An orientation-selected ENDOR and HYSCORE study of the Ni-C active state of Desulfovibrio vulgaris Miyazaki F hydrogenase

Electron nuclear double resonance (ENDOR) and hyperfine sublevel correlation spectroscopy (HYSCORE) are applied to study the active site of catalytic [NiFe]-hydrogenase from Desulfovibrio vulgaris Miyazaki F in the reduced Ni-C state. These techniques offer a powerful tool for detecting nearby magnetic nuclei, including a metal-bound substrate hydrogen, and for mapping the spin density distribution of the unpaired electron at the active site. The observed hyperfine couplings are assigned via comparison with structural data from X-ray crystallography and knowledge of the complete g-tensor in the Ni-C state (Foerster et al. (2003) J Am Chem Soc 125:83–93). This is found to be in good agreement with density functional theory calculations. The two most strongly coupled protons (a_iso=13.7, 11.8 MHz) are assigned to the -CH₂ protons of the nickel-coordinating cysteine 549, and a third proton (a_iso=8.9 MHz) is assigned to a -CH₂ proton of cysteine 546. Using D₂O exchange experiments, the presence of a hydride in the bridging position between the nickel and iron—recently been detected for a regulatory hydrogenase (Brecht et al. (2003) J Am Chem Soc 125:13075–13083)—is experimentally confirmed for the first time for catalytic hydrogenases. The hydride exhibits a small isotropic hyperfine coupling constant (a_iso=–3.5 MHz) since it is bound to Ni in a direction perpendicular to the z-axis of the Ni orbital. Nitrogen signals that belong to the nitrogen N of His-88 have been identified. This residue forms a hydrogen bond with the spin-carrying Ni-coordinated sulfur of Cys-549. Comparison with other hydrogenases reveals that the active site is essentially the same in all proteins, including a regulatory hydrogenase.     

Probing the ground state of the purple mixed valence CuA center in nitrous oxide reductase: a CW ENDOR (X-band) study of the 65Cu, 15N-histidine labeled enzyme and interpretation of hyperfine couplings by molecular orbital calculations

 CW ENDOR (X-band) spectra for the purple mixed-valence [Cu(1.5+)...Cu(1.5+)], S = 1/2, Cu_A site in nitrous oxide reductase were obtained after insertion of ⁶⁵Cu or both ⁶⁵Cu and ¹⁵N-histidine. The ¹⁴N/¹⁵N isotopic substitution allowed for an unambiguous deconvolution of proton and nitrogen hyperfine couplings in the spectra. A single nitrogen coupling with a value of 12.9 ± 0.4 MHz for ¹⁴N was detected. Its anisotropy was characteristic for imidazole bound to copper. A spin density of 3–5% was estimated for the nitrogen donors to Cu_A, indicating that the ground state is ²B_3u. Proton hyperfine structure was detected from four C_β protons of coordinating cysteine residues. Their isotropic and anisotropic parts were deconvoluted by spectral simulation. From the anisotropic couplings a spin density of 16–24% was estimated for each of the cysteine thiolate donors of Cu_A. The [N_HisCu(RS)₂CuN_His]⁺ core structure of Cu_A in nitrous oxide reductase from Pseudomonas stutzeri is predicted to be similar to the crystallographically determined Cu_A* structure (Wilmanns M, Lappalainen P, Kelly M, Sauer-Eriksson E, Saraste M (1995) Proc Natl Acad Sci USA 92 : 11955–11959), but distinct from the Cu_A structure of Paracoccus denitrificans cytochrome c oxidase (Iwata S, Ostermeier C, Ludwig B, Michel H (1995) Nature 376 : 660–669). The angular dependence of the isotropic couplings as a function of the electronic ground state was calculated by the INDO/S method. The Mulliken atomic-spin populations calculated by a gradient-corrected density functional method and the semiempirical INDO/S method were compared with experimentally derived spin populations, and good agreement between theory and experiment was found for both calculations. The ground state of Cu_A is best represented by the resonance structures of the form [Cu^IS^–S^–Cu^II↔ Cu^IS^•S^–Cu^I↔ Cu^IS^–S^•Cu^I↔ Cu^IIS^–S^–Cu^I]. It is proposed that the Cu 4s,p as well as sulfur 3d orbitals play a role in the stabilization of this novel type of cluster. Received: 17 September 1997 / Accepted: 28 October 1997     

