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A coenzyme F430-reducing hydrogenase from Methanobacterium thermoautotrophicum has been purified 25-fold, to approximately 50% homogeneity. Following anaerobic preincubation in high salt under reducing conditions, the purified enzyme exhibits equal catalytic activity (turnover number = 725 s-1) toward the artificial 1-electron acceptor, methyl viologen, and the physiological 2-electron acceptor, 7,8-didemethyl-8-hydroxy-5-deazaflavin (F420). The enzyme had the following Km values (micromolarity) under the described assay conditions: 420 (methyl viologen, pH 9.0), 19 (F420, pH 7.2), 34 (Fo, 7.8-didemethyl-8-hydroxy-5-deazariboflavin, pH 7.2), 10 (H2, Fo as co-substrate, pH 7.2), 2 (H2, methyl viologen as co-substrate, pH 9.0). The native protein is oligomeric (apparent Mr greater than 500,000) and is composed of three distinct subunits with Mr - 40,000, 31,000, and 26,000 in the ratio of 2:2:1, leading to a minimum Mr = 170,000. In addition to 33 atoms of Fe and 24 atoms of acid-labile sulfur, the F420-hydrogenase contains 2.3 mol of FAD/mol of Mr - 170,000. This activity is chromatographically distinct from a smaller methanogen hydrogenase capable of rapid viologen reduction, but which only very slowly reduces 5-deazariboflavins.

A series of plasmids encoding various Klebsiella pneumoniae nif (nitrogen fixation) genes were constructed to determine which were required to produce active iron (Fe) protein in Escherichia coli, a species which does not normally fix nitrogen. The greatest success was achieved with binary plasmid systems that produced nifA regulatory protein under the control of a tac promoter on one plasmid, which then induced synthesis of nifH and nifM proteins from their native promoter sites on a second plasmid. nifH protein, the monomeric subunit of Fe protein, produced in the presence of nifM constituted nearly 10% of the whole cell protein and exhibited the corresponding amount of C2H2-reducing activity in nitrogenase assays conducted in vitro. nifH protein formed in the absence of nifM constituted 4.7% of the whole cell protein and exhibited no detectable activity in assays of whole cell extracts. The plasmid-encoded Fe protein was purified to homogeneity and was found to be indistinguishable from that isolated from derepressed wild type K. pneumoniae, having a similar specific activity, approximately 4 Fe/dimer of 68 kDa, and similar epr features. Although these experiments do not exclude the participation of other E. coli gene products in the maturation of nifH protein, they limit the nif-specific genes required for active Fe protein production to nifA, nifH, and nifM. Since nifA is thought to be the required activator protein involved in nif operon transcription, the simplest explanation for these observations is that nifH codes for the peptide of the Fe protein, while nifM acts to convert this nifH peptide to the functioning Fe protein of nitrogenase. In the absence of nifM, only an inactive nifH polypeptide is produced.

The difference between the magnetic susceptibilities of native and thionine-oxidized molybdenum-iron protein from Azotobacter vinelandii nitrogenase was measured by the nuclear magnetic resonance method. Reversible oxidation of the MoFe protein by 4 to 8 electron eq of thionine/mol made the protein more paramagnetic than it was in the native state. The NMR susceptibility results were analyzed in terms of a model for the spin states of the iron centers in the MoFe protein based on low temperature electron paramagnetic resonance and Mössbauer spectral studies. The model proposes that the native protein contains 2 "M" centers (S = 3/2) and 4 "P" centers (S = 0)/mol and that the oxidized protein has diamagnetic M centers and paramagnetic P centers with S greater than or equal to 3/2. Assuming that this model holds at 280 K, the NMR susceptibility results show that the effective magnetic moment of the oxidized P centers is larger than that of the native M centers. Based on an analysis in terms of spin only magnetic moments, the susceptibility results suggest that the P centers in the oxidized protein are S = 5/2 systems.

