Electron Nuclear Double Resonance Spectra Research Articles

Structural analysis of the dominant axial Fe3+ center in LiNbO3 crystal by electron nuclear double resonance

Measurements of the angular dependencies of Electron Nuclear Double Resonance (ENDOR) were used for the analysis of the substitution site and local environment of the dominant axial Fe3+ center in LiNbO3 crystals. All components of hyperfine and quadrupole tensors for several shells of Li and Nb nuclei were determined. Since the found isotropic part of the hyperfine interaction of the iron electrons with the Li nuclei was small, it was justified to approximate the anisotropic part by the dipole-dipole interaction of electron and nuclear spins. From the comparison of the calculated and observed ENDOR spectra of the Li nuclei, it was derived that the Fe3+ ion occupies a Li site but is shifted from the regular Li position by 0.009 nm toward the octahedral structural vacancy. There is no evidence for the presence of any charge compensators in the nearest Li and Nb shells, which indicates a distant mechanism of excess charge compensation. ENDOR of Nb nuclei shows that there is a large isotropic hyperfine interaction due to the significant indirect transferred interaction of Fe3+ with nearest off axis Nb nuclei via O2− and polarization of Nb inner electron shells.

Q-Band Electron-Nuclear Double Resonance Reveals Out-of-Plane Hydrogen Bonds Stabilize an Anionic Ubisemiquinone in Cytochrome bo3 from Escherichia coli.

The respiratory cytochrome bo3 ubiquinol oxidase from Escherichia coli has a high-affinity ubiquinone binding site that stabilizes the one-electron reduced ubisemiquinone (SQH), which is a transient intermediate during the electron-mediated reduction of O2 to water. It is known that SQH is stabilized by two strong hydrogen bonds from R71 and D75 to ubiquinone carbonyl oxygen O1 and weak hydrogen bonds from H98 and Q101 to O4. In this work, SQH was investigated with orientation-selective Q-band (∼34 GHz) pulsed 1H electron-nuclear double resonance (ENDOR) spectroscopy on fully deuterated cytochrome (cyt) bo3 in a H2O solvent so that only exchangeable protons contribute to the observed ENDOR spectra. Simulations of the experimental ENDOR spectra provided the principal values and directions of the hyperfine (hfi) tensors for the two strongly coupled H-bond protons (H1 and H2). For H1, the largest principal component of the proton anisotropic hfi tensor Tz' = 11.8 MHz, whereas for H2, Tz' = 8.6 MHz. Remarkably, the data show that the direction of the H1 H-bond is nearly perpendicular to the quinone plane (∼70° out of plane). The orientation of the second strong hydrogen bond, H2, is out of plane by ∼25°. Equilibrium molecular dynamics simulations on a membrane-embedded model of the cyt bo3 QH site show that these H-bond orientations are plausible but do not distinguish which H-bond, from R71 or D75, is nearly perpendicular to the quinone ring. Density functional theory calculations support the idea that the distances and geometries of the H-bonds to the ubiquinone carbonyl oxygens, along with the measured proton anisotropic hfi couplings, are most compatible with an anionic (deprotonated) ubisemiquinone.

EPR of Structural Phase Transition in Manganese- and Copper-Doped Formate Framework of [NH3(CH2)4NH3][Zn(HCOO)3]2

Electron paramagnetic resonance (EPR) and pulse electron–nuclear double resonance (ENDOR) spectroscopies are applied to investigate the structural phase transition and the low temperature phase in manganese- and copper-doped [NH3(CH2)4NH3][Zn(HCOO)3]2 dense metal–organic framework. Continuous-wave (CW) EPR measurements indicate successful incorporation of Mn2+ and Cu2+ ions at the Zn2+ lattice sites. Pulse ENDOR spectrum reveals at least four different proton species in the vicinity of the Mn2+ center showing excellent agreement with the X-ray diffraction experiments. Temperature-dependent CW EPR spectra demonstrate that Mn2+ and Cu2+ local paramagnetic probes are sensitive to the phase transition in the studied compounds. The analysis of the temperature dependence of the Cu2+ hyperfine coupling parameter reveals a first-order phase transition at Tc = 235 K into an antiferroelectric phase that is close to the tricritical point. The obtained logarithmic divergence of the line width of the Mn2+ EPR spectrum...

