Publications

• The heterodisulfide reductase from Methanobacterium thermoautotrophicum contains sequence motifs characteristic of pyridine-nucleotide-dependent thioredoxin reductases.

  Hedderich R., Koch J., Linder D., Thauer R.K.

  The genes hdrA, hdrB and hdrC, encoding the three subunits of the iron-sulfur flavoprotein heterodisulfide reductase, have been cloned and sequenced. HdrA (72.19 kDa) was found to contain a region of amino acid sequence highly similar to the FAD-binding domain of pyridine-nucleotide-dependent disu ... >> More

  The genes hdrA, hdrB and hdrC, encoding the three subunits of the iron-sulfur flavoprotein heterodisulfide reductase, have been cloned and sequenced. HdrA (72.19 kDa) was found to contain a region of amino acid sequence highly similar to the FAD-binding domain of pyridine-nucleotide-dependent disulfide oxidoreductases. Additionally, 110 amino acids C-terminal to the FAD-binding consensus, a short polypeptide stretch (VX2CATID) was detected which shows similarity to the region of thioredoxine reductase that contains the active-site cysteine residues (VX2CATCD). These findings suggest that HdrA harbors the site of heterodisulfide reduction and that the catalytic mechanism of the enzyme is similar to that of pyridine-nucleotide-dependent thioredoxin reductase. HdrA was additionally found to contain four copies of the sequence motif CX2CX2CX3C(P), indicating the presence of four [4Fe-4S] clusters. Two such sequence motifs were also present in HdrC (21.76 kDa), the N-terminal amino acid sequence of which showed sequence similarity to the gamma-subunit of the anaerobic glycerol-3-phosphate dehydrogenase of Escherichia coli. HdrC is therefore considered to be an electron carrier protein that contains two [4Fe-4S] clusters. HdrB (33.46 kDa) did not show sequence similarity to other known proteins, but appears to possess a C-terminal hydrophobic alpha-helix that might function as a membrane anchor. Although hdrB and hdrC are juxtaposed, these genes are not near hdrA. << Less

  Eur. J. Biochem. 225:253-261(1994) [PubMed] [EuropePMC]

• Physiological role of the F420-non-reducing hydrogenase (Mvh) from Methanothermobacter marburgensis.

  Stojanowic A., Mander G.J., Duin E.C., Hedderich R.

  F(420)-non-reducing hydrogenase (Mvh) from Methanothermobacter marburgensis is a [NiFe] hydrogenase composed of the three subunits MvhA, MvhG, and MvhD. Subunits MvhA and MvhG form the basic hydrogenase module conserved in all [NiFe] hydrogenases, whereas the 17-kDa MvhD subunit is unique to Mvh. ... >> More

  F(420)-non-reducing hydrogenase (Mvh) from Methanothermobacter marburgensis is a [NiFe] hydrogenase composed of the three subunits MvhA, MvhG, and MvhD. Subunits MvhA and MvhG form the basic hydrogenase module conserved in all [NiFe] hydrogenases, whereas the 17-kDa MvhD subunit is unique to Mvh. The function of this extra subunit is completely unknown. In this work, the physiological function of this hydrogenase, and in particular the role of the MvhD subunit, is addressed. In cells of Mt. marburgensis from Ni(2+)-limited chemostat cultures the amount of Mvh decreased about 70-fold. However, the amounts of mvh transcripts did not decrease in these cells as shown by competitive RT-PCR, arguing against a regulation at the level of transcription. In cells grown in the presence of non-limiting amounts of Ni(2+), Mvh was found in two chromatographically distinct forms-a free form and in a complex with heterodisulfide reductase. In cells from Ni(2+)-limited chemostat cultures, Mvh was only found in a complex with heterodisulfide reductase. The EPR spectrum of the purified enzyme reduced with sodium dithionite was dominated by a signal with g(zyx)=2.006, 1.936 and 1.912. The signal could be observed at temperatures up to 80 K without broadening, indicative of a [2Fe-2S] cluster. Subunit MvhD contains five cysteine residues that are conserved in MvhD homologues of other organisms. Four of these conserved cysteine residues can be assumed to coordinate the [2Fe-2S] cluster that was detected by EPR spectroscopy. The MvhG subunit contains 12 cysteine residues, which are known to ligate three [4Fe-4S] clusters. Data base searches revealed that in some organisms, including the Methanosarcina species and Archaeoglobus fulgidus, a homologue of mvhD is fused to the 3' end of an hdrA homologue, which encodes a subunit of heterodisulfide reductase. These data allow the conclusion that the only function of Mvh is to provide reducing equivalents for heterodisulfide reductase and that the MvhD subunit is an electron transfer protein that forms the contact site to heterodisulfide reductase. << Less

  Arch. Microbiol. 180:194-203(2003) [PubMed] [EuropePMC]

• A hydrogenase-linked gene in Methanobacterium thermoautotrophicum strain delta H encodes a polyferredoxin.

  Reeve J.N., Beckler G.S., Cram D.S., Hamilton P.T., Brown J.W., Krzycki J.A., Kolodziej A.F., Alex L., Orme-Johnson W.H., Walsh C.T.

  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 ... >> More

  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. << Less

  Proc. Natl. Acad. Sci. U.S.A. 86:3031-3035(1989) [PubMed] [EuropePMC]

• VhuD facilitates electron flow from H2 or formate to heterodisulfide reductase in Methanococcus maripaludis.

  Costa K.C., Lie T.J., Xia Q., Leigh J.A.

