PURIFICATION AND CHARACTERIZATION OF MEMBRANE-BOUND HYDROGENASE FROM METHANOSARCINA-BARKERI MS

Hydrogenase was solubilized from the membrane of acetate-grown Methanosarcina barkeri MS and purification was carried out under aerobic conditions. The enzyme was reactivated under reducing conditions in the presence of H-2. The enzyme showed a maximal activity of 120 +/- 40 mu mol H-2 oxidized.min(-1).mg(-1) with methyl viologen as an electron acceptor, a maximal hydrogen production rate of 45 +/- 4 mu mol H-2.min(-1).mg(-1) with methyl viologen as electron donor, and an apparent K-m for hydrogen oxidation of 5.6 +/- 1.7 mu M. The molecular weight estimated by gel filtration was 98,000. SDS-PAGE showed the enzyme to consist of two polypeptides of 57,000 and 35,000 present in a 1:1 ratio. The native protein contained 8 +/- 2 mol Fe, 8 +/- 2 mol S2-, and 0.5 mol Ni/mol enzyme. Cytochrome b was reduced by hydrogen in a solubilized membrane preparation. The hydrogenase did not couple with autologous F-420 or ferredoxin, nor with FAD, FMN, or NAD(P)(+). The physiological function of the membrane-bound hydrogenase in hydrogen consumption is discussed.