Spin Density Distribution in a Nitroxide Biradical Containing 13C-Enriched Acetylene Groups in the Bridge: DFT Calculations and EPR Investigation

A specially synthesized nitroxide biradical R6-13C≡C-p-C6H4–C≡13C-R6, B3, where R6 = 1-oxyl-2,2,6,6-tetramethyl-1,2,3,6-tetrahydropyridine nitroxide, has been studied by electron paramagnetic resonance spectroscopy, and electron-nuclear double resonance (ENDOR). Spin density distribution and hyperfine splitting (hfs) constant on 13C atoms calculations for biradical B3 were carried out using B3LYP and PBE0 functionals and several different basis sets including N07 family and were compared with the experimental value of the hfs constant on 13C atoms, measured from ENDOR spectra of B3. The mechanism of the intramolecular electron spin exchange in B3 biradical is discussed.

Electronic Structure of a Cu(II)-Alkoxide Complex Modeling Intermediates in Copper-Catalyzed Alcohol Oxidations.

In the copper-catalyzed oxidation of alcohols to aldehydes, a Cu(II)-alkoxide (Cu(II)-OR) intermediate is believed to modulate the αC-H bond strength of the deprotonated substrate to facilitate the oxidation. As a structural model for these intermediates, we characterized the electronic structure of the stable compound Tp(tBu)Cu(II)(OCH2CF3) (Tp(tBu) = hydro-tris(3-tert-butyl-pyrazolyl)borate) and investigated the influence of the trifluoroethoxide ligand on the electronic structure of the complex. The compound exhibits an electron paramagnetic resonance (EPR) spectrum with an unusually large gzz value of 2.44 and a small copper hyperfine coupling Azz of 40 × 10(-4) cm(-1) (120 MHz). Single-crystal electron nuclear double resonance (ENDOR) spectra show that the unpaired spin population is highly localized on the copper ion (≈68%), with no more than 15% on the ethoxide oxygen. Electronic absorption and magnetic circular dichroism (MCD) spectra show weak ligand-field transitions between 5000 and 12,000 cm(-1) and an intense ethoxide-to-copper charge transfer (LMCT) transition at 24,000 cm(-1), resulting in the red color of this complex. Resonance Raman (rR) spectroscopy reveals a Cu-O stretch mode at 592 cm(-1). Quantum chemical calculations support the interpretation and assignment of the experimental data. Compared to known Cu(II)-thiolate and Cu(II)-alkylperoxo complexes from the literature, we found an increased σ interaction in the Cu(II)-OR bond that results in the spectroscopic features. These insights lay the basis for further elucidating the mechanism of copper-catalyzed alcohol oxidations.

Role of the Proximal Cysteine Hydrogen Bonding Interaction in Cytochrome P450 2B4 Studied by Cryoreduction, Electron Paramagnetic Resonance, and Electron-Nuclear Double Resonance Spectroscopy.

