Jm. Kemner et Jg. Zeikus, PURIFICATION AND CHARACTERIZATION OF MEMBRANE-BOUND HYDROGENASE FROM METHANOSARCINA-BARKERI MS, Archives of microbiology, 161(1), 1994, pp. 47-54
Hydrogenase was solubilized from the membrane of acetate-grown Methano
sarcina barkeri MS and purification was carried out under aerobic cond
itions. The enzyme was reactivated under reducing conditions in the pr
esence of H-2. The enzyme showed a maximal activity of 120 +/- 40 mu m
ol H-2 oxidized.min(-1).mg(-1) with methyl viologen as an electron acc
eptor, 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 estim
ated by gel filtration was 98,000. SDS-PAGE showed the enzyme to consi
st of two polypeptides of 57,000 and 35,000 present in a 1:1 ratio. Th
e native protein contained 8 +/- 2 mol Fe, 8 +/- 2 mol S2-, and 0.5 mo
l 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 physiologica
l function of the membrane-bound hydrogenase in hydrogen consumption i
s discussed.