Probing magnetic properties of the reduced [2Fe-2S] cluster of the ferredoxin from Arthrospira platensis by 1H ENDOR spectroscopy

The 1H electron nuclear double resonance (ENDOR) spectra in frozen solutions of the reduced [2Fe-2S] cluster in ferredoxin from Arthrospira (Spirulina) platensis have been measured at low temperatures (5-20 K) and simulated using orientational selection methods. The analysis confirmed the existence of a single paramagnetic species with iron valence states II and III connected uniquely to the cluster irons. The experimental ENDOR spectra were fitted to a model including the spin distribution on the centre, the orientation of the g-matrix, and the isotropic and anisotropic hyperfine couplings of the nearest protons in the crystallographically determined structure. In order to partially simulate ENDOR line shapes, a statistical distribution of the corresponding torsion angles between the Fe(III) centre and one of the beta-CH2 protons was introduced. From the analysis, four of the larger hyperfine couplings found were assigned to the cysteine beta-protons near the Fe(III) ion of the cluster, with isotropic hyperfine couplings ranging from 1.6 to 4.1 MHz. The spin distribution on the two iron ions was estimated to be +1.85 for the Fe(III) ion and -0.9 for the Fe(II) ion. The Fe(III) ion was identified as being coordinated to the cysteine ligands Cys49 and Cys79, confirming previous NMR results. The direction of the g-tensor with respect to the cluster was deduced. The g1-g2 plane is parallel to the planes through each iron and its adjacent cysteine sulfurs; the g2-g3 plane is nearly perpendicular to the latter planes and deviates by 25 degrees from the FeSSFe plane. The g1 direction is dominated by the bonding geometry of Fe(II) and does not align with the Fe(II)-Fe(III) vector.     

Hydrogenases in the "active" state: determination of g-matrix axes and electron spin distribution at the active site by 1H ENDOR spectroscopy

Hydrons and electrons are substrates for the enzyme hydrogenase, but cannot be observed in X-ray crystal structures. High-resolution 1H electron nuclear double resonance (ENDOR) spectroscopy offers a means to detect the distribution of protons and unpaired electrons. ENDOR spectra were recorded from frozen solutions of the nickel-iron hydrogenases of Desulfovibrio gigas and Desulfomicrobium baculatum, in the "active" state ("Ni-C" EPR signal) and analyzed by orientationally selective simulation methods. The experimental spectra were fitted using a structural model of the nickel-iron centre based on crystallographic results, allowing for differences in electron spin distribution as well as the spatial orientation of the g-matrix ( g-tensor), and anisotropic and isotropic hyperfine couplings of the protons nearest to the nickel ion. ENDOR signals, detected after complete deuterium exchange, were assigned to six protons of the cysteines bound to nickel. The assignment took advantage of the substitution of a selenium for a sulfur ligand, which occurs naturally between the [NiFeSe] and [NiFe] hydrogenases from Dm. baculatum and D. gigas, respectively, and was found to affect just two signals. The four signals with the largest hyperfine couplings, including isotropic contributions from 4.5 to 13.5 MHz, were assigned to the beta-methylene protons of the two terminal cysteine ligands, one of which is substituted by seleno-cysteine in [NiFeSe] hydrogenase. The electron spin is delocalized onto the nickel (50%) and its sulfur ligands, with a higher proportion on the terminal than the bridging ligands. The g-matrix was found to align with the active site in such a way that the g1- g2 plane is nearly coplanar (18.3 degrees) with the plane defined by nickel and three sulfur atoms, and the g2 axis deviates by 22.9 degrees from the vector between nickel and iron. Significantly for the reaction of the enzyme, direct evidence for the binding of hydrons at the active site was obtained by the detection of H/D-exchangeable ENDOR signals.     