The genes mvhDGA, which encode the subunit polypeptides of the methyl viologen-reducing hydrogenase in Methanobacterium thermoautotrophicum strain delta H, have been cloned and sequenced. These genes, together with a fourth open reading frame designated mvhB, are tightly linked and appear to form an operon that is transcribed starting 42 base pairs upstream of mvhD. The organization and sequences of the mvhG and mvhA genes indicate a common evolutionary ancestry with genes encoding the small and large subunits of hydrogenases in eubacterial species. The product of the mvhB gene is predicted to contain six tandomly repeated bacterial-ferredoxin-like domains and, therefore, is predicted to be a polyferredoxin that could contain as many as 48 iron atoms in 12 Fe4S4 clusters.

Two techniques have been applied to the determination of the number and type (2-Fe, 4-Fe) of iron-sulfur centers in the iron-sulfur flavoprotein succinate dehydrogenase [succinate:(acceptor) oxidoreductase, EC 1.3.99.1]. One procedure uses p-CF3C6H4SH as an extrusion reagent and Fourier transform 19F nuclear magentic resonance as the method of detection and quantitation of extruded cores of these centers in the form of [Fe2S2(SRF)4]2- and [Fe4S4(SRF)4]2- (RF = p-C6H4CF3). The second procedure, interprotein core transfer, involves thiol displacement of iron-sulfur cores followed by specific core transfer to the apoproteins of Bacillus polymyxa ferredoxin and adrenodoxin. Detection and quantitation are accomplished by electron paramagnetic resonance of reduced proteins at low temperatures. Both procedures clearly show that succinate dehydrogenase contains two dimeric (Fe2S2) and one tetrameric (Fe4S4) centers per mole of histidyl flavin, accounting for all eight nonheme iron and eight labile sulfur atoms found by chemical analysis. These results remove uncertainties created by the less than stoichiometric amounts of binuclear centers detected by electron paramagnetic resonance after dithionite reduction and provide secure characterization of the iron-sulfur centers in this enzyme.

Recently Hagen et al. (Hagen, W. R., Wassink, H., Eady, R. R., Smith, B. E., and Haaker, H. (1987) Eur. J. Biochem. 169, 457-465) reported the observation of S = 7/2 EPR signals for thionin-oxidized nitrogenase MoFe protein. Here we have studied the protein from Azotobacter vinelandii and Klebsiella pneumoniae with Mössbauer and EPR spectroscopies, with the following results: when the MoFe protein is oxidized by addition of stoichiometric amounts (6-8 equivalents) of dissolved thionin, the well characterized P-cluster state Pox results. Pox has an as yet undetermined, but half-integer electronic spin; however, the state is EPR-silent. In contrast, oxidation by addition of a large excess of solid thionin powder, the method used by Hagen et al., yields mixtures with variable proportions of two oxidized P-cluster forms, namely the familiar Pox and the new state Pox(S = 7/2) observed by Hagen et al. The Mössbauer data suggest that Pox and Pox(S = 7/2) are isoelectronic. The two states, however, have distinct electronic structures; the Mössbauer spectra of Pox exhibit the characteristic trapped-valence Fe2+ site, whereas the spectra of Pox(S = 7/2) lack this feature. Hagen et al. have proposed two new P-cluster models. We conclude that one of the models is incompatible with the Mössbauer data and that the basic assumptions of the other model are not supported by the available data. Finally, the Mössbauer data show that either oxidation method puts the cofactor centers into the diamagnetic state Mox.

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.

The sulfur content of N-methylformamide solutions of cofactor from Clostridium pasteurianum nitrogenase has been determined to be 11.9 (+/- 0.9) mol per mol of molybdenum. This number was determined radiochemically, using iron-molybdenum cofactor isolated from molybdenum-iron protein from bacteria grown on 35SO4. A total of 3.2 (+/- 0.2) mol of sulfur per mol of molybdenum was found to be present in cysteine and methionine, probably arising from contaminating proteins not intrinsic to the cofactor. Combined with accumulated evidence that is discussed, these results lead to an updated stoichiometry of MoFe6S8 or 9, not MoFe6S4 as previously thought, for this cluster.