Crystallographic studies have shown that the F429H mutation of cytochrome P450 2B4 introduces an H-bond between His429 and the proximal thiolate ligand, Cys436, without altering the protein fold but sharply decreases the enzymatic activity and stabilizes the oxyferrous P450 2B4 complex. To characterize the influence of this hydrogen bond on the states of the catalytic cycle, we have used radiolytic cryoreduction combined with electron paramagnetic resonance (EPR) and (electron-nuclear double resonance (ENDOR) spectroscopy to study and compare their characteristics for wild-type (WT) P450 2B4 and the F429H mutant. (i) The addition of an H-bond to the axial Cys436 thiolate significantly changes the EPR signals of both low-spin and high-spin heme-iron(III) and the hyperfine couplings of the heme-pyrrole (14)N but has relatively little effect on the (1)H ENDOR spectra of the water ligand in the six-coordinate low-spin ferriheme state. These changes indicate that the H-bond introduced between His and the proximal cysteine decreases the extent of S → Fe electron donation and weakens the Fe(III)-S bond. (ii) The added H-bond changes the primary product of cryoreduction of the Fe(II) enzyme, which is trapped in the conformation of the parent Fe(II) state. In the wild-type enzyme, the added electron localizes on the porphyrin, generating an S = (3)/2 state with the anion radical exchange-coupled to the Fe(II). In the mutant, it localizes on the iron, generating an S = (1)/2 Fe(I) state. (iii) The additional H-bond has little effect on g values and (1)H-(14)N hyperfine couplings of the cryogenerated, ferric hydroperoxo intermediate but noticeably slows its decay during cryoannealing. (iv) In both the WT and the mutant enzyme, this decay shows a significant solvent kinetic isotope effect, indicating that the decay reflects a proton-assisted conversion to Compound I (Cpd I). (v) We confirm that Cpd I formed during the annealing of the cryogenerated hydroperoxy intermediate and that it is the active hydroxylating species in both WT P450 2B4 and the F429H mutant. (vi) Our data also indicate that the added H-bond of the mutation diminishes the reactivity of Cpd I.

Cross-Polarization Electron-Nuclear Double Resonance Spectroscopy.

Magnetic nuclei in the proximity of a paramagnetic center can be polarized through electron-nuclear cross-polarization and detected in electron-nuclear double resonance (ENDOR) spectroscopy. This principle is demonstrated in a single-crystal model sample as well as on a protein, the β2 subunit of E.coli ribonucleotide reductase (RNR), which contains an essential tyrosyl radical. ENDOR is a fundamental technique to detect magnetic nuclei coupled to paramagnetic centers. It is widely employed in biological and materials sciences. Despite its utility, its sensitivity in real samples is about one to two orders of magnitude lower than conventional electron paramagnetic resonance, thus restricting its application potential. Herein, we report the performance of a recently introduced concept to polarize nuclear spins and detect their ENDOR spectrum, which is based on electron-nuclear cross polarization (eNCP). A single-crystal study permits us to disentangle eNCP conditions and CP-ENDOR intensities, providing the experimental foundation in agreement with the theoretical prediction. The CP-ENDOR performance on a real protein sample is best demonstrated with the spectra of the essential tyrosyl radical in the β2 subunit of E.coli RNR.

How Bonding in Manganous Phosphates Affects their Mn(II)-(31)P Hyperfine Interactions.

Manganous phosphates have been postulated to play an important role in cells as antioxidants. In situ Mn(II) electron-nuclear double resonance (ENDOR) spectroscopy has been used to measure their speciation in cells. The analyses of such ENDOR spectra and the quantification of cellular Mn(II) phosphates has been based on comparisons to in vitro model complexes and heuristic modeling. In order to put such analyses on a more physical and theoretical footing, the Mn(II)-(31)P hyperfine interactions of various Mn(II) phosphate complexes have been measured by 95 GHz ENDOR spectroscopy. The dipolar components of these interactions remained relatively constant as a function of pH, esterification, and phosphate chain length, while the isotropic contributions were significantly affected. Counterintuitively, although the manganese-phosphate bonds are weakened by protonation and esterification, they lead to larger isotropic values, indicating higher unpaired-electron spin densities at the phosphorus nuclei. By comparison, extending the phosphate chain with additional phosphate groups lowers the spin density. Density functional theory calculations of model complexes quantitatively reproduced the measured hyperfine couplings and provided detailed insights into how bonding in Mn(II) phosphate complexes modulates the electron-spin polarization and consequently their isotropic hyperfine couplings. These results show that various classes of phosphates can be identified by their ENDOR spectra and provide a theoretical framework for understanding the in situ (31)P ENDOR spectra of cellular Mn(II) complexes.

