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) to ammonia (NH). All three utilize the same reductive elimination mechanism: an intermediate with two metal-bound hydrides reductively-eliminates hydrogen gas (H) in a reaction coupled to binding and activation of N. Nonetheless, the three isozymes show dramatically different relative rates of H formation and N 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 (CH) is the major reduction product with a maximum specific activity of ~7.5 nmol CH/nmol VFe protein/min at 1 atm CO. The major product of CO reduction for Fe‑nitrogenase is methane (CH) with a maximum specific activity of ~4.8 nmol CH/nmol FeFe protein/min at 0.05 atm CO. The rate of CH production by Fe‑nitrogenase progressively increases to a maximum at 0.05 atm CO and then declines by ~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.