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X-ray absorption spectroscopy of nickel in the hydrogenase from Desulfovibrio gigas, Scott, Robert A., Wallin Sten A., Czechowski Melvin, Dervartanian D. V., Legall Jean, Peck Harry D., and Moura Isabel , Journal of the American Chemical Society, 1984/10/01, Volume 106, Number 22, p.6864-6865, (1984) AbstractWebsite
Molybdenum EXAFS of the Desulfovibrio gigas Mo(2Fe-2S) protein--structural similarity to "desulfo" xanthine dehydrogenase, Cramer, S. P., Moura J. J., Xavier A. V., and Legall J. , J Inorg Biochem, Apr, Volume 20, Number 4, p.275-80, (1984) AbstractWebsite

The molybdenum EXAFS of the Mo(2Fe-2S) protein from Desulfovibrio gigas has been examined using fluorescence detection and synchrotron radiation. In the oxidized form the molybdenum environment is found to contain two terminal oxo groups and two long (2.47 A) Mo-S bonds. Evidence was also found for an oxygen or nitrogen donor ligand at 1.90 A. Addition of dithionite to the oxidized enzyme results in loss of a terminal oxo group, perhaps due to protonation. In addition, a 0.1 A contraction in the Mo-S bond lengths is observed. The behavior of both oxidized and dithionite-treated forms is similar to that observed previously with "desulfo" xanthine oxidase.

Purification, characterization and redox properties of hydrogenase from Methanosarcina barkeri (DSM 800), Fauque, G., Teixeira M., Moura I., Lespinat P. A., Xavier A. V., Dervartanian D. V., Peck, H. D. Jr., Legall J., and Moura J. G. , Eur J Biochem, Jul 2, Volume 142, Number 1, p.21-8, (1984) AbstractWebsite

A soluble hydrogenase from the methanogenic bacterium, Methanosarcina barkeri (DSM 800) has been purified to apparent electrophoretic homogeneity, with an overall 550-fold purification, a 45% yield and a final specific activity of 270 mumol H2 evolved min-1 (mg protein)-1. The hydrogenase has a high molecular mass of approximately equal to 800 kDa and subunits with molecular masses of approximately equal to 60 kDa. The enzyme is stable to heating at 65 degrees C and to exposure to air at 4 degrees C in the oxidized state for periods up to a week. The overall stability of this enzyme is compared with other hydrogenase isolated from strict anaerobic sulfate-reducing bacteria. Ms. barkeri hydrogenase shows an absorption spectrum typical of a non-heme iron protein with maxima at 275 nm, 380 nm and 405 nm. A flavin component, identified as FMN or riboflavin was extracted under acidic conditions and quantified to approximately one flavin molecule per subunit. In addition to this component, 8-10 iron atoms and 0.6-0.8 nickel atom were also detected per subunit. The electron paramagnetic resonance (EPR) spectrum of the native enzyme shows a rhombic signal with g values at 2.24, 2.20 and approximately equal to 2.0. probably due to nickel which is optimally measured at 40 K but still detectable at 77 K. In the reduced state, using dithionite or molecular hydrogen as reductants, at least two types of g = 1.94 EPR signals, due to iron-sulfur centers, could be detected and differentiated on the basis of power and temperature dependence. Center I has g values at 2.04, 1.90 and 1.86, while center II has g values at 2.08, 1.93 and 1.85. When the hydrogenase is reduced by hydrogen or dithionite the rhombic EPR species disappears and is replaced by other EPR-active species with g values at 2.33, 2.23, 2.12, 2.09, 2.04 and 2.00. These complex signals may represent different nickel species and are only observable at temperatures higher than 20 K. In the native preparation, at high temperatures (T greater than 35 K) or in partially reduced samples, a free radical due to the flavin moiety is observed. The EPR spectrum of reduced hydrogenase in 80% Me2SO presents an axial type of spectrum only detectable below 30 K.

