Lorentz Center - Electron Paramagnetic Resonance at High Field and High Frequency: Technology and Applications from 10 May 2004 through 12 May 2004
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    Electron Paramagnetic Resonance at High Field and High Frequency: Technology and Applications
    from 10 May 2004 through 12 May 2004

 

         180 GHz PULSED EPR AND 95 GHz ENDOR FOR THE MECHANISTIC STUDIES OF ENZYME CATALYSIS

 

   Marina Bennati1, Melanie Hertel1, Vasyl Denisenkov1, Thomas Prisner1, Hans.-R. Kalbitzer2, Norbert Weiden3, K.-Peter Dinse3, Reiner Hedderich4, Matthias Boll5

 

1Institute of Physical and Theoretical Chemistry, J. Goethe University of Frankfurt,Germany 2Department of Biophysics, University of Regensburg, Germany, 3Institute of Physical Chemistry III, DarmstadtUniversity of Technology, Germany, 4Max Planck Institute for Microbiology, Marburg, Germany, 5Institute of Biology II, University of Freiburg, Germany

 

 

High frequency pulsed EPR and ENDOR have emerged as essential techniques to study intermediate states in enzyme catalysis, such as transient radical intermediates or enzyme-substrate interactions. In this contribution, we report new results for the application potential of these techniques on representative enzymes as found in the aromatic metabolism of anaerobic bacteria, methanogenesis and human cells.

 

Benzoyl-CoA reductase is a key enzyme in the aromatic metabolism of anaerobic bacteria. Recent stopped flow UV-VIS and X-band EPR studies had shown that the reaction mechanism of this enzyme occurs via formation of radical intermediates, however identification of the species remained elusive. We investigated the reaction with 180 GHz pulsed EPR in combination with freeze-quenching techniques. The feasibility to separate the EPR spectra of the observed species, according to their relaxation behaviour, by pulse techniques allowed to determine the g-values and provided evidence for sulfur centered radical intermediates. The results allowed the proposal for a new enzymatic mechanism.

 

Heterodisulfide reductase catalyses the reduction of the heterodisulfide CoM-S-S-CoB in the final step of methanogenesis, a process called disulfide respiration. The reaction has been proposed to occur via a new mechanism of complex interaction between the substrate and a putative FeS cluster, that stabilizes a thiyl radical intermediate. We applied 57Fe pulsed ENDOR spectroscopy at W-band and we found direct evidence for a 4Fe4S cluster with unusual magnetic properties, likely arising from the binding of the substrate. Additionally the W-band spectra, recorded with standard Davies ENDOR sequence, display a completely polarized pattern of absorptive and emissive features. The analysis of the pattern allows to extract the sign of the hyperfine couplings.

 

Small G-proteins, like Ras, act as molecular switches in cellular signaling pathways controlling cell proliferation and differentiation. Point mutations of the Ras protein lead to malfunction of the whole cell and are found in 25-30% of human tumors. In the active site, the GDP/GTP substrate is bound to a Mg2+/Mn2+ ion, however details of the binding site and their implication on the switching mechanism are still controversial. We used 95 GHz pulsed ENDOR to detect the hfc interactions of Mn2+ with 17O, 31P and 13C nuclei in the ligand sphere. Particularly, the ENDOR spectra on the uniformly and selectively labeled 13C wild-type protein and as compared with the oncogenic G12V mutant allow to discuss a new structural model.

 

 

 

Magneto-Orientation of Photosynthetic Cyanobacteria Studied

by High-Field EPR: Mechanism and Applications

 

 

Gerd Kothe,a Oleg G. Poluektov,b Ulrich Heinen,a Thomas Berthold,a and Marion C. Thurnauerb

 

aDepartment of Physical Chemistry, University of Freiburg, D-79104 Freiburg/Germany

bChemistry Division, Argonne National Laboratory, Illinois 60439/USA

 

 

Magnetic field induced orientation of photosynthetic reaction centers has been studied by time-resolved D-band EPR of photo-excited cyanobacteria. Analysis of the observed spin polarized EPR spectra reveals that membrane proteins are the major source for the anisotropy of the diamagnetic susceptibility. From the magnetic field dependence of the degree of orientation, a value for the anisotropy of the diamagnetic susceptibility has been extracted. The value corresponds to the susceptibility anisotropy of a cyanobacterial cell, demonstrating that whole cells are aligned in the magneto-orientation process [1]. The cell order parameter from EPR compares favorably with corresponding data from electron microscopy obtained under similar experimental conditions [2].

