KINETIC-ANALYSIS OF THE INTERACTION OF NITRIC-OXIDE WITH THE MEMBRANE-ASSOCIATED, NICKEL AND IRON-SULFUR-CONTAINING HYDROGENASE FROM AZOTOBACTER-VINELANDII

The effects of nitric oxide (NO) on the membrane-associated form of the nickel and iron-sulfur-containing hydrogenase from Azotobacter vinelandii have been investigated. In the presence of H-2 and an electron acceptor (turnover conditions), NO acts as a noncompetitive inhibitor vs. methylene blue (K(i) = 12-mu-M). There is no element of competition between NO and H-2, implying that the site of NO action is not the H-2-activating site of the hydrogenase. When the membrane-associated hydrogenase is incubated under non-turnover conditions, the enzyme is irreversibly inactivated by NO in a time-dependent process. The inactivation is a non-saturable, pseudo-first-order process which is consistent with a direct chemical reaction between NO and the hydrogenase. Kinetic evidence is presented which is compatible with an interaction between NO and a redox-active component other than the H-2-activating site on the enzyme. The complex inhibition pattern of NO has been interpreted in terms of two distinct interactions of NO with iron-sulfur centers of the hydrogenase.