CO as a substrate and inhibitor of H+ reduction for the Mo-, V-, and Fe-nitrogenase isozymes

Three known nitrogenase isozymes, Mo-, V-, and Fe-, catalyze biological reduction of dinitrogen (N-2) to ammonia (NH3). All three utilize the same reductive elimination mechanism: an intermediate with two metal-bound hydrides reductively-eliminates hydrogen gas (H-2) in a reaction coupled to binding and activation of N-2. Nonetheless, the three isozymes show dramatically different relative rates of H-2 formation and N-2 reduction, revealing important differences in reactivity with substrates. Carbon monoxide (CO) has been characterized as both an inhibitor and substrate for Mo- and V-nitrogenases, but not for the Fe-nitrogenase. Here, we present a comparative study of the reactivity of the three isozymes with CO, examining CO both as a substrate and as an inhibitor of proton (H+) reduction under steady-state conditions. For Mo-nitrogenase, there is neither detectable reduction of CO nor inhibition of H+ reduction. Fe- and V-nitrogenase show CO reduction and inhibition of H+ reduction that depends on the CO partial pressure. For V-nitrogenase, ethylene (C2H4) is the major reduction product with a maximum specific activity of similar to 7.5 nmol C2H4/nmol VFe protein/min at 1 atm CO. The major product of CO reduction for Fe-nitrogenase is methane (CH4) with a maximum specific activity of similar to 4.8 nmol CH4/nmol FeFe protein/min at 0.05 atm CO. The rate of CH4 production by Fe-nitrogenase progressively increases to a maximum at 0.05 atm CO and then declines by similar to 90% with increasing CO partial pressure up to 1 atm. CO does not inhibit proton reduction in Mo-nitrogenase but shows 16% inhibition for V-nitrogenase and 35% for Fe-nitrogenase.