Multi-frequency (S, X, Q and W-band) EPR and ENDOR Study of Vanadium(IV) Incorporation in the Aluminium Metal-Organic Framework MIL-53.

Doping the well-known metal-organic framework MIL-53(Al) with vanadium(IV) ions leads to significant changes in the breathing behaviour and might have repercussions on the catalytic behaviour as well. To understand the properties of such a doped framework, it is necessary to determine where dopant ions are actually incorporated. Electron paramagnetic resonance (EPR) and electron-nuclear double resonance (ENDOR) are applied to reveal the nearest environment of the paramagnetic vanadium(IV) dopant ions. EPR spectra of as-synthesised vanadium-doped MIL-53 are recorded at S-, X-, Q- and W-band microwave frequencies. The EPR spectra suggest that at low dopant concentrations (1.0-2.6 mol %) the vanadium(IV) ions are well dispersed in the matrix. Varying the vanadium dopant concentration within this range or the dopant salt leads to the same dominant EPR component. In the ENDOR spectra, hyperfine (HF) interactions with (1) H, (27) Al and (51) V nuclei are observed. The HF parameters extracted from simulations strongly suggest that the vanadium(IV) ions substitute Al in the framework.

A Microfluidic Rapid Freeze Quench Apparatus for High Field EPR Measurements

Rapid freeze quench (RFQ) EPR is a well-established technique for trapping reaction intermediates. A major difficulty in using commercial RFQ-EPR combined with standard X-band EPR spectroscopy is the relatively large amount of sample needed for each time point, and the associated amount that is wasted in the dead volume of the tubes and mixer. This is particularly prohibitive when one would like to couple RFQ with high resolution EPR techniques such as ENDOR (electron-nuclear double resonance) and DEER (electron-electron double resonance) that provide electron-nuclear and electron-electron distances and are less sensitive than standard continuous wave EPR. We have developed a dedicated microfluidic RFQ (RFQ) apparatus for W-band measurements, optimized for the small W-band sample size and a minimal sample amount for a series of ∼7 time points collected in triplicates (∼200 l of 0.03-0.1 mM labeled protein). The mixer is based on a recent published design[1] with a modified sample ejection system and cold trap. It current time window is 5-90 msec and its performance has been demonstrated on the reduction of nitroxide with dithionite[2]. The current state of the RFQ apparatus is demonstrated on : (i) the ATPase activity of an RNA helicase. Here Mg2+ was substituted with Mn2+ and the 31P ENDOR spectrum was recorded. The ADP and ATP spectra have significantly different lineshapes and can therefore be used to probe the hydrolysis state. (ii) Conformational changes induced in a protein through ligand binding as detected by DEER. 1. Egawa T, Durand JL, Hayden EY, Rousseau DL, Yeh S-R. Anal Chem. 2009;81(4):1622-7. 2. R. Kaufmann, D. Goldfarb.. J Magn Reson. 2013;230:220-6.

ATPase site configuration of the RNA helicase DbpA probed by ENDOR spectroscopy.

Electron-nuclear double resonance (ENDOR) is a method that probes the local structure of paramagnetic centers via their hyperfine interactions with nearby magnetic nuclei. Here we describe the use of this technique to structurally characterize the ATPase active site of the RNA helicase DbpA, where Mg(2+)-ATP binds. This is achieved by substituting the EPR (electron paramagnetic resonance) silent Mg(2+) ion with paramagnetic, EPR active, Mn(2+) ion. (31)P ENDOR provides the interaction of the Mn(2+) with the nucleotide (ADP, ATP and its analogs) through the phosphates. The ENDOR spectra clearly distinguish between ATP- and ADP-binding modes. In addition, by preparing (13)C-enriched DbpA, (13)C ENDOR is used to probe the interaction of the Mn(2+) with protein residues. This combination allows tracking structural changes in the Mn(2+) coordination shell, in the ATPase site, in different states of the protein, namely with and without RNA and with different ATP analogs. Here, a detailed description of sample preparation and the ENDOR measurement methodology is provided, focusing on measurements at W-band (95 GHz) where sensitivity is high and spectral interpretations are relatively simple.

