Solubilization, Purification and Properties of Particulate Hydrogenase from Desulfovibrio vulgaris

Particulate hydrogenase [EC 1.12.2.1] of Desulfovibrio vulgaris has been solubilized and purified to a nearly homogeneous state. Its isoelectric point was about 6.25. It contained about 8 atoms Fe per mole. This enzyme is specific to cytochrome c3 as an electron carrier in the H2 evolution and H2 consumption reactions. pKm for ferrocytochrome c3 in the H2 evolution was 4.15. In this reaction, reduced methyl viologen could replace ferrocytochrome c3 to a limited extent, but the reaction with this artificial electron carrier was stimulated by cytochrome c3. “pKm” for this stimulating effect of cytochrome c3 was 5.90. The enzyme had, thus, two Kms for ferrocytochrome c3 in the H2 evolution reaction. In the H2 consumption reaction, Km for Ht was less than 8 mmHg. The enzyme catalyzed the hydrogen-deuterium exchange reaction in the absence of cytochrome cs. This reaction was also stimulated by cytochrome c3. No significant isotope effect was observed in the H2 evolution and H2 consumption reactions catalyzed by hydrogenase.