Interconversion from 3Fe into 4Fe clusters in the presence of Desulfovibrio gigas cell extracts, Moura, J. J., Legall J., and Xavier A. V. , Eur J Biochem, Jun 1, Volume 141, Number 2, p.319-22, (1984) AbstractWebsite

Desulfovibrio gigas ferredoxin II (FdII) contains a single 3Fe cluster [Huynh, B.H., Moura, J.J.G., Moura, I., Kent, T.A., LeGall, J., Xavier, A.V., and Munck, E. (1980) J. Biol. Chem. 255, 3242-3244]. In the oxidized state the protein exhibits an intense electron paramagnetic resonance (EPR) signal at g = 2.02. Upon one-electron reduction the center becomes EPR silent. In the presence of D. gigas crude cell extracts, devoid of acidic electron carriers and supplemented with pyruvate and FdII, an EPR signal typical of reduced [4Fe-4S] centers is obtained. The appearance of this signal correlates with the beginning of stimulation of the phosphoroclastic reaction as judged by the production of H2. These results, supported by the occurrence of easy chemical conversion of the 3Fe cluster of D. gigas ferredoxin into 4Fe structures [Moura, J.J.G., Moura, I., Kent, T.A., Lipscomb, J.D., Huynh, B.H., LeGall, J., Xavier, A.V., and Munch, E. (1982) J. Biol. Chem. 257, 6259-6267], suggest that cluster conversion takes place in conditions close to the situation in vivo. This cluster interconversion is discussed in the context of some of the relevant metabolic pathways of Desulfovibrio spp.

NMR and electron-paramagnetic-resonance studies of a dihaem cytochrome from Pseudomonas stutzeri (ATCC 11607) (cytochrome c peroxidase), Villalain, J., Moura I., Liu M. C., Payne W. J., Legall J., Xavier A. V., and Moura J. J. , Eur J Biochem, Jun 1, Volume 141, Number 2, p.305-12, (1984) AbstractWebsite

A dihaem cytochrome (Mr 37 400) with cytochrome c peroxidase activity was purified from Pseudomonas stutzeri (ATCC 11 607). The haem redox potentials are far apart: one of the haems is completely ascorbate-reducible and the other is only reduced by dithionite. The coordination, spin states and redox properties of the covalently bound haems were probed by visible, NMR and electron paramagnetic resonance (EPR) spectroscopies in three oxidation states. In the oxidized state, the low-temperature EPR spectrum of the native enzyme is a complex superimposition of three components: (I) a low-spin haem indicating a histidinyl-methionyl coordination; (II) a low-spin haem indicating a histidinyl-histidinyl coordination; and (III) a minor high-spin haem component. At room temperature, NMR and optical studies indicate the presence of high-spin and low-spin haems, suggesting that for one of the haems a high-spin to low-spin transition is observed when temperature is decreased. In the half-reduced state, the component I (high redox potential) of the EPR spectrum disappears and induces a change in the g-values and linewidth of component II; the high-spin component II is no longer detected at low temperature. Visible and NMR studies reveal the presence of a high-spin ferric and a low-spin (methionyl-coordinated) ferrous state. The NMR data fully support the haem-haem interaction probed by EPR. In the reduced state, the NMR spectrum indicates that the low-potential haem is high-spin ferrous.

NMR studies of a dihaem cytochrome from Pseudomonas perfectomarinus (ATCC 14405), Moura, I., Liu M. C., Legall J., Peck, H. D. Jr., Payne W. J., Xavier A. V., and Moura J. J. , Eur J Biochem, Jun 1, Volume 141, Number 2, p.297-303, (1984) AbstractWebsite

Pseudomonas perfectomarinus (ATCC 14405) dihaem cytochrome c552 was studied by 300-MHz proton magnetic resonance. Some of the haem resonances were assigned in the fully reduced and fully oxidized states. No evidence was found for methionine haem axial coordination. The oxidation-reduction equilibrium was studied in detail. Due to the large difference in mid-point redox potential between the two haems (+174 mV, for haem II and -180 mV for haem I) an intermediate oxidation state could be obtained containing reduced haem I and oxidized haem II. In this way the total paramagnetic shift at different oxidation levels could be decomposed in the intrinsic and extrinsic contributions. It was found that the two haems interact. The rate of electron exchange is slow on the NMR time scale. The redox equilibria are discussed for four possible redox species in solution.