 

Time-resolved high-frequency and multifrequency EPR of the electron transfer intermediates in photosystem I reveal new details of structure and function that could not be obtained without the magneto-orientation effect [3]. We expect that these EPR techniques in combination with a magneto-oriented sample will continue to advance our understanding of the particular interactions in photosynthetic proteins that result in control and optimization of the charge separation process.

 

[1] U. Heinen, O.G. Poluektov, E. Stavitski, T. Berthold, E. Ohmes, S.L. Schlesselman, J.R. Golecki, G.J. Moro, H. Levanon, M.C. Thurnauer and G. Kothe, J. Phys. Chem. B 2004, in

press.

[2] U. Heinen, J.R. Golecki, O.G. Poluektov, T. Berthold, S.L. Schlesselman, D. Frezzato, E. Ohmes, G.J. Moro, M.C. Thurnauer and G. Kothe, Appl. Magn. Reson. 2004, in press.

[3] M.C. Thurnauer, O.G. Poluektov and G. Kothe in: “Photosystem I” (J.H. Golbeck, ed.); Kluwer Academic Publishers, Dordrecht, 2004, in press.

 

 

 

 

Structure and Dynamics of Photosynthetic Reaction Center Proteins as Studied by High-Frequency EPR Spectroscopy

 

Oleg G. Poluektov, Lisa M. Utschig, Marion C.Thurnauer

Chemistry Division, Argonne National Laboratory, Argonne, IL, USA

 

Protein structure along with protein conformational dynamics play an important role in controlling the functional activity of biological systems. It is well-documented that a number of charge transfer reactions in proteins are gated by conformational transitions. Thus, studies of not only the static structure, but also the dynamic structure of proteins are necessary for understanding the function of biological supramolecular assemblies. Several applications of the high-frequency EPR technique will be discussed in this presentation.

 

In the first application information on protein dynamics was obtained using the spin label technique. A cysteine specific nitroxide spin label MTSL has been successfully covalently bound to the bacterial reaction center (RC) protein. The multifrequency EPR study of spin-labeled protein has the potential to be sensitive to subtle movements of the protein that can not be observed in the static crystal structure. Application of the pulsed technique for the characterization of libration dynamics will be discussed. The data obtained provide a foundation for future studies, with the goal of correlating protein motions with photosynthetic charge transfer reactions.

 

The second application involves structural determination with pulsed high-frequency EPR methodologies. When the primary electron transfer pathway from the water-oxidation complex in photosystem II (PS II) is inhibited, chlorophyll (ChlZ and ChlD), β-carotene (Car) and cytochrome b559 are alternate electron donors that are believed to function in a photoprotection mechanism. High-frequency EPR spectroscopy, together with deuteration of PS II, yields resolved Car+ and Chl+ EPR signals. The Car+ and Chl+ cation radicals exhibit dipolar-enhanced relaxation rates in the presence of high-spin (S=2) Fe(II) that are eliminated when the Fe(II) is low-spin (S=0). The relaxation enhancements of the T1 time by the nonheme Fe(II) allow us to determine possible binding sites for the Car cofactors in PS II..

 

This work was supported by the U.S. Department of Energy, Office of Basic Energy Sciences, Division of Chemical Science, under Contract W-31-109-Eng-38.

 

 

Probing the wave function of shallow Li and Na donors in ZnO nanoparticles using

ENDOR spectroscopy at 95 GHz.

 

 

Jan Schmidt

 

Huygens Laboratory, Leiden University, P.O. Box 9504, 2300 RA Leiden, The Netherlands

 

 

EPR and ENDOR experiments at 95 GHz on ZnO nanoparticles reveal the presence of shallow donors related to interstitial Li and Na atoms. The shallow character of the wave function is evidenced by the multitude of 67Zn ENDOR lines and further by the hyperfine interactions with the 7Li and 23Na nuclei that are much smaller than for atomic lithium and sodium. In the case of the Li-doped nanoparticles an increase of the hyperfine interaction with the 7Li nucleus and with the 1H nuclei in the Zn(OH)2 capping layer is observed when reducing the size of the nanoparticles. This effect is caused by the confinement of the shallow-donor 1s-type wave function that has a Bohr radius of about 1.5 nm, i.e., comparable to the dimension of the nanoparticles. By using trial functions for the shape of the electronic wave function a satisfactory fit is produced for the variation of the spin density on the 7Li and 1H nuclei with the dimension of the nanoparticles.