EPR, ESE, and pulsed ENDOR study of the nitrogen donors in 15R SiC grown under carbon‐rich conditions

X‐band field‐sweep electron spin echo and pulsed electron nuclear double resonance (ENDOR) spectroscopy were used to study n‐type 15R SiC wafers grown under carbon (C)‐rich conditions with the aim to verify the recently proposed concept that nitrogen (N) donors substitute both carbon (C) and silicon (Si) sites and may occupy nonequivalent positions at Si sites. It was found that besides the 14N ENDOR spectra of the C substituting quasicubic “k1”, “k2”, and “k3” positions five doublet lines due to 14N nuclei at other lattice positions were observed in the ENDOR spectrum of highly compensated C‐rich n‐type 15R SiC. Three of them with the hyperfine interaction (HFI) constants 34.66, 32.20, and 29.77 MHz were attributed to the N donors substituting “k1”, “k2”, and “k3” positions at Si sites. Two other additional ENDOR spectra were explained by the presence of the C antisite (CSi) defects in C‐rich 15R SiC, which leads to the formation of CSiNC complexes with donor levels close to the conduction‐band minimum and isotropic 14N HFI constants of 21.58 and 25.94 MHz. The energy levels of N donors have been evaluated by comparing the number of ENDOR spectra observed in C‐rich 15R SiC with different degrees of compensation.

DFT and ENDOR Study of Bixin Radical Cations and Neutral Radicals on Silica-Alumina.

Bixin, a carotenoid found in annatto (Bixa orellana), is unique among natural carotenoids by being water-soluble. We stabilized free radicals from bixin on the surface of silica-alumina (Si-Al) and characterized them by pulsed electron-nuclear double resonance (ENDOR). DFT calculations of unpaired electron spin distribution for various bixin radicals predict the EPR hyperfine couplings. Least-square fitting of experimental ENDOR spectra by spectra calculated from DFT hyperfine couplings characterized the radicals trapped on Si-Al. DFT predicts that the trans bixin radical cation is more stable than the cis bixin radical cation by 1.26 kcal/mol. This small energy difference is consistent with the 26% trans and 23% cis radical cations in the ENDOR spectrum. The remainder of the ENDOR spectrum is due to several neutral radicals formed by loss of a H(+) ion from the 9, 9', 13, or 13' methyl group, a common occurrence in all water-insoluble carotenoids previously studied. Although carboxyl groups of bixin strongly affect its solubility relative to other natural carotenoids, they do not alter properties of its free radicals based on DFT calculations and EPR measurements which remain similar to typical water-insoluble carotenoids.

A DFT, X- and W-band EPR and ENDOR Study of Nitrogen-Centered Species in (Nano)Hydroxyapatite

Incorporation of the nitrogen-containing impurities in hydroxyapatite (HAp) powders with the sizes of the crystallites of (20–50) nm was studied using first-principles modeling combined with the multi-frequency (9 and 94 GHz) electron paramagnetic resonance (EPR) methods. It is shown that the observed EPR spectra are undoubtedly due to the presence of the bulk radiation-induced NO3 2− radicals. This conclusion is based on spin-polarized density functional theory calculations of spectroscopic parameters within gauge-including projector augmented wave framework followed by the exact comparison of the simulated EPR and electron–nuclear double resonance spectra with the experimental findings. In addition, a comprehensive analysis of the simulated properties allows us to suggest that the paramagnetic centers preferably occupy PO4 3− sites in the HAp structure.

