The reduction of the heterodisulfide (CoM-S-S-HTP) of coenzyme M (H-S-CoM) and N-7-mercaptoheptanoylthreonine phosphate (H-S-HTP) with H, 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 H, :heterodisulfide oxidoreductase complex of F4,,,-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 F,,,-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 heterodisulfide-reductase activity. The stimulatory effects of potassium phosphate, a membrane component, uracil derivatives and coenzyme F,,, on the H, :heterodisulfide-oxidoreductase activity of the purified complex are described.

The reduction of CoM‐S‐S‐HTP, the heterodisulfide of coenzyme M (H‐S‐CoM) and N‐7‐mercaptoheptanoylthreonine phosphate (H‐S‐HTP), with H2 is an energy‐conserving step in methanogenic archaea. We report here that in Methanosarcina barkeri this reaction is catalyzed by a membrane‐bound multienzyme complex, designated H2:heterodisulfide oxidoreductase complex, which was purified to apparent homogeneity. The preparation was found to be composed of nine polypeptides of apparent molecular masses 46 kDa, 39 kDa, 28 kDa, 25 kDa, 23 kDa, 21 kDa, 20 kDa, 16 kDa, and 15 kDa and to contain 3.2 nmol cytochrome b, 70 to 80 nmol non‐heme iron and acidlabile sulfur, 5 nmol Ni, and 0.6 nmol FAD per mg protein. The 23 kDa polypeptide possessed heme‐derived peroxidase activity indicating that this polypeptide is the cytochrome b. The purified H2:heterodisulfide oxidoreductase complex catalyzed the reduction of CoM‐S‐S‐HTP with H2 at a specific activity of 6 U/mg protein (1 U = 1 μmol · min−1), the reduction of benzylviologen with H2 at a specific activity of 66 U/mg protein and the reduction of CoM‐S‐S‐HTP with reduced benzylviologen at a specific activity of 24 U/mg protein. The complex did not mediate the reduction of coenzyme F420 with H2 nor the oxidation of reduced coenzyme F420 with CoM‐S‐S‐HTP. The reduced cytochrome b in the enzyme complex could be oxidized by CoM‐S‐S‐HTP and re‐reduced by H2. The specific rates of cytochrome oxidation and reduction were too high to be resolved under our experimental conditions. The findings suggest that the H2: heterodisulfide oxidoreductase complex is composed of a F420‐non‐reducing hydrogenase, a cytochrome b and heterodisulfide reductase and that cytochrome b is a redox carrier in the electron transport chain involved in CoM‐S‐S‐HTP reduction with H2.

Heterodisulfide reductase catalyzes the terminal step in the energy-conserving electron-transport chain in methanogenic Archaea. The heterodisulfide reductase activity of the membrane fraction of methanol-grown Methanosarcina barkeri was solubilized by Chaps. Chromatography on Q-Sepharose and Superdex-200 yielded a high-molecular-mass fraction (> 700 kDa) which was dissociated by dodecyl P-D-maltoside. After chromatography on Q-Sepharose, an active heterodisulfide reductase preparation was obtained which was composed of only two different subunits of apparent molecular masses 46 kDa and 23 kDa. For each 69 kDa, the enzyme contained 0.6 rnol cytochrome b, 0.2 rnol FAD, 20 rnol non-heme iron and 20 mol acid-labile sulfur. The 23-kDa subunit possessed heme-derived peroxidase activity, showing that this polypeptide is the cytochrome b. The purified enzyme contained the cytochrome b in the reduced form. Upon addition of the heterodisulfide of coenzyme M and N-7-mercaptoheptanoylthreonine phosphate the cytochrome was instantaneously oxidized, indicating that the cytochrome b served as electron donor for heterodisulfide reduction.An energy-conserving step in the metabolism of all methanogenic Archaea (Ferry, 1993), which grow on H, and CO, (4H,+CO,-CH,+ 2H,O), is the reduction of the heterodisulfide of coenzyme M (H-S-CoM) and N-7-mercaptoheptanoylthreonine phosphate (H-S-HTP) with molecular hydrogen, which is coupled with electrogenic proton translocation (Blaut et al., 3992;Weiss and Thauer, 1993). H, + COM-S-S-HTP + H-S-CoMfH-S-HTPAGO' = -40 k.J/mol (CoM-S-S-HTP, the heterodisulfide of H-S-CoM and H-S-HTP). The reaction is catalyzed by a membrane-associated H, :heterodisulfide oxidoreductase complex which has been purified and characterized from HJCO, grown Methanobacterium thermoautotrophicum (Setzke et al., 1994) and from acetate-grown Methanosarcina barkeri (Heiden et al., 1993). The purified H, : heterodisulfide oxidoreductase complex from M. thermoautotrophicum is composed of six different subunits of apparent molecular masses 80, 51, 41, 36, 21 and 17 kDa and contains in each 250 kDa, 0.6 mol nickel, 0.9 mol FAD and approximately 26 mol non-heme iron and 22 mol acid-labile sulfur. The complex can be dissociated by Chaps into a hydrogenase and a heterodisulfide reductase subcomplex, each composed of three different subunits. The heterodisulfide reductase subcomplex, which contains the FAD and approximately 18 rnol non-heme iron and acid-labile sulfur, is composed of the 80-, 36-and 21-kDa subunits (Hedderich et al., 1990), and the hydrogenase subcomplex, which contains the nickel and approximately 10 mol nonheme iron and acid-labile sulfur, is composed of the 51-, 41-and 17-kDa subunits (Hedderich et al., 1990;Setzke et al., 1994). From DNA sequence data it can be concluded that the 80-kDa polypeptide is the catalytic subunit of the heterodisulfide reductase subcomplex (unpublished results).The purified H, :heterodisulfide oxidoreductase complex from M. barkeri is composed of nine different polypeptides of a...