Mechanism of Nitrogen Fixation: Use of Carbon Monoxide to Probe Nitrogenase Activity.

The inhibitor CO was used to investigate the mechanism of Mo and V nitrogenase. While low pressures of CO enhanced, high pressures inhibited V nitrogenase-catalyzed C$\rm\sb2H\sb6$ formation. This is not the case for $\rm C\sb2H\sb4$ formation, where the rate of electron flux determined if CO acted as an enhancer or inhibitor. A two-site model is proposed, in which CO binding to one site enhances, and CO binding to both sites inhibits product formation. Replacement of CO(g) by Ar or $\rm C\sb2H\sb2$ resulted in correlated decay of hi-CO and development of lo-CO EPR signals, suggesting that the two signals arise from one metal cluster. The detection of CO binding to the lo-CO form of the enzyme in the absence of turnover suggests that CO stabilizes a MoFe protein cluster or turnover state and enhances binding of additional CO molecules. $\sp{13}$CO and $\sp{57}$Fe ENDOR studies of Mo nitrogenase in moderate flux demonstrated that (i) lo-CO Mo nitrogenase has one bound CO molecule and hi-CO enzyme has two; (ii) the second CO binds to the same cluster that has one bound CO; and (iii) CO binds to the FeMo cofactor and not to the P cluster.