The polysulphide reductase (formerly sulphur reductase) of Wolinella succinogenes is a component of the phosphorylative electron transport system with polysulphid as the terminal acceptor. Using an antiserum raised against the major subunit (PsrA, 85 kDa) of the enzyme, the corresponding gene (psr A) was cloned from a λ‐gene bank. The N‐terminal amino acid sequence of PsrA mapped within the psr A gene product, which also contained an apparent signal peptide. Downstream of the Psr A gene two more open reading frames (psrB and psrC) were found. the three genes may form a transcriptional unit with the transcription start site in front of psr A. The three genes were present only once on the genome. PsrA is a hydrophilic protein homologous to the largest subunits of six prokaryotic molybdoenzymes. PsrB is predicted to be hydrophilic, to contain ferredoxin‐like cysteine clusters and to be homologous to the smaller hydrophilic subunits of four molybdoenzymes. PsrC is predicted to be a hydrophobic protein that could possibly serve as the membrane anchor of the enzyme.

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The genes coding for methyl coenzyme M reductase were cloned from a genomic library ofMethanobacterium thermoautotrophicum Marburg into Escherichia coli by using plasmid expression vectors. When introduced into E. coli, the reductase genes were expressed, yielding polypeptides identical in size to the three known subunits of the isolated enzyme, a, 13, and y. The polypeptides also reacted with the antibodies raised against the respective enzyme subunits. In M. thermoautotrophicum, the subunits are encoded by a gene cluster whose transcript boundaries were mapped. Sequence analysis revealed two more open reading frames of unknown function located between two of the methyl coenzyme M reductase genes.Methanogenic bacteria are archaebacteria, which gain their energy by anaerobic formation of methane (6). Independent of the methanogenic substrate, the final step of methanogenesis is the reduction of methyl coenzyme M (CoM) to methane and CoM. This reaction, which is coupled to the synthesis of ATP in vivo (7,20), is catalyzed by the methyl CoM reductase (MCR) (3,12). This enzyme amounts to about 10% of the total protein (3, 12) and has been isolated from a large number of methanogenic bacteria (3,18,21 Q359 (19) hsdR supE tonA (P2) Y1089 (42) AlacUl69 Alon araDI39 strA hflA150 chr::TnlO (pMC9) Y1090 (42) A1acUl69 Alon araD139 strA supF trpC22::TnlO (pMC9) BNN97 (41) hsdR supE thr leu thi lacYI tonA21 (Xgtll) pUC8 (26, 39) XEMBL4 (13) Agtll (37) previously (23), using formaldehyde gels. Transfer of the DNA to GeneScreen sheets essentially followed the procedure described by Thomas (37) with the modifications given in the GeneScreen manual by the supplier. The DNA probe was labeled by nick translation as described in reference 24, using a-32P-labeled deoxynucleoside triphosphates to a specific activity of 0.5 x 108 to 1 x 108 cpm/,Lg of DNA. Hybridization and further processing of the filters were performed as described in reference 15.Antisera against MCR subunits. Antisera were raised in rabbits against the subunits of purified MCR, which were eluted from an SDS-polyacrylamide gel after their electrophoretic separation. The specificities were checked in Western blot (immunoblot) experiments, using total extracts of E. coli or M. thermoautotrophicum cells or purified MCR. No reactions were observed with E. coli extracts. The antisera were found to react with one polypeptide band each in the M. thermoautotrophicum extract, which corresponded in size to the respective subunit reacting with the same antiserum.Screening of M. thermoautotrophicum genomic libraries. Two DNA libraries were constructed. First, an EcoRI total digest of M. thermoautotrophicum DNA was ligated to Agtll DNA, which was digested with EcoRI and treated with calf intestinal phosphatase (41). After in vitro packaging (42), bacteriophages were plated on E. coli Y1090 and screened with antisera against the isolated subunits after induction with isopropyl-1-D-thiogalactopyranoside as described below. Positive phages were lysogenized in E. coli Y1089 an...

Methyl-coenzyme-M reductase from Methanobacterium thermoautotrophicum (strain Marburg) was purified to a stage where, besides the a, /j' and y subunits, no additional polypeptides were detectable in the preparation. Under appropriate conditions the enzyme was found to catalyze the reduction of methyl-CoM with 7 -mercaptoheptanoylthreonine phosphate (H-S-HTP) to CH4 at a specific rate of 2.5 pmol . min-' . mg protein-'. This finding contradicts a recent report that methyl-CoM reductase is only active when some contaminating proteins are present.The two polypeptides encoded by the open reading frames ORF, and ORF, of the methyl-CoM reductase transcription unit did not co-purify with the a, /I and y subunits. They were neither required nor did they stimulate the activity under the assay conditions. 3-Bromopropanesulfonate (apparent Ki = 0.05 pM) and 2-azidoethanesulfonate (apparent Ki = 1 pM) were found to be two new competitive inhibitors of methyl-CoM reductase. Both inhibitors were considerably more effective than the "classical" 2-bromoethanesulfonate (apparent Ki = 4 pM).Methane is formed in methanogenic bacteria from methylcoenzyme M by reduction with 7-mercaptoheptanoylthreonine phosphate [ I , 21: CH3-S-CoM + H-S-HTP +CH4

The formate dehydrogenase of Wolinella succinogenes is a membraneous molybdo-enzyme which is involved in phosphorylative electron transport. The gene (fdhA) encoding the largest subunit was isolated from a gene bank by immunological screening. The fdhA gene was located in an apparent transcriptional unit (fdhA,B,C,D) together with three more structural genes. The N-terminal sequences of three polypeptides present in the isolated enzyme were found to map within the fdhA, B and C structural genes. A polypeptide corresponding to fdhD was not detected in the enzyme preparation. This suggested that the functional formate dehydrogenase was made up of three or four different subunits. The genes fdhA and C encode larger preproteins which differ from the corresponding mature proteins by N-terminal signal peptides. The N-terminal half of the mature FdhA is homologous to the larger subunits of the formate dehydrogenases of E. coli (formate-hydrogenlyase linked) and Methanobacterium formicicum as well as to three bacterial reductases containing molybdenum. It harbours a conserved cysteine cluster and two more domains which may be involved in binding the molybdenum cofactor. FdhB may represent an iron-sulphur protein, twelve cysteine residues of which are arranged in two clusters which are typical of ligands of the iron-sulfur centers in ferredoxins. FdhC is a hydrophobic protein with four predicted transmembrane segments, which appears to be identical with the cytochrome b present in the isolated formate dehydrogenase. It may form the membrane anchor of the enzyme and react with the bacterial menaquinone.