NMR studies of electron transfer mechanisms in a protein with interacting redox centres: Desulfovibrio gigas cytochrome c3, Santos, H., Moura J. J., Moura I., Legall J., and Xavier A. V. , Eur J Biochem, Jun 1, Volume 141, Number 2, p.283-96, (1984) AbstractWebsite

The proton NMR spectra of the tetrahaem cytochrome c3 from Desulfovibrio gigas were examined while varying the pH and the redox potential. The analysis of the NMR reoxidation pattern was based on a model for the electron distribution between the four haems that takes into account haem-haem redox interactions. The intramolecular electron exchange is fast on the NMR time scale (larger than 10(5) s-1). The NMR data concerning the pH dependence of the chemical shift of haem methyl resonances in different oxidation steps and resonance intensities are not compatible with a non-interacting model and can be explained assuming a redox interaction between the haems. A complete analysis at pH* = 7.2 and 9.6, shows that the haem-haem interacting potentials cover a range from -50 mV to +60 mV. The midpoint redox potentials of some of the haems, as well as some of their interacting potentials, are pH-dependent. The physiological relevance of the modulation of the haem midpoint redox potentials by both the pH and the redox potential of the solution is discussed.

Nuclear-magnetic-resonance studies of Desulfuromonas acetoxidans cytochrome c551.5 (c7), Moura, J. G., Moore G. R., Williams R. J., Probst I., Legall J., and Xavier A. V. , Eur J Biochem, Nov 2, Volume 144, Number 3, p.433-40, (1984) AbstractWebsite

1H nuclear magnetic resonance (NMR) spectroscopy has been used to examine cytochrome c551.5 (c7) from the sulfur reducer, Desulfuromonas acetoxidans. This protein contains three hemes. Two stable oxidation states (the fully oxidized and the fully reduced) as well as intermediate oxidation states were studied. The axial ligands of the iron were found to be neutral histidines. The redox properties of cytochrome c7 were examined and good quantitative agreement found between the NMR results and previously reported redox potential measurements. The properties of cytochrome c7 are discussed together with those of the homologous tetraheme cytochromes c3 isolate from sulfate-reducing bacteria.

Cobalt containing B12 cofactors from methanogenic bacteria - spectroscopic characterization, Lino, A. R., Xavier A. V., Moura I., Legall J., and Ljungdahl P. O. , Rev Portuguesa de Química, Volume 27, p.175-177, (1985) Abstract
Nickel X-ray absorption spectroscopy of Desulvovibrio gigas hydrogenase, Scott, R. A., Czechowski M., Dervartanian D. V., Legall J., Peck Jr H. D., and Moura I. , Rev Portuguesa de Química, Volume 27, p.67-70, (1985) Abstract
Electron paramagnetic resonance studies on the mechanism of activation and the catalytic cycle of the nickel-containing hydrogenase from Desulfovibrio gigas, Teixeira, M., Moura I., Xavier A. V., Huynh B. H., Dervartanian D. V., Peck, H. D. Jr., Legall J., and Moura J. J. , J Biol Chem, Jul 25, Volume 260, Number 15, p.8942-50, (1985) AbstractWebsite

Desulfovibrio gigas hydrogenase (EC is a complex enzyme containing one nickel, one 3Fe, and two [Fe4S4] clusters (Teixeira, M., Moura, I., Xavier, A. V., Der Vartanian, D. V., LeGall, J., Peck, H. D., Jr., Huynh, B. H., and Moura, J. J. G. (1983) Eur. J. Biochem. 130, 481-484). This hydrogenase belongs to a class of enzymes that are inactive "as isolated" (the so-called "oxygen-stable hydrogenases") and must go through an activation process in order to express full activity. The state of characterization of the active centers of the enzyme as isolated prompted us to do a detailed analysis of the redox patterns, activation profile, and catalytic redox cycle of the enzyme in the presence of either the natural substrate (H2) or chemical reductants. The effect of natural cofactors, as cytochrome C3, was also studied. Special focus was given to the intermediate redox species generated during the catalytic cycle of the enzyme and to the midpoint redox potentials associated. The available information is discussed in terms of a "working hypothesis" for the mechanism of the [NiFe] hydrogenases from sulfate reducing organisms in the context of activation process and catalytic cycle.