 

High-Field EPR on Transfer and Channel-Forming Proteins in Action:

Structure and Dynamics of Molecular Switches

 

Klaus Möbius

 

Dept. of Physics, Free University Berlin

 

In photosynthetic organisms and channel-forming toxins the respective electron and ion

transfer processes across cell membranes are vectorial in nature. For these transfer processes

to be unidirectional, subtle cofactor-protein interactions and/or conformational changes of

specific protein segments are functionalized as molecular switches. For pore formation

through cell-protecting membranes by bacterial toxins, such as colicins, even massive

refolding of dedicated protein domains is necessary. To explore the location of such

molecular switches and to understand their function, site-specific mutants have been studied

to reveal functionally important subdomains and to characterize their structure and dynamics

in the transient states of their biological action.

 

In the lecture a report will be given about our recent high-field EPR experiments at 95 GHz

and 360 GHz on tailor-made site-directed mutants of spin-labeled bacteriorhodopsin and

Colicin A as well as of bacterial photosynthetic reaction centers. The results obtained at

successively higher Zeeman fields - in conjunction with results from X-band EPR - provide

important information on molecular switches beyond that revealed by X-ray crystallography.

The work presented was performed in collaboration with Martin Fuchs, Michael Fuhs, Martin

Plato, Anton Savitsky and Alexander Schnegg in cooperation with the groups of H.-J.

Steinhoff (Osnabrück), W. Lubitz (Mülheim/Ruhr), T.F. Prisner (Frankfurt/M), D. Duché

(Marseille), Y.A. Grishin (Novosibirsk), and A.A. Dubinskii (Moscow).

 

Financial support by the Deutsche Forschungsgemeinschaft (Sfb 498, SPP 1051) is gratefully

acknowledged.

 

 

W-band EPR study of pure and beryllium doped cubic boron nitride crystals.

Etienne Goovaerts*, Sergiu V. Nistor**, Daniela Ghica** and Takashi Taniguchi***

 

* Department of Physics-Campus Drie Eiken, University of Antwerp,

BE-2610 Antwerpen(Wilrijk), Belgium

** National Institute for Materials Physics, P.O. Box MG-7 Magurele-Bucuresti,

RO-077125 Romania

*** National Institute for Materials Science, 1-1 Namiki, Tsukuba, 305-0044 Japan

 

Cubic boron nitride (cBN) is a synthetic material of high interest as wide bandgap semiconductor for special applications. Relatively straightforward p- and n-type doping has been demonstrated, and recently the production of p-n junctions and UV-emitters based on them have been reported [1,2]. Until now, very little progress has been made to understand the structure of point defects, essential in controlling the materials properties. For most characterization methods, an important obstacle was the very small size of available single crystals, which are produced by high-pressure high-temperature methods. We will report here on the results of an electron paramagnetic resonance (EPR) investigation of several oriented single crystals of cBN, nominally pure and Be-doped, taking advantage of the high absoute sensitivity and g-factor resolution at 95 GHz (in the W-band). The correlation between observed EPR spectra and differences in the composition, growth conditions and illumination history of the cBN crystals gives evidence for the existence of a number of new paramagnetic defects. A general discussion of their structural characteristics will be given.

 

 [1]. R. H. Wentorf, Jr., J. Chem. Phys. 36, 1990 (1962); O. Mishima, Mater. Sci. Forum 54&55, 313 (1990); [2]. T. Taniguchi, S. Koizumi, K. Watanabe, I. Sakaguchi, T. Sekiguchi and

S. Yamaoka, Diam. & Rel. Mater. 12, 1098 (2003) and references cited therein.

 

Structure of Catalytically Active Radicals in Enzymes from Combined Single Crystal High-Field EPR/ENDOR and X-Ray Diffraction Studies

F. Lendzian a, M. Galander a, M. Högbom b, P. Nordlund, J. C. Fontecilla-Camps c

a Max-Volmer-Laboratory, Technical University Berlin, PC14, 10623 Berlin, Germany.

b Arrhenius Laboratories, Stockholm University, SE-10691 Stockholm, Sweden.

c Institute de Biologogie Structurale, CNRS, 38027 Grenoble Cedex 1, France 

Radicals derived from amino acids or from cofactors, which function as active intermediates in the catalytic mechanism, are found in an increasing number of enzymes. However, the structures of enzymes are in general known only for the inactive states of their catalytic centers. We present here an approach, where structure information on the nature and orientation of the radical state is obtained from single crystal high-field EPR and ENDOR on catalytically competent radicals, which can be generated with low yields in single crystals of enzymes. Comparison with the X-ray structure of the respective enzyme in the inactive state reveals then information on the conformational changes induced by formation of the active radical state, which is important for the understanding of the catalytic mechanism.

