Purification and catalytic properties of Ech hydrogenase from Methanosarcina barkeri


R. Hedderich, Max-Planck-Institut für terrestrische Mikrobiologie, Karl-von-Frisch-Straße, D-35043 Marburg, Germany. Fax: + 49 6421178299, Tel.: + 49 6421178230, E-mail: hedderic@mailer.uni-marburg.de


Methanosarcina barkeri has recently been shown to produce a multisubunit membrane-bound [NiFe] hydrogenase designated Ech (Escherichia coli hydrogenase 3) hydrogenase. In the present study Ech hydrogenase was purified to apparent homogeneity in a high yield. The enzyme preparation obtained only contained 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 enzyme catalyzed the H2 dependent reduction of a M. barkeri 2[4Fe-4S] ferredoxin with a specific activity of 50 U·mg protein−1 at pH 7.0 and exhibited an apparent Km for the ferredoxin of 1 µm. The enzyme also catalyzed hydrogen formation with the reduced ferredoxin as electron donor at a rate of 90 U·mg protein−1 at pH 7.0. The apparent Km for the reduced ferredoxin was 7.5 µm. Reduction or oxidation of the ferredoxin proceeded at similar rates as the reduction or oxidation of oxidized or reduced methylviologen, respectively. The apparent Km for H2 was 5 µm. The kinetic data strongly indicate that the ferredoxin is the physiological electron donor or acceptor of Ech hydrogenase. Ech hydrogenase amounts to about 3% of the total cell protein in acetate-grown, methanol-grown or H2/CO2-grown cells of M. barkeri, as calculated from quantitative Western blot experiments. The function of Ech hydrogenase is ascribed to ferredoxin-linked H2 production coupled to the oxidation of the carbonyl-group of acetyl-CoA to CO2 during growth on acetate, and to ferredoxin-linked H2 uptake coupled to the reduction of CO2 to the redox state of CO during growth on H2/CO2 or methanol.