Mechanistic Insights into Nitrogenase FeMo-Cofactor Catalysis through a Steady-State Kinetic Model.

Mo-nitrogenase catalyzes the challenging N-to-NH reduction. This complex reaction proceeds through a series of intermediate states (E) of its active site FeMo-cofactor. An understanding of the kinetics of the conversion between E states is central to defining the mechanism of nitrogenase. Here, rate constants of key steps have been determined through a steady-state kinetic model with fits to experimental data. The model reveals that the rate for H formation from the early electron populated state E(2H) is much slower than that from the more reduced E(4H) state. Further, it is found that the competing reactions of H formation and N binding at the E(4H) state occur with equal rate constants. The H-dependent reverse reaction of the N binding step is found to have a rate constant of 5.5 ± 0.2 (atm H) s (7.2 ± 0.3 (mM H) s). Importantly, the reduction of N bound to FeMo-cofactor proceeds with a rate constant of 1 ± 0.1 s, revealing a previously unrecognized slow step in the Mo-nitrogenase catalytic cycle associated with the chemical transformation of N to 2 NH. Finally, the populations of E states under different reaction conditions are predicted, providing a powerful tool to guide the spectroscopic and mechanistic studies of Mo-nitrogenase.