Ferredoxin II from Desulphovibrio gigas is a tetrameric protein containing a novel iron-sulphur cluster consisting of three iron atoms. The low-temperature magnetic circular dichroism (MCD) spectra of the oxidized and dithionite-reduced forms of ferredoxin II have been measured over the wavelength range approx. 300-800 nm. Both oxidation levels of the cluster are shown to be paramagnetic, although only the oxidized form gives an EPR signal. MCD magnetization curves have been constructed over the temperature range approx. 1.5-150 K and at fields between 0 and 5.1 Tesla. The curve for the oxidized protein can be fitted to a ground state of spin S = 1/2 with an isotropic g factor of 2.01. There is evidence for the thermal population of a low-lying electronic state above 50 K. The reduced protein gives a distinctive set of magnetization curves that are tentatively assigned to a ground state of S = 2, with a predominantly axial zero-field distortion that leaves the doublet Ms = +/-2 lowest in energy. The zero-field components have a maximum energy spread of approx. 15 cm-1. which places an upper limit of 4 cm-1 on the axial zero-field parameter D. The MCD spectra of the oxidized and reduced forms of the cluster are quite distinctive from one another. The spectra of the oxidized state are also different from those of oxidized high-potential iron protein from Chromatium and should provide a useful criterion for distinguishing between four- and three-iron clusters in their highest oxidation levels.

Hydrogenase from Desulfovibrio desulfuricans (ATCC No. 27774) grown in unenriched and in enriched 61Ni and 57Fe media has been purified to apparent homogeneity. Two fractions of enzymes with hydrogenase activity were separated and were termed hydrogenase I and hydrogenase II. they were shown to have similar molecular weights (77,600 for hydrogenase I and 75,500 for hydrogenase II), to be composed of two polypeptide chains, and to contain Ni and non-heme iron. Because of its higher specific activity (152 versus 97) hydrogenase II was selected for EPR and Mossbauer studies. As isolated, hydrogenase II exhibits an "isotropic" EPR signal at g = 2.02 and a rhombic EPR signal at g = 2.3, 2.2, and 2.0. Isotopic substitution of 61Ni proves that the rhombic signal is due to Ni. Combining the Mossbauer and EPR data, the isotropic g = 2.02 EPR signal was shown to originate from a 3Fe cluster which may have oxygenous or nitrogenous ligands. In addition, the Mossbauer data also revealed two [4Fe-4S]2+ clusters iun each molecule of hydrogenase II. The EPR and Mossbauer data of hydrogenase I were found to be identical to those of hydrogenase II, indicating that both enzymes have common metallic centers.

We have obtained the iron K-edge extended X-ray adsorption fine structure spectra of the 3Fe ferredoxin II of Desulfovibrio gigas in the oxidized and reduced states. For both states, interpretation of the EXAFS data suggests that the Fe-S first shell coordination distance is near 2.25 A, in agreement with crystallographic studies of model compounds and proteins containing 2Fe-2S and 4Fe-4S centers, as well as with a recent crystallographic study of Azotobacter vinelandii ferredoxin I (Ghosh, D., Furey, W., Jr., O'Donnell, S., and Stout, C. D. (1981) J. Biol. Chem. 256, 4185-4192). The apparent Fe-Fe distance we obtain for the desulfovibrio protein (2.7 A) also agrees with similar distances seen in other Fe-S centers, except with the 3Fe cluster in the Azotobacter vinelandii ferredoxin I structure, for which an Fe-Fe distance of 4.2 A was reported. We conclude that either the two 3Fe ferredoxins have substantially different core dimensions, a possibility apparently unique to 3Fe centers among known Fe-S systems in proteins, or that one (or more) of the structural studies is in substantial error.