Purification and characterization of three proteins from a halophilic sulfate-reducing bacterium,<i>Desulfovibrio salexigens</i&gt, Czechowski, M., Fauque G., Galliano N., Dimon B., Moura I., Moura J. J. G., Xavier A. V., Barato B. A. S., Lino A. R., and Legall J. , Journal of Industrial Microbiology & Biotechnology, Volume 1, Number 3, p.139-147, (1986) AbstractWebsite
Evidence for the formation of a cobalt-iron-sulfur (CoFe3S4) cluster in Desulfovibrio gigas ferredoxin II, Moura, Isabel, Moura Jose J. G., Munck Eckard, Papaefthymiou Vasilios, and Legall Jean , Journal of the American Chemical Society, 1986/01/01, Volume 108, Number 2, p.349-351, (1986) AbstractWebsite
Resonance Raman spectra of rubredoxin: new assignments and vibrational coupling mechanism from iron-54/iron-56 isotope shifts and variable-wavelength excitation, Czernuszewicz, Roman S., Legall Jean, Moura Isabel, and Spiro Thomas G. , Inorganic Chemistry, 1986/02/01, Volume 25, Number 5, p.696-700, (1986) AbstractWebsite
Low-spin sulfite reductases: a new homologous group of non-heme iron-siroheme proteins in anaerobic bacteria, Moura, I., Lino A. R., Moura J. J., Xavier A. V., Fauque G., Peck, H. D. Jr., and Legall J. , Biochem Biophys Res Commun, Dec 30, Volume 141, Number 3, p.1032-41, (1986) AbstractWebsite

Two new low molecular weight proteins with sulfite reductase activity, isolated from Methanosarcina barkeri (DSM 800) and Desulfuromonas acetoxidans (strain 5071), were studied by EPR and optical spectroscopic techniques. Both proteins have visible spectra similar to that of the low-spin sulfite reductase of Desulfovibrio vulgaris strain Hildenborough and no band at 715 nm, characteristic of high-spin Fe3+ complexes in isobacteriochlorins is observed. EPR shows that as isolated the siroheme is in a low-spin ferric state (S = 1/2) with g-values at 2.40, 2.30 and 1.88 for the Methanosarcina barkeri enzyme and g-values at 2.44, 2.33 and 1.81 for the Desulfuromonas acetoxidans enzyme. Chemical analysis shows that both proteins contain one siroheme and one [Fe4S4] center per polypeptidic chain. These results suggest that the low molecular weight, low-spin non-heme iron siroheme proteins represent a new homologous class of sulfite reductases common to anaerobic microorganisms.

Redox properties and activity studies on a nickel-containing hydrogenase isolated from a halophilic sulfate reducer Desulfovibrio salexigens, Teixeira, M., Moura I., Fauque G., Czechowski M., Berlier Y., Lespinat P. A., Legall J., Xavier A. V., and Moura J. J. , Biochimie, Jan, Volume 68, Number 1, p.75-84, (1986) AbstractWebsite

A soluble hydrogenase from the halophilic sulfate reducing bacterium Desulfovibrio salexigens, strain British Guiana (NCIB 8403) has been purified to apparent homogeneity with a final specific activity of 760 mumoles H2 evolved/min/mg (an overall 180-fold purification with 20% recovery yield). The enzyme is composed of two non-identical subunits of molecular masses 62 and 36 kDa, respectively, and contains approximately 1 Ni, 12-15 Fe and 1 Se atoms/mole. The hydrogenase shows a visible absorption spectrum typical of an iron-sulfur containing protein (A400/A280 = 0.275) and a molar absorbance of 54 mM-1cm-1 at 400 nm. In the native state (as isolated, under aerobic conditions), the enzyme is almost EPR silent at 100 K and below. However, upon reduction under H2 atmosphere a rhombic EPR signal develops at g-values 2.22, 2.16 and around 2.0, which is optimally detected at 40 K. This EPR signal is reminiscent of the nickel signal C (g-values 2.19, 2.16 and 2.02) observed in intermediate redox states of the well characterized D. gigas nickel containing hydrogenase and assigned to nickel by 61 Ni isotopic substitution (J.J.G. Moura, M. Teixeira, I. Moura, A.V. Xavier and J. Le Gall (1984), J. Mol. Cat., 23, 305-314). Upon longer incubation with H2 the "2.22" EPR signal decreases. During the course of a redox titration under H2, this EPR signal attains a maximal intensity around--380 mV. At redox states where this "2.22" signal develops (or at lower redox potentials), low temperature studies (below 10 K) reveals the presence of other EPR species with g-values at 2.23, 2.21, 2.14 with broad components at higher fields. This new signal (fast relaxing) exhibits a different microwave power dependence from that of the "2.22" signal, which readily saturates with microwave power (slow relaxing). Also at low temperature (8 K) typical reduced iron-sulfur EPR signals are concomitantly observed with gmed approximately 1.94. The catalytic properties of the enzyme were also followed by substrate isotopic exchange D2/H+ and H2 production measurements.

