Hydrogenase from the hydrogen-oxidizing bacterium Alcaligenes eutrophus Z I: Catalytic activity-quaternary structure relationship

Abstract The relationship between the quaternary structure and catalytic activity of the soluble hydrogenase (hydrogen:NAD + oxidoreductase, EC 1.12.1.2) from the hydrogen-oxidizing bacterium. Alcaligenes eutrophus strain Z I, has been investigated. The enzyme specific activity depends on both the protein concentration and preincubation conditions. The hydrogenase activity with NAD or methyl viologen as substrates is more sensitive to dilution than the NADH-dehydrogenase activity of the enzyme. The molecular weight of hydrogenase determined by gel-filtration decreases from 160000 to 90000, while the enzyme concentration falls from 4.5 to 0.044 mg/ml. Coenzymes change both the apparent molecular weight and concentration dependence of the hydrogenase specific activity. Gel-filtration under dissociation conditions reveals an enzyme fragment with a molecular weight of 60000 which is active in NADH-dehydrogenase reaction, but does not catalyse the hydrogenase activity. Gradient polyacrylamide gel electrophoresis experiments reveal only one component with a molecular weight of 180 000-150 000 which stains for both hydrogenase and NADH-dehydrogenase activity. Under dissociation conditions the activity band splits into several bands corresponding to molecular weights from 6000 to 200000 only in the case of NADH-dehydrogenase. It is proposed that the native enzyme can undergo reversible dissociation. It is hypothesized that hydrogenase contains a special subunit with a molecular weight of about 60000, which may determine the NADH-dehydrogenase activity of the enzyme.