate buffer, pH 7.0, were recorded at 25 °C with an Ultrospec 2100 pro
spectrophotometer (Biochrom Ltd., Cambridge, England). Anaerobic experiments were
made in a Thunberg–type spectrophotometer cuvette (Soffieria Vetro, Sassari, Italy).
Solutions were subjected to several cycles of evacuation followed by flushing with
Argon.
CHAPTER VI – MATERIALS AND METHODS
177
Fluorescence Spectra - Fluorescence spectra were obtained using a Perkin–Elmer
LS–3 spectrofluorimeter (Perkin–Elmer Ltd, Buckinghamshire, UK).
129Xe NMR Experiments - Samples of native lentil AO in 1 mM sodium phosphate
buffer, pH 7.0, 20% D2O, were degassed using three freeze–pump–thaw cycles,
pressurized with Xenon gas into 5 mm Wilmad high–pressure tubes (OD 5 mm and ID
7.1 mm, OD 5 mm and ID 2.2 mm; Buena, NJ), and allowed to equilibrate for 48 hours. 129Xe NMR spectra were recorded on a Varian VXR-300 spectrometer (Palo Alto, CA) at
a resonance frequency of 82.968 MHz. Chemical shift measurements were carried out at
25.0 ± 0.1 °C, using 30° pulse lengths (7 ms), 2 s repetition times and spectral width of
20 kHz. The obtained chemical shifts were referred to the 129Xe chemical shift of Xenon
(8 atm overpressure) dissolved in 1 mM sodium phosphate buffer, pH 7.0, 20% D2O,
used as reference standard. The 129Xe NMR spectrum, due to the noisy signals, allows the
determination of each resonance peak with an estimate accuracy of 0.1 ppm. 129Xe NMR
spin lattice relaxation time of native and Copper–free lentil AO were measured using the
inversion recovery method, with an acquisition time of 1 s and a recycling delay of 3T1.
Assays of Products - Benzylamine oxidase activity was measured in 1 mM
sodium phosphate buffer, pH 7.0, by monitoring the increase in absorbance at 250 nm
using an ε250 = 12.8 mM-1 cm-1 for benzaldehyde37. Ammonia production was checked
from the amount of NADH consumed in the presence of glutamate dehydrogenase and
hydrogen peroxide formation was detected with the peroxidase/4-hydroxy-3-
methoxyphenylacetic acid method38.
α-Aminoadipic-δ-semialdehyde (also 2-amino-6-oxo-hexanoic acid or, more
commonly, allysine) residue was derivatized to a decarboxylated fluoresceinamine
(AASF) and determined by high performance liquid chromatography (HPLC)40,41.
CHAPTER VI – MATERIALS AND METHODS
178
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PAPERS PUBLISHED
180
PAPERS PUBLISHED DURING THE DOCTORATE
1. R. Medda, S. Longu, R. Anedda, A. Padiglia, A. Mura, M. Casu, and G. Floris AN UNEXPECTED FORMATION OF THE SPECTROSCOPIC CuI–SEMIQUINONE RADICAL BY
XENON–INDUCED SELF–CATALYSIS OF A COPPER QUINOPROTEIN
Biochimie, 88 (2006) 827-835.
2. Matteo Ceccarelli, Paolo Ruggerone, Roberto Anedda, Mariano Casu, Antonella Fais, Benedetta Era, Maria Carla Sollaino and Marcella Corda
STRUCTURE-FUNCTION RELATIONSHIP IN A VARIANT HAEMOGLOBIN: A COMBINED
COMPUTATIONAL-EXPERIMENTAL APPROACH
BIOPHYSICAL JOURNAL, 91 (2006) 3529-3541
3. A. Mura, F. Pintus, R. Anedda, M. Casu, A. Padiglia, G. Floris and R. Medda IMPORTANT LYSINE RESIDUE IN COPPER/QUINONE CONTAINING AMINE OXIDASES.
FEBS Journal 274(10), (2007) 2585-2595 4. R. Anedda, C. Cannas, M. Casu, A. Musinu, G. Piccaluga
A TWO-STAGE CITRIC ACID - SOL/GEL SYNTHESIS OF ZnO/SiO2 NANOCOMPOSITES:
STUDY OF PRECURSORS AND FINAL PRODUCTS
Journal Nanoparticle Research, in press (2007) 5. Matteo Ceccarelli, Roberto Anedda, Mariano Casu and Paolo Ruggerone
CO ESCAPE FROM MYOGLOBIN WITH METADYNAMICS SIMULATIONS Proteins - Structure, function and bioinformatics, in press (2007) 6. Fais A, Anedda R, Porcu S, Casu M, Ruggerone P, Ceccarelli M, Sollaino M, Galanello R, Corda M
IDENTIFICATION AND MOLECULAR CHARACTERIZATION OF A NEW DOUBLE VARIANT
HEMOGLOBIN (Hb G-Philadelphia/Duarte α268Asn→Lysβ2
62Ala→Pro) Submitted (2007)
7. R. Anedda, D.V. Soldatov, I.L. Moudrakovski, M. Casu, J.A. Ripmeester A NEW APPROACH TO CHARACTERIZE SORPTION IN MATERIALS WITH FLEXIBLE MICROPORES
Submitted (2007)
8. Long-Li Lai, Chun-Han Wu, Roberto Anedda, Kuang-Lieh Lu, Yu-Shen Wen, Dao-Wen Luo, Kung-Lung Cheng, Zhi Yu, Kui Yu, and John A. Ripmeester
UNUSUAL INCORPORATION OF CD(SCN)64- BY THE VIOLOGEN TEMPLATES VIA
THE HOST-GUEST SELF-ASSEMBLY AND SUBSEQUENT REACTION OF ONE OF THE REPRESENTATIVE ON THE SOLID STATE
Submitted (2007)