The metal-binding site of imidazole glycerol phosphate dehydratase; EPR and ENDOR studies of the oxo-vanadyl enzyme

 The apo protein of imidazole glycerol phosphate dehydratase (IGPD) from Saccharomyces cerevisiae combines stoichiometrically with certain specific divalent metal cations to assemble the catalytically active form comprising 24 protein subunits and tightly bound metal. VO²⁺ ions react similarly but, uniquely, result in a metallo-protein (VO-IGPD) with neither catalytic activity nor the ability to bind to the reaction intermediate analogue, 2-hydroxy-3-(1,2,4-triazol-1-yl) propylphosphonate. Since VO²⁺ apparently assembles the quaternary structure correctly, it is used in the present study as a spin probe to investigate the metal centre coordination environment by electron paramagnetic resonance (EPR) and electron nuclear double resonance (ENDOR) spectroscopy. At neutral pH, the EPR spectrum of VO-IGPD reveals at least three distinct VO²⁺ sub-spectra with one predominant at low pH. The spin Hamiltonian parameters for some of the sub-spectra are consistent with ⁵¹V having nitrogen in the inner-sphere equatorial coordination environment from, most probably, multiple coordinating histidines. Further evidence for inner-sphere nitrogen ligands is obtained from ENDOR spectroscopy. The spectra of the low rf region show signals from interactions with ¹⁴N which are consistent with couplings to the imino nitrogen of coordinated histidine residues. In addition a number of proton ENDOR line pairs are resolved. Of the few that disappear upon exchange of the protein into D₂O, one most likely originates from the exchangeable proton of the N-H group of a coordinated histidine imidazole. ¹H-ENDOR line pairs from non-exchangeable protons with splittings of approximately 3 MHz can be attributed to imidazole carbon protons. Thus, most of the couplings observed by ENDOR are consistent with being from the imidazole heterocycle of one or more histidine ligands. Received: 27 June 1996 / Accepted: 14 March 1997     

The [NiFe] hydrogenase from Allochromatium vinosum studied in EPR-detectable states: H/D exchange experiments that yield new information about the structure of the active site

In this study we report on thus-far unobserved proton hyperfine couplings in the well-known EPR signals of [NiFe] hydrogenases. The preparation of the enzyme in several highly homogeneous states allowed us to carefully re-examine the Ni(u)*, Ni(r)*, Ni(a)-C* and Ni(a)-L* EPR signals which are present in most [NiFe] hydrogenases. At high resolution (modulation amplitude 0.57 G), clear indications for hyperfine interactions were observed in the g(z) line of the Ni(r)* EPR signal. The hyperfine pattern became more pronounced in 2H2O. Simulations of the spectra suggested the interaction of the Ni-based unpaired electron with two equivalent, non-exchangeable protons (A1,2=13.2 MHz) and one exchangeable proton (A3=6.6 MHz) in the Ni(r)* state. Interaction with an exchangeable proton could not be observed in the Ni(u)* EPR signal. The identity of the three protons is discussed and correlated to available ENDOR data. It is concluded that the NiFe centre in the Ni(r)* state contains a hydroxide ligand bound to the nickel, which is pointing towards the gas channel rather than to iron.     

Determination of ligand conformation in reduced [2Fe-2S] ferredoxin from cysteine β-proton hyperfine couplings

 The isotropic hyperfine couplings of cysteine β-protons in iron-sulfur clusters of proteins provide information about the structure and conformation of the clusters if their magnetic resonance peaks can be resolved and assigned. The application of two-dimensional ESEEM (HYSCORE) spectroscopy to the reduced [2Fe-2S] cluster in ferredoxin from red marine algae Porfira umbilicalis is described. After deuterium substitution of the exchangeable protons, highly-resolved, orientationally-selected HYSCORE spectra show cross-peaks from strongly coupled, nonexchangeable protons. When cross-peaks from all the HYSCORE spectra are linearized and transformed to a common nuclear Zeeman frequency, they fall along five straight lines. Four of these sets of peaks are assigned to β-protons of the cysteine ligands. The isotropic and anisotropic hyperfine couplings for these protons are extracted from the slopes and intercepts of these lines. Two rescaling procedures are examined for the conversion of the experimentally measured isotropic couplings from different irons in [2Fe-2S] and [4Fe-4S] clusters. The couplings from P. umbilicalis appear to fit the same empirical dependence on Fe-S-C-H dihedral angle as do the couplings from a [4Fe-4S] model cluster. A method to assign protons for proteins of unknown structure is proposed that yields the correct assignment as derived from the crystal structure of the highly homologous protein from Spirulina platensis. The conformations of the cysteines in the reduced protein, derived without any adjustable parameters from this procedure and the empirical functions, are consistent with those reported for the latest refinement of the crystal structure of the oxidized protein. Received: 24 September 1997 / Accepted: 28 October 1997     