We have recently shown (Lindahl, P. A., Day, E. P., Kent, T. A., Orme-Johnson, W. H., and Münck, E. (1985) J. Biol. Chem. 260, 11160-11173) that the [4Fe-4S]1+ cluster of the native Fe protein can exist in two forms characterized by different cluster spin: an S = 1/2 state exhibiting a g = 1.94 type EPR signal and an S = 3/2 state yielding signals at g approximately 5.8 and 5.1. We have now extended our study of the Fe protein to include the MgATP- and MgADP-bound forms. The [4Fe-4S]1+ cluster of the nucleotide-bound Fe protein exists in a similar S = 1/2, S = 3/2 spin mixture. The S = 3/2 cluster exhibits a broad EPR signal at g approximately 4.8. In spectra of the MgATP-bound protein, we have also observed a g = 4.3 signal from an S = 5/2 state (D = 1 − 3 cm−1, E/D approximately 0.32). Various experiments strongly suggest that this signal does not originate from adventitiously bound Fe3+. The g = 4.3 signal may arise from approximately 2% of the [4Fe-4S]1+ clusters when MgATP is protein-bound. We have also discovered substoichiometric amounts of what appears to be ADP in some nominally nucleotide-free Fe protein samples. MgATP binds to Fe protein in the presence of perturbing solvents, resulting in EPR spectra similar to those of MgATP-bound samples in aqueous solutions; MgADP binding, on the other hand, results in signals more typical of the solvent state. Spectra of samples frozen during turnover of the nitrogenase system exhibit a mixture of spin states. Characterization of the Fe protein EPR signature described here should aid future mechanistic and nucleotide-binding studies.

Previous Mössbauer and EPR studies of the MoFe protein (approximately 30 Fe and 2 Mo) of nitrogenase have revealed the presence of two unique clusters, namely, the P-clusters (presumably of the Fe4S4 type) and the molybdenum- and iron-containing cofactors (or M-clusters). Mössbauer components D (approximately 10-12 Fe) and Fe2+ (approximately 4 Fe) represent subsites of the P-clusters while component S (approximately 2 Fe) appeared to belong to a separate, unidentified cluster. In order to refine the analyses of Mössbauer spectra, we have constructed an isotopic hybrid of the Klebsiella pneumoniae protein which contains 57Fe-enriched P-clusters and 56Fe-enriched M-clusters. The highly resolved 57Fe Mössbauer spectra of this hybrid show that component S behaves spectroscopically like the P-cluster sites D and Fe2+ in oxidized and reduced MoFe protein. This suggests that S is a subset of the P-clusters rather than a different cluster type. The present study shows, for the first time, that the Debye-Waller factors of different P-cluster subsites have a different temperature dependence. Thus, the Fe2+/D absorption ratio is 4.0:10.0 at 4.2 K and 4.0:11.6 at 173 K. We propose that the reduced MoFe protein contains two pairs of P-clusters: one pair containing one Fe2+ and three D-sites and the other one Fe2+, two D, and one S-site. We have argued previously that the oxidized P-clusters occur in pairs as well.

We have studied the Fe protein (Av2) of the Azotobacter vinelandii nitrogenase system with Mössbauer and EPR spectroscopies and magnetic susceptometry. In the oxidized state the protein exhibits Mössbauer spectra typical of diamagnetic [4Fe-4S]2+ clusters. Addition of Mg.ATP or Mg.ADP causes a pronounced decline in the quadrupole splitting of the Mössbauer spectra of the oxidized protein. Our studies show that reduced Av2 in the native state is heterogeneous. Approximately half of the molecules contain a [4Fe-4S]1+ cluster with electronic spin S = 1/2 and half contain a [4Fe-4S]1+ cluster with spin S = 3/2. The former yields the characteristic g = 1.94 EPR signal whereas the latter exhibits signals around g = 5. The magnetization of reduced Av2 is dominated by the spin S = 3/2 form of its [4Fe-4S]1+ clusters. These results explain a long standing puzzle, namely why the integrated spin intensity of the g = 1.94 EPR signal is substantially less than 1 spin/4 Fe atoms. In 50% ethylene glycol, 90% of the clusters are in the spin S = 1/2 form whereas, in 0.4 M urea, 85% are in the S = 3/2 form. In 0.4 M urea, the EPR spectrum of reduced Av2 exhibits well defined resonances at g = 5.8 and 5.15, which we assign to the S = 3/2 system. The EPR and Mössbauer studies yield a zero-field splitting of 2D approximately equal to -5 cm-1 for this S = 3/2 state.