Below 30 K, oxidized Desulfovibrio gigas hydrogenase presents an intense electron paramagnetic resonance (EPR) signal centered at g = 2.02, typical of an iron-sulfur center. In addition a rhombic EPR signal, attributed to Ni(III) species, is also observed [LeGall, J., Ljungdahl, P., Moura, I., Peck, H.D., Jr, Xavier, A.V., Moura, J.J.G., Teixeira, M., Huynh, B.H., and DerVartanian, D.V. (1982) Biochem. Biophys. Res. Commun. 106, 610-616; and Cammack, R., Patil, D., Aguirre, R., and Hatchikian, E.C., (1982) FEBS Lett. 142, 289-292]. At higher temperatures (77 K) the iron-sulfur EPR signal is broader and all the EPR features of the rhombic nickel signal can easily be observed. We have now obtained additional information concerning the redox properties of these EPR active centers, using an EPR redox titration method in the presence of dye mediators at pH = 8.5. The mid-point potential was determined to be -70 mV for the Fe,S cluster and -220 mV for the Ni center. Intermediate oxidation states were obtained upon partial reduction with either dithionite or hydrogen. Although upon dithionite reduction the centers are reduced in the order of decreasing mid-point reduction potentials, under a hydrogen atmosphere the nickel center reduces preferentially. This suggests a catalytic involvement of the nickel redox center in the binding of hydrogen. Preliminary Mossbauer studies on Desulfovibrio gigas hydrogenase reveal the presence of a paramagnetic 3 Fe center and two 4 Fe centers. The 3 Fe center is responsible for the g = 2.02 EPR signal but the two 4 Fe centers have been so far undetectable by EPR.

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.

Desulfovibrio gigas hydrogenase (EC 1.12.2.1) 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.

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.

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)

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.

The molybdenum iron-sulphur protein originally isolated from Desulfovibrio gigas by Moura, Xavier, Bruschi, Le Gall, Hall & Cammack [(1976) Biochem. Biophys. Res. Commun. 72, 782-789] has been further investigated by e.p.r. spectroscopy of molybdenum(V). The signal obtained on extended reduction of the protein with sodium dithionite has been shown, by studies at 9 and 35 HGz in 1H2O and 2H2O and computer simulations, to have parameters corresponding to those of the Slow signal from the inactive desulpho form of various molybdenum-containing hydroxylases. Another signal obtained on brief reduction of the protein with small amounts of dithionite was shown by e.p.r. difference techniques to be a Rapid type 2 signal, like that from the active form of such enzymes. In confirmation that the protein is a molybdenum-containing hydroxylase, activity measurements revealed that it had aldehyde:2,6-dichlorophenol-indophenol oxidoreductase activity. No such activity towards xanthine or purine was observed. Salicylaldehyde was a particularly good substrate, and treatment of the protein with it also gave rise to the Rapid signal. Molybdenum cofactor liberated from the protein was active in the nit-1 Neurospora crassa nitrate reductase assay. It is concluded that the protein is a form of an aldehyde oxidase or dehydrogenase. From the intensity of the e.p.r. signals and from enzyme activity measurements, 10-30% of the protein in the sample examined appeared to be in the functional form. The evolutionary significance of the protein, which may represent a primitive form of the enzyme rather than a degradation product, is discussed briefly.

The nickel tetrahedral sulfur-coordinated core formed upon metal replacement of the native iron in Desulfovibrio sp. rubredoxins is shown to mimic the reactivity pattern of nickel-containing hydrogenases with respect to hydrogen production, deuterium-proton exchange, and inhibition by carbon monoxide.

Three types of hydrogenases have been isolated from the sulfate-reducing bacteria of the genus Desulfovibrio. They differ in their subunit and metal compositions, physico-chemical characteristics, amino acid sequences, immunological reactivities, gene structures and their catalytic properties. Broadly, the hydrogenases can be considered as 'iron only' hydrogenases and nickel-containing hydrogenases. The iron-sulfur-containing hydrogenase ([Fe] hydrogenase) contains two ferredoxin-type (4Fe-4S) clusters and an atypical iron-sulfur center believed to be involved in the activation of H2. The [Fe] hydrogenase has the highest specific activity in the evolution and consumption of hydrogen and in the proton-deuterium exchange reaction and this enzyme is the most sensitive to CO and NO2-. It is not present in all species of Desulfovibrio. The nickel-(iron-sulfur)-containing hydrogenases [( NiFe] hydrogenases) possess two (4Fe-4S) centers and one (3Fe-xS) cluster in addition to nickel and have been found in all species of Desulfovibrio so far investigated. The redox active nickel is ligated by at least two cysteinyl thiolate residues and the [NiFe] hydrogenases are particularly resistant to inhibitors such as CO and NO2-. The genes encoding the large and small subunits of a periplasmic and a membrane-bound species of the [NiFe] hydrogenase have been cloned in Escherichia (E.) coli and sequenced. Their derived amino acid sequences exhibit a high degree of homology (70%); however, they show no obvious metal-binding sites or homology with the derived amino acid sequence of the [Fe] hydrogenase. The third class is represented by the nickel-(iron-sulfur)-selenium-containing hydrogenases [( NiFe-Se] hydrogenases) which contain nickel and selenium in equimolecular amounts plus (4Fe-4S) centers and are only found in some species of Desulfovibrio. The genes encoding the large and small subunits of the periplasmic hydrogenase from Desulfovibrio (D.) baculatus (DSM 1743) have been cloned in E. coli and sequenced. The derived amino acid sequence exhibits homology (40%) with the sequence of the [NiFe] hydrogenase and the carboxy-terminus of the gene for the large subunit contains a codon (TGA) for selenocysteine in a position homologous to a codon (TGC) for cysteine in the large subunit of the [NiFe] hydrogenase. EXAFS and EPR studies with the 77Se-enriched D. baculatus hydrogenase indicate that selenium is a ligand to nickel and suggest that the redox active nickel is ligated by at least two cysteinyl thiolate and one selenocysteine selenolate residues.(ABSTRACT TRUNCATED AT 400 WORDS)