EPR and ENDOR studies of the water oxidizing complex of Photosystem II

A comparative study of X-band EPR and ENDOR of the S₂ state of photosystem II membrane fragments and core complexes in the frozen state is presented. The S₂ state was generated either by continuous illumination at T=200 K or by a single turn-over light flash at T=273 K yielding entirely the same S₂ state EPR signals at 10 K. In membrane fragments and core complex preparations both the multiline and the g=4.1 signals were detected with comparable relative intensity. The absence of the 17 and 23 kDa proteins in the core complex preparation has no effect on the appearance of the EPR signals. ¹H-ENDOR experiments performed at two different field positions of the S₂ state multiline signal of core complexes permitted the resolution of four hyperfine (hf) splittings. The hf coupling constants obtained are 4.0, 2.3, 1.1 and 0.6 MHz, in good agreement with results that were previously reported (Tang et al. (1993) J Am Chem Soc 115: 2382–2389). The intensities of all four line pairs belonging to these hf couplings are diminished in D₂O. A novel model is presented and on the basis of the two largest hfc's distances between the manganese ions and the exchangeable protons are deduced. The interpretation of the ENDOR data indicates that these hf couplings might arise from water which is directly ligated to the manganese of the water oxidizing complex in redox state S₂.Abbreviations cw continuous wave - ENDOR electron nuclear double resonance - EPR electron paramagnetic resonance - hf hyperfine - hfc hyperfine coupling - MLS multiline signal - PS II Photosystem II - rf radio frequency - WOC water oxidizing complex     

Electron nuclear double resonance of cytochrome oxidase: nitrogen and proton ENDOR from the 'copper' EPR signal.

Proton ENDOR resonances have been found from at least two different protons with fairly large and isotropic couplings of about 12 and 19 MHz. It is possible that such protons are attached to carbons that are one bond removed from the point of ligation to copper. A number of weakly coupled protons with anisotropic couplings have also been seen. None of the protons, either weakly or strongly coupled, appears to exchange with 2H2O. We have obtained nitrogen ENDOR from at least one nitrogen with a hyperfine coupling large enough for the nitrogen to be a ligand of copper. We have not yet demonstrated experimentally ENDOR characteristic of the copper nucleus itself.     

Electron nuclear double resonance and electron paramagnetic resonance study on the structure of the NO-ligated heme alpha 3 in cytochrome c oxidase

The techniques of EPR and electron nuclear double resonance (ENDOR) were used to probe structure and electronic distribution at the nitric oxide (NO)-ligated heme alpha 3 in the nitrosylferrocytochrome alpha 3 moiety of fully reduced cytochrome c oxidase. Hyperfine and quadrupole couplings to NO (in both 15NO and 14NO forms), to histidine nitrogens, and to protons near the heme site were obtained. Parallel studies were also performed on NO-ligated myoglobin and model NO-heme-imidazole systems. The major findings and interpretations on nitrosylferrocytochrome alpha 3 were: 1) compared to other NO-heme-imidazole systems, the nitrosylferrocytochrome alpha3 gave better resolution of EPR and ENDOR signals; 2) at the maximal g value (gx = 2.09), particularly well resolved NO nitrogen hyperfine and quadrupole couplings and mesoproton hyperfine couplings were seen. These hyperfine and quadrupole couplings gave information on the electronic distribution on the NO, on the orientation of the g tensor with respect to the heme, and possibly on the orientation of the FeNO plane; 3) a combination of experimental EPR-ENDOR results and EPR spectral simulations evidenced a rotation of the NO hyperfine tensor with respect to the electronic g tensor; this implied a bent Fe-NO bond; 4) ENDOR showed a unique proton not seen in the other NO heme systems studied. The magnitude of this proton's hyperfine coupling was consistent with this proton being part of a nearby protein side chain that perturbs an axial ligand like NO or O2.     