Escherichia coli NADPH-sulfite reductase can be dissociated into an oligomeric flavoprotein and a monomeric hemoprotein (HP) subunit in 4 M urea. HP catalyzes stoichiometric 6-electron reductions of SO32- (to S2-) and of NO2-, as well as 2-electron reduction of NH2OH, with reduced methyl viologen (MV+) as reductant. While Vmax values are highest with the nitrogenous substrates, Km for SO32- is 2 to 3 orders of magnitude less than the Km for NO2- or NH2OH. EPR spectroscopic and chemical analyses show that HP contains one siroheme and one Fe4S4 center per polypeptide. The heme is in the high spin Fe3+ state in HP as isolated. Near-quantitative reduction of the Fe4S4 center to a state yielding a g = 1.94 type of EPR spectrum by S2O42- and/or MV+ could be achieved if HP was converted to either the CN- or CO complex or treated with 80% dimethyl sulfoxide. HP binds one SO32- or CN- per peptide. Binding of these ligands, as well as CO, appears to be mutually exclusive and to involve the heme. The heme Fe3+/Fe2+ potential is shifted from -340 mV in the free HP to -155 mV in the HP-CN- complex. The potential of the Fe4S4 center is approximately 70 mV more negative in the CN- as opposed to the CO-ligated HP (-420 mV), a result which indicates the presence of heme-Fe4S4-ligand interaction in the HP complexes.

Methanogens catalyze the hydrogen-dependent eight-electron reduction of carbon dioxide to methane. Two of the key catalysts in the eight-electron reduction pathway are the nickel-containing enzymes F420-reducing hydrogenase and methyl reductase. In the present study, the structures of these archaebacterial enzymes from Methanobacterium thermoautotrophicum delta H have been determined by electron microscopy. By negative stain techniques, F420 hydrogenase was found to be a ring structure with a diameter of 15.7 nm and an inner channel 4 nm in diameter. Shadow-casting experiments demonstrated that the rings were 8.5 nm deep, indicating a holoenzyme molecular weight of 8.0 X 10(5). Methyl reductase appeared to be an oligomeric complex of dimensions 8.5 by 9 by 11 nm, with a central stain-penetrating region. The morphology and known subunit composition suggest a model in which the subunits are arranged as an eclipsed pair of open trimers. Methyl reductase was also found in the form of larger aggregates and in paracrystalline arrays derived from highly concentrated solutions. The extremely large size of F420 hydrogenase and the methyl reductase supramolecular assemblies may have relevance in vivo in the construction of multiprotein arrays that function in methane biogenesis.

The chemical characterization, EPR properties, and mechanism of pyruvate:flavodoxin (ferredoxin) oxidoreductase from Klebsiella pneumoniae and Clostridium thermoaceticum have been investigated. A simple, specific, and sensitive assay and an efficient purification (based on the high affinity of these enzymes for a dye attached to agarose) are reported. The observed iron content of 8 atoms/subunit is twice that reported by others, whereas the contents of lipoate and flavin are less than 0.1 mol/subunit, in agreement with previous reports. Spectroscopic evidence suggests that the iron is present in Fe4S4(2+,1+) clusters. Reduction of the enzyme requires the presence of CoA as well as 1.1 pyruvate/subunit, which is very nearly the theoretical amount required the reduce two Fe4S(2+,1+) clusters. In the absence of CoA, stoichiometric amounts of pyruvate are decarboxylated, but the Fe/S centers are not reduced. We conclude that the K. pneumoniae and C. thermoaceticum enzymes are adapted to rapid reduction of low potential 1-e- carriers, similar to the pyruvate oxidoreductase of Halobacterium (Kerscher, L., and Oesterhelt, D. (1977) FEBS Lett. 83, 197-201), but different in that an Fe/S center-radical pair is used in the latter enzyme in place of the pair of Fe4S4 centers we find. The K. pneumoniae and C. thermoaceticum oxidoreductases appear to be mechanistically closely related to the Clostridium acidiurici enzyme (Uyeda, K., and Rabinowitz, J. C. (1971) J. Biol. Chem. 246, 3111-3119), differing as a class from the lipoate-containing, pyridine nucleotide-reducing enzyme present in aerobes (Reed, L. J. (1974) Accts. Chem. Res. 2, 740-746). The function of the Klebsiella enzyme is to supply electrons to nitrogenase. This is accomplished in vitro with purified components via a nif-specific flavodoxin or other low potential 1-e- carriers such as viologen dyes or ferredoxins. The in vivo molar ratio of nitrogenase to the physiological reduction system, estimated from activity measurements of individual components in crude extracts, was 0.4:0.03:2:1 pyruvate oxidoreductase:flavodoxin:nitrogenase component II:nitrogenase component I.

