Mechanistic Insights into Nitrogenase FeMo-Cofactor Catalysis through a Steady-State Kinetic Model
2022
Harris, Derek F. | Badalyan, Artavazd | Seefeldt, Lance C.
Mo-nitrogenase catalyzes the challenging N₂-to-NH₃ reduction. This complex reaction proceeds through a series of intermediate states (Eₙ) of its active site FeMo-cofactor. An understanding of the kinetics of the conversion between Eₙ 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 H₂ formation from the early electron populated state E₂(2H) is much slower than that from the more reduced E₄(4H) state. Further, it is found that the competing reactions of H₂ formation and N₂ binding at the E₄(4H) state occur with equal rate constants. The H₂-dependent reverse reaction of the N₂ binding step is found to have a rate constant of 5.5 ± 0.2 (atm H₂)⁻¹ s–¹ (7.2 ± 0.3 (mM H₂)⁻¹ s–¹). Importantly, the reduction of N₂ 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 N₂ to 2 NH₃. Finally, the populations of Eₙ states under different reaction conditions are predicted, providing a powerful tool to guide the spectroscopic and mechanistic studies of Mo-nitrogenase.
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