 

Two examples are presented. i) In ribonucleotide reductase from E. coli a significant displacement of the active tyrosyl radical was observed (see Figure), which is discussed in the context of the catalytic proton-coupled electron transfer [1]. ii) Recently the X-ray structure of the enzyme pyruvate ferredoxin oxidoreductase with the proposed active substrate- and cofactor- derived intermediate hydroxy-ethyl thiamine pyrophosphate radical has been reported [2]. Based on single crystal high-field EPR/ENDOR data, different suggested radical structures are discussed.  

[1] M. Högbom, M. Galander, M. Andersson, M. Kolberg, W. Hofbauer, G. Lassmann, P. Nordlund, and F. Lendzian (2003) Proc. Natl. Acad. Sci. USA, 100 (6) 3209-3214. [2] E. Chabričre, X. Vernčde, B. Guigliarelli, M.-H. Charon, E.C. Hatchikian, J.C. Fontecilla-Camps (2001) Science, 294, 2559-2563.

 

 

 

A Multifrequency Study of Spin Probe Dynamics in Polystyrene

 

 

Bercu, V.1); Leporini, D.1);  Martinelli, M.2), Pardi, L.A.2)

 

 

1)Dipartimento di Fisica “Enrico Fermi”, Universitŕ di Pisa, via .F .Buonarroti 2,I-56127 Pisa, IT

2)Istituto per i Processi Chimico-Fisici, CNR, Area Ricerca Pisa via G .Moruzzi 1, I-56124, Pisa, IT

 

An HF2EPR study of dynamic processes of spin probe in polymers will be presented. A multifrequency approach at High Frequency leads to increased spectral resolution and enhanced orientation selectivity, which in turn allows for the determination of subtle differences in the rotational dynamics of the spin probe.1,2

The paramagnetic spin probe was 2,2,6,6-Tetramethypiperidin-1-yloxy (TEMPO) in polystyrene.

The adjustable parameters used for the simulation of the spectra were: the correlation time t, a jump angle f and the magnetic spin hamiltonian parameters of the spin probe. 3

Owing to the good resolution of the HF2EPR line shape we were able to find with a very good precision the spin Hamiltonian parameters and to determine the rotational model.

Moreover using HF2EPR spectroscopy it was found that correlation time is described by a distribution rather than by a single value of the correlation time t.

 

 

1)Budil, D. E.; Earle, K. A. ;Freed, J. H. J. Phys. Chem. 1993, 97, 1294.

2)Cramer, S.;Bauer, C.;Jeschke, G.;Spiess, H. W. Appl. Magn. Reson. 2001; 21 (3-4) : 495-506.

3)Giordano, M.;Grigolini, P.;Leporini, D.;Marin, P. Adv.Chem.Phys.1985, 62,321.

4) Bercu, V.; Leporini, D.; Martinelli, M., Pardi, L.A submitted

 

 

Electron delocalization and dimerization in solid

C59N doped C60 fullerene

 

A. Rockenbauer1, G. Csányi2, F. Fülöp3,

S. Garaj4, L. Korecz1, R. Lukács3, F. Simon3† L. Forró4, S. Pekker5, A. Jánossy3

 

1Chemical Research Center, Institute of Chemistry, P.O. Box 17 H-1525 Budapest, Hungary

2TCM Group, Cavendish Laboratory, University of Cambridge, Madingley Road, CB3 0HE, United Kingdom

3Budapest  University of  Technology and Economics, Institute of Physics, and Solids in Magnetic Fields Research Group of the Hungarian Academy of Sciences, P.O. Box 91, H-1521 Budapest, Hungary

4École Polytechnique Fédérale de Lausanne, Institut de Génie Atomique, CH-1015 Lausanne, Switzerland

5 Research Institute for Solid State Physics and Optics, Hungarian Academy of Sciences Budapest P.O. Box 49 H-1525 Hungary

 

Pure C60 fullerene is an insulating solid with an energy gap of about 1.5 eV between the valence and conduction bands. It is natural to expect that suitably doped fullerenes maybe semiconductors. However, there is no experimental evidence for this as so far no donors or acceptors could be introduced into C60 in a controllable way. Highly conducting alkali fullerid compounds and polymers are well known, but these are “line compounds” with well defined stoichiometry. The production of dilute solid solutions of monomeric C59N in C60 (C59N:C60) in macroscopic quantities[6] opens a new possibility. The distortion of the cage by the substitution of a N atom for C is small and a filled electron state 1.5 eV above the last filled shell is the main difference between C59N and C60.