The production of deuterated methane by Methanobacterium thermoautotrophicum in H2O-D2O mixtures was examined by high-resolution mass spectrometry. The hydrogen in the methane arose solely from water and not from hydrogen gas. Hydrogen gas served only as an electron source in methanogenesis. A whole-cell product isotope discrimination of 1.5 favoring hydrogen over deuterium was observed in methane production in 81 atom% deuterated water. The distribution of deuterated methane species is described by a simple model of the overall reaction.

The crystal structure of Clostridium acidi-urici ferredoxin has been determined using multiple wavelength anomalous diffraction (MAD) techniques at 5.0-A resolution. The electron density map shows striking similarity to a map of Peptococcus aerogenes ferredoxin computed at the same resolution from the atomic coordinates reported by Adman et al. (Adman, E. T., Sieker, L. C., and Jensen, L. H. (1973) J. Biol. Chem. 248, 3987-3996). Such similarity is expected from the high degree of identity between amino acid sequences of the two proteins. The use of MAD methods has in the relatively recent past become a practical possibility due to instrumental advances enabling the collection of accurate data at several wavelengths at synchrotrons and due to theoretical and computational advances that facilitate the analysis of these data for the determination of phases. These methods hold great promise as an alternative to the multiple isomorphous replacement method in macromolecular structure determination. The present report represents one of the first applications of the MAD techniques to the determination of the structure of a protein which was previously unknown in detail.

The electron paramagnetic resonance signals of the soluble P-450 cytochrome from Pseudomonas putidawere observed at temperatures from 4.2 to 80°K. As isolated, P-450 has a signal typical of a low spin ferric-heme compound with sulfur as one of the axial ligands (g= 2.45, 2.26, 1.915). We also detected a minor signal typical of high spin ferric heme (g= 8, 4, 1.8) equivalent to less than 7% of the heme at temperatures below 20°K. On titration with the substrate, (+)-camphor, the low spin signal decreased and the high spin signal increased, maximally representing about 60% of the heme. For reasons not thus far understood, 40% of the heme is not converted to high spin by either (+) or (-)-camphor. The high spin signal has a rhombic character which is stronger than any previously observed with a heme compound (E= 0.33 cm-1; D= 3.8 cm-1; E/D= 0.087).We conclude that P-450camas isolated is equal to or more than 95% in a low spin form probably having sulfur as one of the axial ligands. The binding of substrate displaces this ligand sufficiently to allow for conversion from a low to a high spin form.

The electron paramagnetic resonance of nitrogenase components, separately and together with the other reactants in the nitrogenase system (namely, reductant and Mg·ATP), have been examined at low temperatures (<20°K). The MoFe protein, component I or molybdoferredoxin, in the oxidized (but not oxygen-inactivated) state yields signals with g-values of 4.3, 3.7, and 2.01, and when reduced has no observable electron paramagnetic resonance. The Fe protein, component II, or azoferredoxin, yields a signal with g-values of 2.05, 1.94, and 1.89 in the reduced state that is converted by Mg·ATP into an axial signal with g-values near 2.05 and 1.94, and a second split signal near g = 4.3. The Fe protein has no definite electron paramagnetic resonance in the oxidized (not oxygen-denatured) state under these conditions. The Mg·ATP complex of reduced Fe protein reduces the MoFe protein, whereas dithionite alone does not reduce the MoFe protein. Reoxidation of the system by substrate leads to disappearance of the Fe protein signal and the reappearance of the MoFe protein signal. Thus Mg·ATP, which is hydrolyzed during substrate reduction, converts the Fe protein to a reductant capable of transferring electrons to MoFe protein, after which substrate reduction occurs.