Purification of a cytochrome b containing H2:heterodisulfide oxidoreductase complex from membranes of Methanosarcina barkeri

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Abstract
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 acid-labile 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 mumol.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 benzylviologen with H2 at a specific activity of 66 U/mg protein and the reduction of CoM-S-S-HTP 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.