The periplasmic hydrogenase of Desulfovibrio vulgaris (Hildenbourough NCIB 8303) belongs to the category of [Fe] hydrogenase which contains only iron-sulfur clusters as its prosthetic groups. Amino acid analyses were performed on the purified D. vulgaris hydrogenase. The amino acid composition obtained compared very well with the result derived from the nucleotide sequence of the structural gene (Voordouw, G., Brenner, S. (1985) Eur. J. Biochem. 148, 515-520). Detailed EPR reductive titration studies on the D. vulgaris hydrogenase were performed to characterize the metal centers in this hydrogenase. In addition to the three previously observed EPR signals (namely, the "isotropic" 2.02 signal, the rhombic 2.10 signal, and the complex signal of the reduced enzyme), a rhombic signal with resonances at the g-values of 2.06, 1.96, and 1.89 (the rhombic 2.06 signal) was detected when the samples were poised at potentials between 0 and -250 mV (with respect to normal hydrogen electrode). The midpoint redox potentials for each of the four EPR-active species were determined, and the characteristics of each EPR signal are described. Both the rhombic 2.10 and 2.06 signals exhibit spectral properties that are distinct from a ferredoxin-type [4Fe-4S] cluster and are proposed to originate from the same H2-binding center but in two different conformations. The complex signal of the reduced hydrogenase has been shown to represent two spin-spin interacting ferredoxin-type [4Fe-4S]1+ clusters (Grande, H. J., Dunham, W. R., Averill, B., Van Dijk, C., and Sands, R. H. (1983) Eur. J. Biochem. 136, 201-207). The titration data indicated a strong cooperative effect between these two clusters during their reduction. In an effort to accurately estimate the number of iron atoms/molecule of hydrogenase, plasma emission and chemical methods were used to determine the iron contents in the samples; and four different methods, including amino acid analysis, were used for protein determination. The resulting iron stoichiometries were found to be method-dependent and vary over a wide range (+/- 20%). The uncertainties involved in the determination of iron stoichiometry are discussed.

An EPR redox titration was performed on the tetraheme cytochrome c3 isolated from Desulfovibrio baculatus (strain 9974), a sulfate-reducer. Using spectral differences at different poised redox states of the protein, it was possible to individualize the EPR g-values of each of the four hemes and also to determine the mid-point redox potentials of each individual heme: heme 4 (-70 mV) at gmax = 2.93, gmed = 2.26 and gmin = 1.51; heme 3 (-280 mV) at gmax = 3.41; heme 2 (-300 mV) at gmax = 3.05, gmed = 2.24 and gmin = 1.34; and heme 1 (-355 mV) at gmx = 3.18. A previously described multi-redox equilibria model used for the interpretation of NMR data of D. gigas cytochrome c3 [Santos, H., Moura, J.J.G., Moura, I., LeGall, J. & Xavier, A. V. (1984) Eur. J. Biochem. 141, 283-296] is discussed in terms of the EPR results.