Identification of three classes of hydrogenase in the genus, Desulfovibrio, Prickril, Benet C., He Shao-Hua, Li Ching, Menon Nanda, Choi Eui-Sung, Przybyla Alan E., DerVartanian Daniel V., Peck Jr Harry D., Fauque Guy, Legall Jean, Teixeira Miguel, Moura Isabel, Moura Jose J. G., Patil Daulat, and Huynh Boi H. , Biochemical and Biophysical Research Communications, Volume 149, Number 2, p.369-377, (1987) AbstractWebsite
Isolation and characterization of a rubredoxin and a flavodoxin from Desulfovibrio desulfuricans Berre-Eau, Fauque, Guy D., Moura Isabel, Moura José J. G., Xavier António V., Galliano Nicole, and Legall Jean , Febs Letters, Volume 215, Number 1, p.63-67, (1987) AbstractWebsite
Proton NMR spectra of rubredoxins: new resonances assignable to .alpha.-CH and .beta.-CH2 hydrogens of cysteinate ligands to iron(II), Werth, Mark T., Kurtz Donald M., Moura Isabel, and Legall Jean , Journal of the American Chemical Society, 1987/01/01, Volume 109, Number 1, p.273-275, (1987) AbstractWebsite
Evidence for the formation of a ZnFe3S4 cluster in Desulfovibrio gigas ferredoxin II, Surerus, Kristene K., Munck Eckard, Moura Isabel, Moura Jose J. G., and Legall Jean , Journal of the American Chemical Society, 1987/06/01, Volume 109, Number 12, p.3805-3807, (1987) AbstractWebsite
Moessbauer study of D. gigas ferredoxin II and spin-coupling model for Fe3S4 cluster with valence delocalization, Papaefthymiou, V., Girerd J. J., Moura I., Moura J. J. G., and Muenck E. , Journal of the American Chemical Society, 1987/07/01, Volume 109, Number 15, p.4703-4710, (1987) AbstractWebsite
Nickel-[iron-sulfur]-selenium-containing hydrogenases from Desulfovibrio baculatus (DSM 1743). Redox centers and catalytic properties, Teixeira, M., Fauque G., Moura I., Lespinat P. A., Berlier Y., Prickril B., Peck, H. D. Jr., Xavier A. V., Legall J., and Moura J. J. , Eur J Biochem, Aug 17, Volume 167, Number 1, p.47-58, (1987) AbstractWebsite

The hydrogenase from Desulfovibrio baculatus (DSM 1743) was purified from each of three different fractions: soluble periplasmic (wash), soluble cytoplasmic (cell disruption) and membrane-bound (detergent solubilization). Plasma-emission metal analysis detected in all three fractions the presence of iron plus nickel and selenium in equimolecular amounts. These hydrogenases were shown to be composed of two non-identical subunits and were distinct with respect to their spectroscopic properties. The EPR spectra of the native (as isolated) enzymes showed very weak isotropic signals centered around g approximately 2.0 when observed at low temperature (below 20 K). The periplasmic and membrane-bound enzymes also presented additional EPR signals, observable up to 77 K, with g greater than 2.0 and assigned to nickel(III). The periplasmic hydrogenase exhibited EPR features at 2.20, 2.06 and 2.0. The signals observed in the membrane-bound preparations could be decomposed into two sets with g at 2.34, 2.16 and approximately 2.0 (component I) and at 2.33, 2.24, and approximately 2.0 (component II). In the reduced state, after exposure to an H2 atmosphere, all the hydrogenase fractions gave identical EPR spectra. EPR studies, performed at different temperatures and microwave powers, and in samples partially and fully reduced (under hydrogen or dithionite), allowed the identification of two different iron-sulfur centers: center I (2.03, 1.89 and 1.86) detectable below 10 K, and center II (2.06, 1.95 and 1.88) which was easily saturated at low temperatures. Additional EPR signals due to transient nickel species were detected with g greater than 2.0, and a rhombic EPR signal at 77 K developed at g 2.20, 2.16 and 2.0. This EPR signal is reminiscent of the Ni-signal C (g at 2.19, 2.14 and 2.02) observed in intermediate redox states of the well characterized Desulfovibrio gigas hydrogenase (Teixeira et al. (1985) J. Biol. Chem. 260, 8942]. During the course of a redox titration at pH 7.6 using H2 gas as reductant, this signal attained a maximal intensity around -320 mV. Low-temperature studies of samples at redox states where this rhombic signal develops (10 K or lower) revealed the presence of a fast-relaxing complex EPR signal with g at 2.25, 2.22, 2.15, 2.12, 2.10 and broad components at higher field. The soluble hydrogenase fractions did not show a time-dependent activation but the membrane-bound form required such a step in order to express full activity.(ABSTRACT TRUNCATED AT 400 WORDS)