Electron spin resonance and ab initio electronic structure calculations show an intricate relation between molecular rotation and chemical bonding in the dilute solid solution of azafullerene, C59N in fullerene, C60. Above 700 K, the unpaired electron of C59N is delocalized over several C60 molecules, while at lower temperatures it remains localized within short range. The two ESR active species observed below 350 K are assigned to rigid C59-C60 heterodimers in thermodynamic equilibrium with dissociated rotating molecules. The structural fluctuations between heterodimers and dissociated molecules are accompanied by simultaneous electron spin transfer between C60 and C59N molecules. The calculation confirms that in the C59N-C60 heterodimer the spin density resides mostly on the C60 moiety, while it is almost entirely on C59N in the dissociated case.

 

 

Approaching 180 GHz PELDOR: Set-Up and a First Test

 

Vasyl Denysenkov, Thomas Prisner, and Marina Bennati

 

Institute for Physikalische and Theoretische Chemie, and Center for Biomolecular Magnetic Resonance, J.W. Goethe University, Marie-Curie Str. 11, 60439 Frankfurt am Main, Germany

 

 

For aromatic organic radicals PELDOR experiments at high magnetic field offers the possibility to achieve orientation selective pumping and detection that could allow not only to determine the distance between such paramagnetic species but also the relative orientation with respect to the interconnecting axis. A PELDOR set-up, introduced into a G-band pulsed EPR spectrometer [1], is presented. In the present design, exploiting a single mode cavity, the spectrometer transmitter produces preparation, detecting and pumping pulses of equal output power of 20 mW at both frequencies, that leads to an inversion bandwidth of approximately 8 MHz. The PELDOR unit is introduced into the spectrometer transmitter at the 45-GHz stage, before the frequency multiplication chain, to avoid the introduction of additional less efficient high frequency components. The new set-up has been tested with a RNR-R2 tyrosyl sample using a 3-pulse PELDOR sequence [2]. Experimental results are discussed and compared to previously measured X-band data [3].

 

Ref.:

  1. M. Rohrer, G. Brügmann, B. Kinzer, and T. F. Prisner, High-field/high-frequency EPR spectrometer operating in pulsed and continuous mode at 180 GHz, Appl. Magn. Reson. 21, 257 – 274 (2001).
  2. A. D. Milov, A. B. Ponomarev, and Yu. D. Tsvetkov, Electron-electron double resonance in electron spin echo model biradical systemsand the sensitised photolysis of decalin, Chem. Phys. Lett, 110, 67 (1984).
  3. M. Bennati, A. Weber, J. Antonic, D. Perlstein, J. Robblee, and J. Stubbe, Pulsed ELDOR spectroscopy measures the distance between the two tyrosyl radicals in the ribonucleotide reductase, J. Am. Chem. Soc. 125, 14988-14989 (2003).

 

 

High-Field ENDOR at 180 GHz

 

M. M. Hertel, M. Bennati, V. Denysenkov & T. F. Prisner

 

a Institut für Physikalische und Theoretische Chemie and Center for Biomolecular Magnetic Resonance, Johann Wolfgang Goethe-Universität Frankfurt, Marie-Curie-Str. 11, D-60439 Frankfurt a.M., Germany.

 

 

One major advantage of high-field/frequency ENDOR (Electron Nuclear DOuble Resonance) is the increased nuclear Zeeman splitting which brings about the separation of ENDOR signals from different nuclei as well as significant simplification of the ENDOR spectra which become of first order. Another advantage is the possibility to determine the sign of hyperfine coupling constants exploiting of the large thermal polarization at high fields and low temperatures. Furthermore, the increased resolution of g anisotropy at high fields allows the selective excitation of specific molecular orientations for ENDOR experiments. For high-spin systems such as Mn2+ [1], high-field ENDOR offers, apart from the increased sensitivity, crucial advantages, one of them being the reduction of forbidden transitions at high fields as well as the narrowing of the central transitions (mS= - ˝ « + ˝).

