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

Abstract

The effects of O{sub 2} on membrane-bound Azotobacter vinelandii hydrogenase in both the membrane-associated and purified states were characterized. O{sub 2} was a rapid-equilibrium, reversible inhibitor both of H{sub 2} oxidation/electron acceptor reduction activity and of the exchange activity. O{sub 2} inhibition was noncompetitive versus the electron acceptor methylene blue and uncompetitive versus the substrate H{sub 2}. O{sub 2} inhibition was rapidly reversed by removal of the O{sub 2}. In addition to an O{sub 2}-independent, reversible inhibition of hydrogenase, a time-dependent, irreversible inactivation by O{sub 2} of both H{sub 2} oxidation and exchange activities for this hydrogenase was demonstrated. The irreversible inactivation followed a first-order process. The inactivation by O{sub 2} was protected against by the substrate H{sub 2} in a concentration-dependent manner. This is the first report of H{sub 2} protection from O{sub 2}-dependent, irreversible inactivation for any hydrogenase. The competitive inhibitor CO did not affect the irreversible inactivation by O{sub 2} or the protection by H{sub 2}. A model is proposed that outlines various oxidation states of this hydrogenase and the effects of O{sub 2} on each state.