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

Mo-nitrogenase catalyzes the challenging N 2 -to-NH 3 reduction. This complex reaction proceeds through a series of intermediate states (E n ) of its active site FeMo-cofactor. An understanding of the kinetics of the conversion between E n 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 2 formation from the early electron populated state E 2 (2H) is much slower than that from the more reduced E 4 (4H) state. Further, it is found that the competing reactions of H 2 formation and N 2 binding at the E 4 (4H) state occur with equal rate constants. The H 2 -dependent reverse reaction of the N 2 binding step is found to have a rate constant of 5.5 ± 0.2 (atm H 2 ) -1 s -1 (7.2 ± 0.3 (mM H 2 ) -1 s -1 ). Importantly, the reduction of N 2 bound to FeMo-cofactor proceeds with a rate constant of 1 ± 0.1 s -1 , revealing a previously unrecognized slow step in the Mo-nitrogenase catalytic cycle associated with the chemical transformation of N 2 to 2 NH 3 . Finally, the populations of E n states under different reaction conditions are predicted, providing a powerful tool to guide the spectroscopic and mechanistic studies of Mo-nitrogenase.