The development of a high-field 180 GHz pulsed and CW EPR spectrometer has been reported recently by Rohrer et al. [2]. The design of an implemented ENDOR setup is presented here and its performance demonstrated on various systems. Mims and Davies ENDOR were carried out on a [Mn(H2O)6]2+ complex as a model system for measurements on the Ras protein, a GTP-hydrolyzing protein, which is found mutated in 20-30% of human tumors (for a review see [3]). First ENDOR spectra of the p21ras·GDP·Mn2+ complex were acquired allowing the determination of hyperfine couplings to surrounding protons at the active site.

 

-------

[1] G. H. Reed and G. D. Markham (1984), Biol. Magn. Reson. 6, 73-142

[2] M. Rohrer, O. Brügmann, B. Kinzer, T. F. Prisner (2001), Appl. Magn. Reson. 21, 257-274

[3] A. Wittinghofer and H. Waldmann (2000), Angew. Chem. Int. Ed. 39, 4192-4214

 

 

High-Field EPR Studies of Proteins Involved in Oxidative Stress.

Sun Un

Service de Bioénergétique, DBJC, CNRS URA 2096, CEA Saclay, 91191 Gif-sur-Yvette, Cedex France.

 

Oxygen is required for aerobic biological energy production and is produced from water by the large photosynthetic protein complex Photosystem II (PSII).  Paradoxically, oxygen can also be harmful since it is the source of the so-called reactive oxygen species (ROS) such as hydrogen peroxide and the  superoxide and hydroxide radicals. These ROS are associated with various damage pathways, diseases and aging. Various redox proteins have evolved to cope with this "oxidative stress" and these harmful molecules. Using high-field EPR, in conjunction with a variety of biological and computational methods, we have been examining proteins involved with both aspects of oxygen.  Recent findings in low temperature proton-coupled electron transfer in PSII will be discussed.  We have been able to photooxidize a tyrosine residue at 1.8K and using high-field EPR watch the relaxation of the protein environment via charge motion. The second topic will be manganese binding in superoxide dismutase (SOD), a protein that controls superoxide levels. The Mn(II) zero-field interaction appears to be a very sensitive probe of the protein environment and we are investigating how it can be related to the structure and function of SOD.

 

 

Distance and orientation of paramagnetic centres in proteins

as determined by multi-frequency pulsed EPR spectroscopy

 

T. F. Prisner, S. Lyubenova, M. Penning de Vries,

K. Siddiqui# and B. Ludwig#

 

Institute of Physical and Theoretical Chemistry and Center of Biological Magnetic Resonance

# Biochemistry Department, Johann Wolfgang Goethe University Frankfurt am Main

 

Dipolar measurements can be used to investigate the distance of paramagnetic species in proteins in the distance range of 2-6 nm. If one of the paramagnetic molecules is very fast relaxing and has a very broad spectral width, as FeS centres or hemes, dipolar relaxation measurements are superior to PELDOR measurements. We performed such relaxation measurements at various magnetic fields (0.3-6.4 T) to determine the orientation and the distance of the first electron acceptor in cytochrome c oxidase, a binuclear CuA center, to the next electron acceptor molecule, a heme a and also to the bound cytochrome c552 substrate. In both cases the fast relaxing heme enhances the transverse relaxation rates of the binuclear CuA center (S=1/2) by almost an order of magnitude. This enhancement is absent, in the second case, if a non-binding cytochrome c substrate is used instead, demonstrating that the relaxation enhancement is related to specific binding of the substrate to the binding pocket and not due to unspecific binding or concentration effects. A detailed analysis of the temperature dependence of this dipolar relaxation enhancement allows to analyse the dipolar coupling strength and separate contributions from different heme molecules. High field relaxation measurements allow to further determine the orientation of the dipolar vector with respect to the known G tensor axis system of the binuclear Cu center. This approach allows to measure distances within enzymes between the paramagnetic cofactors, like metal ions, hemes or FeS clusters as well as binding of coenzymes and substrates with paramagnetic metal ions. The measurements at X-band and G-band will be shown and the obtained structural parameters will be discussed with respect to accuracy and compared with MD simulation and X-ray structure data. The same type of experiments will allow to locate on a similar protein complex, quinol oxidase, the quinone QH binding pocket in the protein, which is yet unknown.   

 

High Field EPR on Copper Proteins: from type I to type II

 

Maria Fittipaldi

Department of Molecular Physics, Huygens Laboratory, Leiden University, P.O. Box 9504, 2300 RA Leiden, The Netherlands

 

Metalloproteins serve various reactions in biological systems. Copper containing metalloproteins are classified in different families according to their function and the spectroscopic features which reflect the specific coordination and geometry of their copper site. Type I copper sites are involved in electron-transfer reactions, while type II sites are catalytically active.

