Purification and characterization of membrane-bound hydrogenase from Methanosarcina barkeri MS

Abstract

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 H2. The enzyme showed a maximal activity of 120±40 μmol H2 oxidized · min−1 · min−1 with methyl viologen as an electron acceptor, a maximal hydrogen production rate of 45±4 μmol H2 · min−1 · mg−1 with methyl viologen as electron donor, and an apparent K m for hydrogen oxidation of 5.6±1.7 μM. 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 F420 or ferredoxin, nor with FAD, FMN, or NAD(P)+. The physiological function of the membrane-bound hydrogenase in hydrogen consumption is discussed.