ENDOR and ESEEM investigation of the Ni-containing superoxide dismutase

Superoxide dismutases (SODs) protect cells against oxidative stress by disproportionating O₂ ⁻ to H₂O₂ and O₂. The recent finding of a nickel-containing SOD (Ni-SOD) has widened the diversity of SODs in terms of metal contents and SOD catalytic mechanisms. The coordination and geometrical structure of the metal site and the related electronic structure are the keys to understanding the dismutase mechanism of the enzyme. We performed Q-band ¹⁴N,^1/2H continuous wave (CW) and pulsed electron–nuclear double resonance (ENDOR) and X-band ¹⁴N electron spin echo envelope modulation (ESEEM) on the resting-state Ni-SOD extracted from Streptomyces seoulensis. In-depth analysis of the data obtained from the multifrequency advanced electron paramagnetic resonance techniques detailed the electronic structure of the active site of Ni-SOD. The analysis of the field-dependent Q-band ¹⁴N CW ENDOR yielded the nuclear hyperfine and quadrupole coupling tensors of the axial N_δ of the His-1 imidazole ligand. The tensors are coaxial with the g-tensor frame, implying the g-tensor direction is modulated by the imidazole plane. X-band ¹⁴N ESEEM characterized the hyperfine coupling of N_ε of His-1 imidazole. The nuclear quadrupole coupling constant of the nitrogen suggests that the hydrogen-bonding between N_ε–H and O_Glu-17 present for the reduced-state Ni-SOD is weakened or broken upon oxidizing the enzyme. Q-band ¹H CW ENDOR and pulsed ²H Mims ENDOR showed a strong hyperfine coupling to the protons(s) of the equatorially coordinated His-1 amine and a weak hyperfine coupling to either the proton(s) of a water in the pocket at the side opposite the axial N_δ or the proton of a water hydrogen-bonded to the equatorial thiolate ligand.     

A single-crystal ENDOR and density functional theory study of the oxidized states of the [NiFe] hydrogenase from Desulfovibrio vulgaris Miyazaki F

The catalytic center of the [NiFe] hydrogenase of Desulfovibrio vulgaris Miyazaki F in the oxidized states was investigated by electron paramagnetic resonance and electron–nuclear double resonance spectroscopy applied to single crystals of the enzyme. The experimental results were compared with density functional theory (DFT) calculations. For the Ni-B state, three hyperfine tensors could be determined. Two tensors have large isotropic hyperfine coupling constants and are assigned to the β-CH₂ protons of the Cys-549 that provides one of the bridging sulfur ligands between Ni and Fe in the active center. From a comparison of the orientation of the third hyperfine tensor with the tensor obtained from DFT calculations an OH⁻ bridging ligand has been identified in the Ni-B state. For the Ni-A state broader signals were observed. The signals of the third proton, as observed for the “ready” state Ni-B, were not observed at the same spectral position for Ni-A, confirming a structural difference involving the bridging ligand in the “unready” state of the enzyme. Electronic Supplementary Material Supplementary material is available for this article at and is accessible for authorized users. Maurice van Gastel and Matthias Stein contributed equally to this work.     

Role of the coordinating histidine in altering the mixed valency of Cu(A): an electron nuclear double resonance-electron paramagnetic resonance investigation

The binuclear Cu(A) site engineered into Pseudomonas aeruginosa azurin has provided a Cu(A)-azurin with a well-defined crystal structure and a CuSSCu core having two equatorial histidine ligands, His120 and His46. The mutations His120Asn and His120Gly were made at the equatorial His120 ligand to understand the histidine-related modulation to Cu(A), notably to the valence delocalization over the CuSSCu core. For these His120 mutants Q-band electron nuclear double resonance (ENDOR) and multifrequency electron paramagnetic resonance (EPR) (X, C, and S-band), all carried out under comparable cryogenic conditions, have provided markedly different electronic measures of the mutation-induced change. Q-band ENDOR of cysteine C(beta) protons, of weakly dipolar-coupled protons, and of the remaining His46 nitrogen ligand provided hyperfine couplings that were like those of other binuclear mixed-valence Cu(A) systems and were essentially unperturbed by the mutation at His120. The ENDOR findings imply that the Cu(A) core electronic structure remains unchanged by the His120 mutation. On the other hand, multifrequency EPR indicated that the H120N and H120G mutations had changed the EPR hyperfine signature from a 7-line to a 4-line pattern, consistent with trapped-valence, Type 1 mononuclear copper. The multifrequency EPR data imply that the electron spin had become localized on one copper by the His120 mutation. To reconcile the EPR and ENDOR findings for the His120 mutants requires that either: if valence localization to one copper has occurred, the spin density on the cysteine sulfurs and the remaining histidine (His46) must remain as it was for a delocalized binuclear Cu(A) center, or if valence delocalization persists, the hyperfine coupling for one copper must markedly diminish while the overall spin distribution on the CuSSCu core is preserved.     