The periplasmic hydrogenase containing equivalent amounts of nickel and selenium plus non-heme iron [NiFeSe) hydrogenase) has been purified from cells of the sulfate reducing bacterium Desulfovibrio baculatus (DSM 1748) grown on a lactate/sulfate medium containing natural Se isotopes and the nuclear isotope, 77Se. Both the 77Se-enriched and unenriched hydrogenases were shown to be free of other hydrogenases and characterized with regard to their Se contents. EPR studies of the reduced nickel signal generated by redox titrations of the enriched and unenriched (NiFeSe) hydrogenases demonstrated that the gx = 2.23 and gy = 2.17 resonances are appreciably broadened by the spin of the 77Se nucleus (I = 1/2). This observation demonstrates unambiguously that the unpaired electron is shared by the Ni and Se atoms and that Se serves as a ligand to the nickel redox center of the (NiFeSe) hydrogenase.

From biphasic stopped-flow kinetic studies it has been established that the two heme centres of cytochrome c4 from Azotobacter vinelandii undergo redox change with [Co(terpy)2]3+/2+ (260 mV) at different rates. Rate constants for oxidation and reduction at pH 7.5 give reduction potentials for the two heme centres in agreement with previous values from spectrophotometric titrations (263 and 317 mV). From NMR studies on the fully reduced protein two sharp methyl methionine resonances are observed at -3.16 and -3.60 ppm, consistent with axial methionine coordination. On titration with [Fe(CN)6]3- the -3.16 ppm resonance is the first to disappear, and is assigned to the less positive reduction potential. Line-broadening effects are observed on partial oxidation, which are dominated by intermolecular processes in an intermediate time-range exchange process. The hemes of the oxidised protein are distinguishable by EPR g-values of 3.64 and 3.22. The former is of interest because it is at an unusually low field for histidine/methionine coordination, and has an asymmetric or ramp shape. The latter assigned to the low potential heme is similar to that of a cytochrome c551. The MCD spectra of the fully oxidised protein are typical of low-spin Fe(III) heme centres, with a negative peak at 710 nm characteristic of methionine coordination, and an NIR peak at 1900 nm characteristic of histidine/methionine (axial) coordination. Of the four histidines per molecule only two undergo diethyl pyrocarbonate (DEPC) modification.

The hydrogenase (EC 1.2.2.1) of Desulfovibrio gigas is a complex enzyme containing one nickel center, one [3Fe-4S] and two [4Fe-4S] clusters. Redox intermediates of this enzyme were generated under hydrogen (the natural substrate) using a redox-titration technique and were studied by EPR and Mossbauer spectroscopy. In the oxidized states, the two [4Fe-4S]2+ clusters exhibit a broad quadrupole doublet with parameters (apparent delta EQ = 1.10 mm/s and delta = 0.35 mm/s) typical for this type of cluster. Upon reduction, the two [4Fe-4S]1+ clusters are spectroscopically distinguishable, allowing the determination of their midpoint redox potentials. The cluster with higher midpoint potential (-290 +/- 20 mV) was labeled Fe-S center I and the other with lower potential (-340 +/- 20 mV), Fe-S center II. Both reduced clusters show atypical magnetic hyperfine coupling constants, suggesting structural differences from the clusters of bacterial ferredoxins. Also, an unusually broad EPR signal, labeled Fe-S signal B', extending from approximately 150 to approximately 450 mT was observed concomitantly with the reduction of the [4Fe-4S] clusters. The following two EPR signals observed at the weak-field region were tentatively attributed to the reduced [3Fe-4S] cluster: (i) a signal with crossover point at g approximately 12, labeled the g = 12 signal, and (ii) a broad signal at the very weak-field region (approximately 3 mT), labeled the Fe-S signal B. The midpoint redox potential associated with the appearance of the g = 12 signal was determined to be -70 +/- 10 mV. At potentials below -250 mV, the g = 12 signal began to decrease in intensity, and simultaneously, the Fe-S signal B appeared. The transformation of the g = 12 signal into the Fe-S signal B was found to parallel the reduction of the two [4Fe-4S] clusters indicating that the [3Fe-4S]o cluster is sensitive to the redox state of the [4Fe-4S] clusters. Detailed redox profiles for the previously reported Ni-signal C and the g = 2.21 signal were obtained in this study, and evidence was found to indicate that these two signals represent two different oxidation states of the enzyme. Finally, the mechanistic implications of our results are discussed.