Nitrogen split interstitial center(N−N)Nin GaN: High frequency EPR and ENDOR study

The nitrogen split interstitial defect introduced by high-energy particle irradiation in n-type GaN has been investigated by very high (up to 324 GHz) frequency electron paramagnetic resonance (EPR) and Q-band electron nuclear double resonance (ENDOR) spectroscopy. The increased resolution of the EPR spectra at 324 GHz has allowed us to determine the g-tensor anisotropy, which is not resolved at X or Q band. The good agreement of the principal values g(xx) = 1.9966, g(yy) = 2.0016, and g(zz) = 2.0036 with the theoretically predicted g tensor confirm the (N-N)(N)(0) defect model. The hyperfine interactions of this defect have been studied byQ-band ENDOR. We observed well-resolved ENDOR lines with distant Ga atoms from which the quadrupole coupling constants and the electrical field gradients were determined and discussed with the help of theoretical values. The observation of ENDOR spectra of the central N and Ga atoms predicted in the 20-90-MHz range required the use of field-frequency ENDOR due to the large linewidth of the ENDOR lines. Our results confirm the importance of the nitrogen split interstitial in particle irradiated GaN similar to the case of diamond and silicon carbide in which the stable configuration at room temperature of the carbon interstitials is also the split interstitial configuration.

Insights into copper coordination in the EcoRI–DNA complex by ESR spectroscopy

The EcoRI restriction endonuclease requires one divalent metal ion in each of two symmetrical and identical catalytic sites to catalyse double-strand DNA cleavage. Recently, we showed that Cu2+ binds outside the catalytic sites to a pair of new sites at H114 in each sub-unit, and inhibits Mg2+-catalysed DNA cleavage. In order to provide more detailed structural information on this new metal ion binding site, we performed W-band (∼94 GHz) and X-band (∼9.5 GHz) electron spin resonance spectroscopic measurements on the EcoRI–DNA–(Cu2+)2 complex. Cu2+ binding results in two distinct components with different gzz and Azz values. X-band electron spin echo envelope modulation results indicate that both components arise from a Cu2+ coordinated to histidine. This observation is further confirmed by the hyperfine sub-level correlation results. W-band electron nuclear double resonance spectra provide evidence for equatorial coordination of water molecules to the Cu2+ ions.

55Mn pulsed ENDOR spectroscopy of Mn2+ ions in ZnO thin films and single crystal

55Mn pulsed electron nuclear double resonance (ENDOR) experiments were performed at X-band on high spin S=5/2 Mn2+ ions incorporated at zinc lattice sites in heteroepitaxial ZnO thin films. The films have been prepared by pulsed laser deposition and the manganese ions were doped during the growth process. We examine how the c/a lattice axes ratio of the ZnO films influences the 55Mn hyperfine (hf) and nuclear quadrupole (nq) coupling parameters of the Mn2+ probe ions. The results are compared with those obtained for Mn2+ ions present as impurities in ZnO single crystals and revealed that the 55Mn nq coupling monitors sensitively the structural distortions in the bonding environment of the Mn2+ ions. The experiments provided the full axially symmetric 55Mn hf and nq interaction tensors. The latter is found to be very sensitive to small axial distortions of the MnO4 tetrahedrons. In particular, the 55Mn pulsed ENDOR spectra of the ZnO:Mn thin films are strongly subjected to strain effects in the nq coupling parameter indicating a variation of the local structural parameters for the heteroepitaxial films. In the analysis of the 55Mn pulsed ENDOR spectra the axial and cubic zero field splitting of the Mn2+ ions was taken into account and intensity effects in the ENDOR spectra due to the zero field splitting effects were discussed.

