Purification and catalytic properties of Ech hydrogenase from Methanosarcina barkeri

Methanosarcina barkeri has recently been shown to produce a multisubunit me mbrane-bound [NiFe] hydrogenase designated Ech (Escherichia coli hydrogenas e 3) hydrogenase. In the present study Ech hydrogenase was purified to appa rent homogeneity in a high yield. The enzyme preparation obtained only cont ained the six polypeptides which had previously been shown to be encoded by the ech operon. The purified enzyme was found to contain 0.9 mol of Ni, 11 .3 mol of nonheme-iron and 10.8 mol of acid-labile sulfur per mol of enzyme . Using the purified enzyme the kinetic parameters were determined. The enz yme catalyzed the H-2 dependent reduction of a M. barkeri 2[3Fe-4S] ferredo xin with a specific activity of 50 U.mg protein(-1) at pH 7.0 and exhibited an apparent K-m for the ferredoxin of 1 mu M. The enzyme also catalyzed hy drogen formation with the reduced ferredoxin as electron donor at a rate of 90 U.mg protein(-1) at pH 7.0. The apparent K-m for the reduced ferredoxin was 7.5 mu M. Reduction or oxidation of the ferredoxin proceeded at simila r rates as the reduction or oxidation of oxidized or reduced methylviologen , respectively. The apparent K-m for H-2 was 5 mu M. The kinetic data stron gly indicate that the ferredoxin is the physiological: electron donor or ac ceptor of Ech hydrogenase. Ech hydrogenase amounts to about 3% of the total cell protein in acetate-grown, methanol-grown or H-2/CO2-grown cells of M. barkeri, as calculated from quantitative Western blot experiments. The fun ction of Ech hydrogenase is ascribed to ferredoxin-linked H-2 production co upled to the oxidation of the carbonyl-group of acetyl-CoA to CO2 during gr owth on acetate, and to ferredoxin-linked H-2 uptake coupled to the reducti on of CO2 to the redox state of CO during growth on H-2/CO2 or methanol.