Methyl‐coenzyme M reductase (= component C) from Methanobacterium thermoautotrophicum (strain Marburg) was highly purified via anaerobic fast protein liquid chromatography on columns of Mono Q and Superose 6. The enzyme was found to catalyze the reduction of methylcoenzyme M (CH3‐S‐CoM) with N‐7‐mercaptoheptanoylthreonine phosphate (H‐S‐HTP = component B) to CH4. The mixed disulfide of H‐S‐CoM and H‐S‐HTP (CoM‐S‐S‐HTP) was the other major product formed. The specific activity was up to 75 nmol min−1 mg protein−1. In the presence of dithiothreitol and of reduced corrinoids or titanium(III) citrate the specific rate of CH3‐S‐CoM reduction to CH4 with H‐S‐HTP increased to 0.5–2 μmol min−1 mg protein−1. Under these conditions the CoM‐S‐S‐HTP formed from CH3‐S‐CoM and H‐S‐HTP was completely reduced to H‐S‐CoM and H‐S‐HTP. Methyl‐CoM reductase was specific for H‐S‐HTP as electron donor. Neither N‐6‐mercaptohexanoylthreonine phosphate (H‐S‐HxoTP) nor N‐8‐mercaptooctanoylthreonine phosphate (H‐S‐OcoTP) nor any other thiol compound could substitute for H‐S‐HTP. On the contrary, H‐S‐HxoTP (apparent Ki=0.1 μM) and H‐S‐OcoTP (apparent Ki= 15 μM) were found to be effectives inhibitors of methyl‐CoM reductase, inhibition being non‐competitive with CH3‐S‐CoM and competitive with H‐S‐HTP.

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Methyl-coenzyme M reductase (MCR) catalyzes the methane-forming step in methanogenic archaebacteria. The reductase has been characterized in detail from Methanobacterium thermoautotrophicum strain Marburg and AH, which grow on H2 and C 0 2 as energy source. During purification of the enzyme we have now discovered a second methyl-coenzyme M reductase (MCR 11) in the two strains, which elutes at lower salt concentration from anion-exchange columns than the enzyme (MCR I) previously characterized. MCR I1 is similar to MCR I in that it is also composed of three different subunits a, p, and y but distinct from MCR I in that the y subunit is 5 kDa smaller, as revealed by sodium dodecyl sulfate/polyacrylamide gel electrophoresis. The N-terminal amino acid sequences of the a, /l, and y subunits of MCR I1 and MCR I were found to be different in several amino acid positions. The respective sequences showed, however, strong similarities indicating that MCR I1 was not derived from MCR I by limited proteolysis. The relative amounts of MCR I and MCR I1 present in the cells were affected by the growth conditions. When the cultures were supplied with sufficient H2 and C 0 2 and the cells grew exponentially, essentially only MCR I1 was found. When growth was limited by the gas supply, MCR I predominated.Methyl-coenzyme M reductase (MCR) catalyzes the reduction of methyl-coenzyme M (CH3-S-CoM) with 7-mercaptoheptanoylthreonine phosphate (H-S-HTP) to methane and the heterodisulfide CoM-S-S-HTP [l -31. CH3-S-CoM + H-S-HTP -+ CH4 + COM-S-S-HTPAGO' = -45 kJ/mol 141 This reaction is the methane-yielding step in the energy metabolism of all methanogenic archaebacteria. It is probably also the rate-limiting step in methanogenesis. The specific activity of methyl-coenzyme M reductase determined in cell extracts was always much lower than that of all the other catabolic enzymes [5].Methyl-coenzyme M reductase is a yellow enzyme with a molecular mass of approximately 300 kDa [6]. It is composed of three different subunits with apparent molecular masses of 65 & 3 kDa, 46 2 kDa, and 35 & 5 kDa, respectively, in an azpzyz arrangement [7, 81 and contains tightly, but not covalently, bound two molecules coenzyme F430 as chromophoric prosthetic group [9]. Coenzyme F430 is a yellow nickel porphinoid of unique structure and properties [lo-121. The molecular mass of the a and p subunits of methylcoenzyme M reductase from different organisms is relatively constant whereas that of the y subunit varies between 30 kDa and 40 kDa. The size of the y subunit has been used as a taxonomic marker [7].We report here on the presence of two genetically distinct methyl-coenzyme M reductases designated MCR I and MCR I1 in Methanobacterium thermoautotrophicum. MCR I is the enzyme previously characterized [l, 31, the genes of which have been cloned and sequenced [16] as evidenced by a comparison of the DNA sequence with the N-terminal amino acid sequences. MCR I1 is the newly found enzyme, which differs from MCR I both in amino acid sequence and y subunit size. Th...

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 reduction of methyl-coenzyme M (CH3SCoM) to methane in methanogenic bacteria is dependent on component B (N-7-mercaptoheptanoyl-O-phospho-L-threonine, HSHTP). We report here that S-methylcomponent B (N-7-(methylthio)heptanoyl-O-phospho-L-threonine, CH3SHTP) can substitute for neither CH3SCoM nor HSHTP in the methyl-CoM reductase reaction. Rather, CH3SHTP proved to be an inhibitor competitive with HSHTP (apparent Ki = 6 gM) and noncompetitive with CH3SCoM. These results make it very unlikely that HSHTP functions as a methyl group carrier. A role for HSHTP as direct electron donor for CH3SCoM reduction to CH4 is proposed.