Nitrogen-containing species in the structure of the synthesized nano-hydroxyapatite

Synthesized by the wet chemical precipitation technique hydroxyapatite powders (HAp) with the sizes of the crystallites of (20-50) nm and 1000 nm were analyzed by different analytical methods. By means of electron paramagnetic resonance (EPR) it is shown that during the synthesis process nitrate anions from the reagents (by-products) could incorporate into the HAp structure. The concentration of the stable NO32- radicals detected after X-ray irradiation of the product at room temperature does not decrease even after annealing up to 400 oC. The relaxation times and EPR parameters of the axially symmetric NO32- paramagnetic centers are measured with high accuracy. Increased sensitivity and resolution of high-frequency EPR (95 GHz) allows to reveal additional paramagnetic centre(s) which origin is discussed. Analyses of electron-nuclear double resonance (ENDOR) spectra from 1H and 31P nuclei and ab-initio density functional theory (DFT) calculations allow suggesting that the paramagnetic centers and nitrate anions as the precursors of NO32- radicals preferably occupy PO43- site in the HAp structure.

Ground state of the singly ionized oxygen vacancy in rutile TiO2

Results from electron paramagnetic resonance (EPR) and electron-nuclear double resonance (ENDOR) experiments are used to establish the model for the ground state of the singly ionized oxygen vacancy in the interior of bulk rutile TiO2 crystals. Hyperfine from 47Ti and 49Ti nuclei show that the unpaired electron in this S = 1/2 defect is localized on one titanium ion adjacent to the oxygen vacancy (i.e., the spin is not shared by two titanium ions). These defects are formed at low temperature (∼35 K) in as-grown oxidized crystals when sub-band-gap 442 nm laser light converts doubly ionized nonparamagnetic oxygen vacancies to the singly ionized paramagnetic charge state. The g matrix is obtained from EPR spectra and the 47Ti and 49Ti hyperfine and nuclear electric quadrupole matrices (A and Q) are obtained from ENDOR spectra. Principal values of the 47Ti and 49Ti hyperfine matrices are 64.54, 11.57, and 33.34 MHz. All the matrices have a principal axis along the [001] direction. In the basal plane, principal axes of the hyperfine and quadrupole matrices also coincide. The principal axes of the g matrix in the basal plane, however, deviate significantly from those of the A and Q matrices, thus indicating mixing of d orbitals due to the low symmetry at the Ti3+ ion site and participation of excited-state orbitals.

Advanced electron paramagnetic resonance on the catalytic iron–sulfur cluster bound to the CCG domain of heterodisulfide reductase and succinate: quinone reductase

Heterodisulfide reductase (Hdr) is a key enzyme in the energy metabolism of methanogenic archaea. The enzyme catalyzes the reversible reduction of the heterodisulfide (CoM-S-S-CoB) to the thiol coenzymes M (CoM-SH) and B (CoB-SH). Cleavage of CoM-S-S-CoB at an unusual FeS cluster reveals unique substrate chemistry. The cluster is fixed by cysteines of two cysteine-rich CCG domain sequence motifs (CX31-39CCX35-36CXXC) of subunit HdrB of the Methanothermobacter marburgensis HdrABC complex. We report on Q-band (34GHz) (57)Fe electron-nuclear double resonance (ENDOR) spectroscopic measurements on the oxidized form of the cluster found in HdrABC and in two other CCG-domain-containing proteins, recombinant HdrB of Hdr from M. marburgensis and recombinant SdhE of succinate: quinone reductase from Sulfolobus solfataricus P2. The spectra at 34GHz show clearly improved resolution arising from the absence of proton resonances and polarization effects. Systematic spectral simulations of 34GHz data combined with previous 9GHz data allowed the unambiguous assignment of four (57)Fe hyperfine couplings to the cluster in all three proteins. (13)C Mims ENDOR spectra of labelled CoM-SH were consistent with the attachment of the substrate to the cluster in HdrABC, whereas in the other two proteins no substrate is present. (57)Fe resonances in all three systems revealed unusually large (57)Fe ENDOR hyperfine splitting as compared to known systems. The results infer that the cluster's unique magnetic properties arise from the CCG binding motif.