Advanced EPR represents a tool to study the electronic structure of the copper site, whose knowledge is a prerequisite for a deeper understanding of the mechanism of the reactions in which copper proteins are involved. The high resolution and sensitivity of high-field single-crystal EPR allows to determine the g-tensor of the copper center. This tensor can be interpreted in terms of the d-orbitals which take part in the molecular orbital describing the unpaired electron. Studies on single crystals and solutions using ESEEM and ENDOR techniques yield the delocalization of the electron spin-density over the nuclei surrounding the copper ion. The availability of isotopically labelled proteins improves the selectivity in probing the spin density beyond the copper ion. These EPR techniques have been used to study the electronic structure of the type I site. The challenge is now to study the reaction complexes (reacting molecule bound to the active site of the protein) of type II copper sites.

 

 

Distance and orientation of paramagnetic centres in proteins

as determined by multi-frequency pulsed EPR spectroscopy

 

T. F. Prisner, S. Lyubenova, M. Penning de Vries,

K. Siddiqui# and B. Ludwig#

 

Institute of Physical and Theoretical Chemistry and Center of Biological Magnetic Resonance

# Biochemistry Department, Johann Wolfgang Goethe University Frankfurt am Main

 

Dipolar measurements can be used to investigate the distance of paramagnetic species in proteins in the distance range of 2-6 nm. If one of the paramagnetic molecules is very fast relaxing and has a very broad spectral width, as FeS centres or hemes, dipolar relaxation measurements are superior to PELDOR measurements. We performed such relaxation measurements at various magnetic fields (0.3-6.4 T) to determine the orientation and the distance of the first electron acceptor in cytochrome c oxidase, a binuclear CuA center, to the next electron acceptor molecule, a heme a and also to the bound cytochrome c552 substrate. In both cases the fast relaxing heme enhances the transverse relaxation rates of the binuclear CuA center (S=1/2) by almost an order of magnitude. This enhancement is absent, in the second case, if a non-binding cytochrome c substrate is used instead, demonstrating that the relaxation enhancement is related to specific binding of the substrate to the binding pocket and not due to unspecific binding or concentration effects. A detailed analysis of the temperature dependence of this dipolar relaxation enhancement allows to analyse the dipolar coupling strength and separate contributions from different heme molecules. High field relaxation measurements allow to further determine the orientation of the dipolar vector with respect to the known G tensor axis system of the binuclear Cu center. This approach allows to measure distances within enzymes between the paramagnetic cofactors, like metal ions, hemes or FeS clusters as well as binding of coenzymes and substrates with paramagnetic metal ions. The measurements at X-band and G-band will be shown and the obtained structural parameters will be discussed with respect to accuracy and compared with MD simulation and X-ray structure data. The same type of experiments will allow to locate on a similar protein complex, quinol oxidase, the quinone QH binding pocket in the protein, which is yet unknown.   

 

 

High Field EPR on Copper Proteins: from type I to type II

 

Maria Fittipaldi

Department of Molecular Physics, Huygens Laboratory, Leiden University, P.O. Box 9504, 2300 RA Leiden, The Netherlands

 

Metalloproteins serve various reactions in biological systems. Copper containing metalloproteins are classified in different families according to their function and the spectroscopic features which reflect the specific coordination and geometry of their copper site. Type I copper sites are involved in electron-transfer reactions, while type II sites are catalytically active.

Advanced EPR represents a tool to study the electronic structure of the copper site, whose knowledge is a prerequisite for a deeper understanding of the mechanism of the reactions in which copper proteins are involved. The high resolution and sensitivity of high-field single-crystal EPR allows to determine the g-tensor of the copper center. This tensor can be interpreted in terms of the d-orbitals which take part in the molecular orbital describing the unpaired electron. Studies on single crystals and solutions using ESEEM and ENDOR techniques yield the delocalization of the electron spin-density over the nuclei surrounding the copper ion. The availability of isotopically labelled proteins improves the selectivity in probing the spin density beyond the copper ion. These EPR techniques have been used to study the electronic structure of the type I site. The challenge is now to study the reaction complexes (reacting molecule bound to the active site of the protein) of type II copper sites.

 

What Is Special About Endofullerenes?

