Oxygen effects on the nickel- and iron-containing hydrogenase from Azotobacter vinelandii

The effects of O2 on membrane-bound Azotobacter vinelandii hydrogenase in both the membrane-associated and purified states were characterized. O2 was a rapid-equilibrium, reversible inhibitor both of H2 oxidation/electron acceptor reduction activity and of the exchange activity. O2 inhibition was noncompetitive versus the electron acceptor methylene blue and uncompetitive versus the substrate H2. O2 inhibition was rapidly reversed by removal of the O2. In addition to an O2-independent, reversible inhibition of hydrogenase, a time-dependent, irreversible inactivation by O2 of both H2 oxidation and exchange activities for this hydrogenase was demonstrated. The irreversible inactivation followed a first-order process. The inactivation by O2 was protected against by the substrate H2 in a concentration-dependent manner. This is the first report of H2 protection from O2-dependent, irreversible inactivation for any hydrogenase. The competitive inhibitor CO did not affect the irreversible inactivation by O2 or the protection by H2. A model is proposed that outlines various oxidation states of this hydrogenase and the effects of O2 on each state