Determination of the proton environment of high stability Menasemiquinone intermediate in Escherichia coli nitrate reductase A by pulsed EPR

Escherichia coli nitrate reductase A (NarGHI) is a membrane-bound enzyme that couples quinol oxidation at a periplasmically oriented Q-site (Q_D) to proton release into the periplasm during anaerobic respiration. To elucidate the molecular mechanism underlying such a coupling, endogenous menasemiquinone-8 intermediates stabilized at the Q_D site (MSQ_D) of NarGHI have been studied by high-resolution pulsed EPR methods in combination with ¹H₂O/²H₂O exchange experiments. One of the two non-exchangeable proton hyperfine couplings resolved in hyperfine sublevel correlation (HYSCORE) spectra of the radical displays characteristics typical from quinone methyl protons. However, its unusually small isotropic value reflects a singularly low spin density on the quinone carbon α carrying the methyl group, which is ascribed to a strong asymmetry of the MSQ_D binding mode and consistent with single-sided hydrogen bonding to the quinone oxygen O1. Furthermore, a single exchangeable proton hyperfine coupling is resolved, both by comparing the HYSCORE spectra of the radical in ¹H₂O and ²H₂O samples and by selective detection of the exchanged deuterons using Q-band ²H Mims electron nuclear double resonance (ENDOR) spectroscopy. Spectral analysis reveals its peculiar characteristics, i.e. a large anisotropic hyperfine coupling together with an almost zero isotropic contribution. It is assigned to a proton involved in a short ∼1.6 Å in-plane hydrogen bond between the quinone O1 oxygen and the Nδ of the His-66 residue, an axial ligand of the distal heme b_D. Structural and mechanistic implications of these results for the electron-coupled proton translocation mechanism at the Q_D site are discussed, in light of the unusually high thermodynamic stability of MSQ_D.     

1H NMR studies of the Fe7S8 ferredoxin from Bacillus schlegelii: a further attempt to understand Fe3S4 clusters

 The oxidized Fe₇S₈ ferredoxin from Bacillus schlegelii, containing both [Fe₃S₄]⁺ and [Fe₄S₄]²⁺ clusters, has been investigated by ¹H NMR spectroscopy. An extensive sequence-specific assignment of the hyperfine-shifted resonances has been obtained by making use of a computer-generated structural model. The pattern and the temperature dependence of the hyperfine shifts of the β-CH₂ protons of the cysteines coordinating the [Fe₃S₄]⁺ cluster are rationalized in terms of magnetic interactions between the iron ions. The same approach holds for the hyperfine coupling with ⁵⁷Fe. It is shown that the magnetic interactions are more asymmetric in Fe₇S₈ ferredoxins than in Fe₃S₄ ferredoxins. The NMR non-observability of the β-CH₂ protons of coordinated cysteines in the one-electron-reduced form has been discussed. Received: 19 June 1996 / Accepted: 2 August 1996     

The cationic plastoquinone radical of the chloroplast water splitting complex: Hyperfine splitting from a single methyl group determines the EPR spectral shape of Signal II

The proposal that EPR Signal II in spinach chloroplasts is due to a plastoquinone cation radical (O'Malley, P.J. and Babcock, G.T. (1983) Biophys. J. 41, 315a) has been investigated in further detail. The similarity in spectral shape between Signal II and the 2-methyl-5-isopropylhydroquinone cation radical is shown to arise from hyperfine coupling to one methyl group for both radicals. A well-resolved four line EPR spectrum of approximate relative intensity 1:3:3:1 for membrane orientation parallel and perpendicular to the applied magnetic field direction also indicates that the partially resolved structure of Signal II is due to hyperfine interaction with one methyl group, i.e., the 2-CH₃ group of the plastoquinone cation radical. The ENDOR band observed for this coupling is similar to that observed for methyl group bands of model quinone radicals. The principal hyperfine tensor values obtained for the methyl group interactions are A_⊥ = 27.2 MHz and $\text{A}{}_{\mathrm{}}\text{= 31.4}\text{MHz}$. The large isotropic coupling value (28.6 MHz) of the plastoquinone cation radical's 2-methyl group in vivo indicates that the antisymmetric orbital is the sole contributor to the spin-density distribution of Signal II. The orientation data also suggest that the plastoquinone cation radical is oriented such that the C-CH₃ bond direction, and hence the aromatic ring plane, lies perpendicular to the membrane plane.     