 

K.-P. Dinse

 

Chem. Dept., Darmstadt University of Technology,

Petersenstr. 20, D-64287 Darmstadt, Germany

 

 

Since trace amounts of metallo-endofullerenes (MEF) were detected more than a decade ago, aspects of ion localization and quantification of charge transfer from the encased metal ion to the cage were intensively studied. Magnetic resonance methods like NMR and EPR have the advantage that the investigation can be performed using highly diluted materials. Invoking multi-frequency EPR it is possible to determine the spin multiplicity of the compounds, thus exploring details of their electronic state. Envisioning MEF as examples of internal charge transfer complexes, sign and size of the exchange coupling between cage and ion will determine the magnetic properties of the ground state of the coupled system. This is important if MEF should be used as molecular magnets.

 

In contrast to MEF, nitrogen or phosphorus encapsulated in fullerenes come close to the realization of a perfect chemical trap, such a confinement being defined as consisting of an interaction-free boundary of high symmetry. The specific properties of such a shielded inert electronic spin make these compounds ideal spin probes. The “high spin” S = 3/2 property of atomic nitrogen furthermore yields an additional tool by which the local symmetry can be investigated. We will also report on recent progress on the route towards the preparation of macroscopic amounts of purified material and first attempts to incorporate N@C60 into carbon nanotubes.

The HiPER Project - High Field nanosecond Pulse ESR

 

Dr G.M.Smith

 

Department of Physics and Astronomy,  St.Andrews University

 

 

The HiPER project is a major new UK collaborative initiative under the Basic Technology Program to develop the technologies that will eventually make possible sub-nanosecond pulse ESR. As a specific objective a 94GHz system will be constructed with single nanosecond p/2 pulses and single nanosecond deadtime, whilst still maintaining flexibility in pulse sequences and  fast averaging capabilities. 

 

This would be a step change in performance for pulse ESR spectrometers potentially opening up many new areas. It would dramatically improve sensitivity for fast relaxing spin systems as well as providing the ability to take molecular snapshots at the earliest stages of the evolution of many important complex chemical reactions,  with nanosecond resolution.   A GHz excitation bandwidth would also permit the excitation of the full spectrum of many important systems including the radicals commonly used in site-directed spin labelling studies, greatly improving sensitivity under conditions where the g-anisotropy is resolved.  This is particularly important at high fields where measurement sensitivity is currently  often constrained by the combination of broadened spectra  and limited   excitation bandwidths (relative to low frequency measurements).

However,  there is a strong impetus to obtain these measurements at high fields to benefit from the increased orientational selectivity, increased sensitivity to fast dynamics,  and improved absolute sensitivity.

 

Nevertheless, the combination of nanosecond p/2 pulses and nanosecond deadtime at 94GHz is a very challenging specification. Indeed  it is simply not possible to construct such a spectrometer using currently available commercial microwave components.  In the talk I will outline some of  these technical challenges as well as indicating some of our proposed development solutions and give an overview of some of the applications that the program is targeting.

 

 


Structure and Dynamics of Proteins from Spin-Label High-Frequency EPR

 

M. Huber

Department of Molecular Physics, Leiden University, 2300 RA Leiden, The Netherlands

 

To understand protein-based biochemistry, information on structural parameters of disordered systems and of the influence of the protein environment on embedded cofactors is of prime importance. From modern EPR techniques and the advances of spin label EPR, major developments can be expected in this area. High-field and pulsed EPR greatly enhance the sensitivity and resolution to determine structural parameters of proteins, and the electronic properties of the protein at the site of the reaction. The techniques of molecular biology (site directed mutagenesis) allow to place the spin label at virtually any position of interest in the protein, thereby providing the necessary paramagnetic probe(s).

Protein dielectric properties in the vicinity of the spin label are obtained by high-field EPR [1] on spin-labelled proteins. In particular, the higher spectral resolution of high-field EPR, from 95 GHz to 275 GHz, allow to measure g-tensor parameters and hyperfine information from which polarity/proticity properties are derived. Dynamical parameters are obtained from the relaxation properties of the spin labels [2], and by high-field EPR also the anisotropy of the motion of the spin label can be determined [3].

 

[1.]Rikard Owenius, Maria Engström, Mikael Lindgren, Martina Huber

J. Phys. Chem. A 105, 10967-10977, (2001)

[2.] M. Huber, M. Lindgren, P. Hammarström, L.‑G. Mĺrtensson, U. Carlsson, G. R. Eaton, S. S. Eaton

Biophysical Chemistry, 94, 245-256 (2001)

[3.] Michelina G. Finiguerra, Irene M.C. van Amsterdam, Sharmini Alagaratnam, Marcellus Ubbink, Martina Huber

Chem. Phys. Lett., 382, 528‑533 (2003)

 

 

 

 



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