  Flavin-based electron bifurcation has recently been characterized as an essential energy conservation mechanism that is utilized by hydrogenotrophic methanogenic Archaea to generate low-potential electrons in an ATP-independent manner. Electron bifurcation likely takes place at the flavin associat ... >> More

  Flavin-based electron bifurcation has recently been characterized as an essential energy conservation mechanism that is utilized by hydrogenotrophic methanogenic Archaea to generate low-potential electrons in an ATP-independent manner. Electron bifurcation likely takes place at the flavin associated with the α subunit of heterodisulfide reductase (HdrA). In Methanococcus maripaludis the electrons for this reaction come from either formate or H2 via formate dehydrogenase (Fdh) or Hdr-associated hydrogenase (Vhu). However, how these enzymes bind to HdrA to deliver electrons is unknown. Here, we present evidence that the δ subunit of hydrogenase (VhuD) is central to the interaction of both enzymes with HdrA. When M. maripaludis is grown under conditions where both Fdh and Vhu are expressed, these enzymes compete for binding to VhuD, which in turn binds to HdrA. Under these conditions, both enzymes are fully functional and are bound to VhuD in substoichiometric quantities. We also show that Fdh copurifies specifically with VhuD in the absence of other hydrogenase subunits. Surprisingly, in the absence of Vhu, growth on hydrogen still occurs; we show that this involves F420-reducing hydrogenase. The data presented here represent an initial characterization of specific protein interactions centered on Hdr in a hydrogenotrophic methanogen that utilizes multiple electron donors for growth. << Less

  J. Bacteriol. 195:5160-5165(2013) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.

• H2: heterodisulfide oxidoreductase complex from Methanobacterium thermoautotrophicum. Composition and properties.

  Setzke E., Hedderich R., Heiden S., Thauer R.K.

  The reduction of the heterodisulfide (CoM-S-S-HTP) of coenzyme M (H-S-CoM) and N-7-mercaptoheptanoylthreonine phosphate (H-S-HTP) with H2 is an energy-conserving step in most methanogenic Archaea. In this study, we show that in Methanobacterium thermoautotrophicum (strain Marburg) this reaction is ... >> More

  The reduction of the heterodisulfide (CoM-S-S-HTP) of coenzyme M (H-S-CoM) and N-7-mercaptoheptanoylthreonine phosphate (H-S-HTP) with H2 is an energy-conserving step in most methanogenic Archaea. In this study, we show that in Methanobacterium thermoautotrophicum (strain Marburg) this reaction is catalyzed by a stable H2-heterodisulfide oxidoreductase complex of F420-non-reducing hydrogenase and heterodisulfide reductase. This complex, which was loosely associated with the cytoplasmic membrane, was purified 17-fold with 80% yield to apparent homogeneity. The purified complex was composed of six different subunits of apparent molecular masses 80, 51, 41, 36, 21 and 17 kDa, and 1 mol complex, with apparent molecular mass 250 kDa, contained approximately 0.6 mol nickel, 0.9 mol FAD, 26 mol non-heme iron and 22 mol acid-labile sulfur. In 25 mM Chaps, the complex partially dissociated into two subcomplexes. The first subcomplex was was composed of the 51-, 41- and 17-kDa subunits; 1 mol trimer contained 0.7 mol nickel, 10 mol non-heme iron and 9 mol acid-labile sulfur and exhibited F420-non-reducing hydrogenase activity. The other subcomplex was composed of the 80-, 36- and 21-kDa subunits; 1 mol trimer contained 0.8 mol FAD, 22 mol non-heme iron and 15 mol acid-labile sulfur and exhibited heterodi-sulfide-reductase activity. The stimulatory effects of potassium phosphate, a membrane component, uracil derivatives and coenzyme F430 on the H2:heterodisulfide-oxidoreductase activity of the purified complex are described. << Less

  Eur. J. Biochem. 220:139-148(1994) [PubMed] [EuropePMC]

• Coupling of ferredoxin and heterodisulfide reduction via electron bifurcation in hydrogenotrophic methanogenic archaea.

  Kaster A.K., Moll J., Parey K., Thauer R.K.

  In methanogenic archaea growing on H(2) and CO(2) the first step in methanogenesis is the ferredoxin-dependent endergonic reduction of CO(2) with H(2) to formylmethanofuran and the last step is the exergonic reduction of the heterodisulfide CoM-S-S-CoB with H(2) to coenzyme M (CoM-SH) and coenzyme ... >> More

  In methanogenic archaea growing on H(2) and CO(2) the first step in methanogenesis is the ferredoxin-dependent endergonic reduction of CO(2) with H(2) to formylmethanofuran and the last step is the exergonic reduction of the heterodisulfide CoM-S-S-CoB with H(2) to coenzyme M (CoM-SH) and coenzyme B (CoB-SH). We recently proposed that in hydrogenotrophic methanogens the two reactions are energetically coupled via the cytoplasmic MvhADG/HdrABC complex. It is reported here that the purified complex from Methanothermobacter marburgensis catalyzes the CoM-S-S-CoB-dependent reduction of ferredoxin with H(2). Per mole CoM-S-S-CoB added, 1 mol of ferredoxin (Fd) was reduced, indicating an electron bifurcation coupling mechanism: 2H(2) + Fd(OX) + CoM-S-S-CoB-->Fd(red)(2-) + CoM-SH + CoB-SH + 2H(+). This stoichiometry of coupling is consistent with an ATP gain per mole methane from 4 H(2) and CO(2) of near 0.5 deduced from an H(2)-threshold concentration of 8 Pa and a growth yield of up to 3 g/mol methane. << Less

  Proc. Natl. Acad. Sci. U.S.A. 108:2981-2986(2011) [PubMed] [EuropePMC]