Protons bound to the Mn cluster in photosystem II oxygen evolving complex detected by proton matrix ENDOR

Protons in the vicinity of the oxygen-evolving manganese cluster in photosystem II were studied by proton matrix ENDOR. Six pairs of proton ENDOR signals were detected in both the S₀ and S₂ states of the Mn-cluster. Two pairs of signals that show hyperfine constants of 2.3/2.2 and 4.0 MHz, respectively, disappeared after D₂O incubation in both states. The signals with 2.3/2.2 MHz hyperfine constants in S₀ and S₂ state multiline disappeared after 3 h of D₂O incubation in the S₀ and S₁ states, respectively. The signal with 4.0 MHz hyperfine constants in S₀ state multiline disappeared after 3 h of D₂O incubation in the S₀ state, while the similar signal in S₂ state multiline disappeared only after 24 h of D₂O incubation in the S₁ state. The different proton exchange rates seem to be ascribable to the change in affinities of water molecules to the variation in oxidation state of the Mn cluster during the water oxidation cycle. Based on the point dipole approximation, the distances between the center of electronic spin of the Mn cluster and the exchangeable protons were estimated to be 3.3/3.2 and 2.7 Å, respectively. These short distances suggest the protons belong to the water molecules ligated to the manganese cluster. We propose a model for the binding of water to the manganese cluster based on these results.     

Characterization of the MCRred2 form of methyl-coenzyme M reductase: a pulse EPR and ENDOR study

Methyl-coenzyme M reductase (MCR), which catalyses the reduction of methyl-coenzyme M (CH(3)-S-CoM) with coenzyme B (H-S-CoB) to CH(4) and CoM-S-S-CoB, contains the nickel porphinoid F430 as prosthetic group. The active enzyme exhibits the Ni(I)-derived axial EPR signal MCR(red1) both in the absence and presence of the substrates. When the enzyme is competitively inhibited by coenzyme M (HS-CoM) the MCR(red1) signal is partially converted into the rhombic EPR signal MCR(red2). To obtain deeper insight into the geometric and electronic structure of the red2 form, pulse EPR and ENDOR spectroscopy at X- and Q-band microwave frequencies was used. Hyperfine interactions of the four pyrrole nitrogens were determined from ENDOR and HYSCORE data, which revealed two sets of nitrogens with hyperfine couplings differing by about a factor of two. In addition, ENDOR data enabled observation of two nearly isotropic (1)H hyperfine interactions. Both the nitrogen and proton data indicate that the substrate analogue coenzyme M is axially coordinated to Ni(I) in the MCR(red2) state.     

Assignment of Proton Endor Resonances of Nitroxyl Spin-Labels in Frozen Solution

Spin-label nitroxyl derivatives of tetramethylpyrroline and tetramethylpyrrolidine in frozen solutions of perdeuterated methanol have been characterized by electron nucleus double resonance (ENDOR spec-troscopy). With use of selectively deuterated derivatives of 2,2,5,5-tetramethylpyrroline-l-oxyl-3-carboxamide, proton ENDOR resonance features have been assigned to the vinylic proton in the five membered pyrrolinyl ring and to the methyl groups. The ENDOR resonance features were analyzed on the basis of their dependence on H₀. Two pairs of resonance features were assigned to the vinylic proton and were shown to correspond to ‖ and ⊥ hyperfine coupling (hfc) components. Six pairs of resonance features were ascribed to the methyl groups. The proton ENDOR spectra of the 3-carboxylic acid spin-label derivatives of tetramethylpyrroline and of tetramethylpyrrolidine compounds exhibited comparable features with nearly identical line splittings. From the observed ENDOR splittings, we have estimated the isotropic hfc component of the vinylic proton in 2,2,5,5-tetramethylpyrroline-l-oxyl-3-carboxamide to be -1.81 ± 0.04 MHz in frozen methanol. On the basis of the anisotropic dipolar hfc components, the electron-to-vinylic proton distance is estimated as 3.78 ± 0.01 Å. in excellent agreement with that of 3.79 Å calculated from X-ray defined coordinates.