Characterization Of Electron-Transfer Proteins From The Nitrogen-Fixing Sulfate-Reducing Bacterium Desulfovibrio-Desulfuricans Berre-Eau, Fauque, G., Moura I., Xavier A. V., Galliano N., Moura J. J. G., and Legall J. , Biochemical Society Transactions, Dec, Volume 15, Number 6, p.1049-1050, (1987) AbstractWebsite
Characterization of the cytochrome system of a nitrogen-fixing strain of a sulfate-reducing bacterium: Desulfovibrio desulfuricans strain Berre-Eau, Moura, I., Fauque G., Legall J., Xavier A. V., and Moura J. J. , Eur J Biochem, Feb 2, Volume 162, Number 3, p.547-54, (1987) AbstractWebsite

Two c-type cytochromes were purified and characterized by electron paramagnetic resonance (EPR) and nuclear magnetic resonance (NMR) spectroscopic techniques, from the sulfate-reducer nitrogen-fixing organism, Desulfovibrio desulfuricans strain Berre-Eau (NCIB 8387). The purification procedures included several chromatographic steps on alumina, carboxymethylcellulose and gel filtration. A tetrahaem and a monohaem cytochrome were identified. The multihaem cytochrome has visible, EPR and NMR spectra with general properties similar to other low-potential bis-histidinyl axially bound haem proteins, belonging to the class of tetrahaem cytochrome c3 isolated from other Desulfovibrio species. The monohaem cytochrome c553 is ascorbate-reducible and its EPR and NMR data are characteristic of a cytochrome with methionine-histidine ligation. Their properties are compared with other homologous proteins isolated from sulfate-reducing bacteria.

On the active sites of the [NiFe] hydrogenase from Desulfovibrio gigas. Mossbauer and redox-titration studies, Huynh, B. H., Patil D. S., Moura I., Teixeira M., Moura J. J., Dervartanian D. V., Czechowski M. H., Prickril B. C., Peck, H. D. Jr., and Legall J. , J Biol Chem, Jan 15, Volume 262, Number 2, p.795-800, (1987) AbstractWebsite

The [NiFe] hydrogenase isolated from Desulfovibrio gigas was poised at different redox potentials and studied by Mossbauer spectroscopy. The data firmly establish that this hydrogenase contains four prosthetic groups: one nickel center, one [3Fe-xS], and two [4Fe-4S] clusters. In the native enzyme, both the nickel and the [3Fe-xS] cluster are EPR-active. At low temperature (4.2 K), the [3Fe-xS] cluster exhibits a paramagnetic Mossbauer spectrum typical for oxidized [3Fe-xS] clusters. At higher temperatures (greater than 20 K), the paramagnetic spectrum collapses into a quadrupole doublet with parameters magnitude of delta EQ magnitude of = 0.7 +/- 0.06 mm/s and delta = 0.36 +/- 0.06 mm/s, typical of high-spin Fe(III). The observed isomer shift is slightly larger than those observed for the three-iron clusters in D. gigas ferredoxin II (Huynh, B. H., Moura, J. J. G., Moura, I., Kent, T. A., LeGall, J., Xavier, A. V., and Munck, E. (1980) J. Biol. Chem. 255, 3242-3244) and in Azotobacter vinelandii ferredoxin I (Emptage, M. H., Kent, T. A., Huynh, B. H., Rawlings, J., Orme-Johnson, W. H., and Munck, E. (1980) J. Biol. Chem. 255, 1793-1796) and may indicate a different iron coordination environment. When D. gigas hydrogenase is poised at potentials lower than -80 mV (versus normal hydrogen electrode), the [3Fe-xS] cluster is reduced and becomes EPR-silent. The Mossbauer data indicate that the reduced [3Fe-xS] cluster remains intact, i.e. it does not interconvert into a [4Fe-4S] cluster. Also, the electronic properties of the reduced [3Fe-xS] cluster suggest that it is magnetically isolated from the other paramagnetic centers.