Kinetic Understanding of N₂ Reduction versus H₂ Evolution at the E₄(4H) Janus State in the Three Nitrogenases

The enzyme nitrogenase catalyzes the reduction of N₂ to ammonia but also that of protons to H₂. These reactions compete at the mechanistically central 'Janus' intermediate, denoted E₄(4H), which has accumulated 4e^-/4H⁺ as two bridging Fe-H-Fe hydrides on the active-site cofactor. This state can lose e^-/H⁺ by hydride protonolysis (HP) or become activated by reductive elimination ( re) of the two hydrides and bind N₂ with H₂ loss, yielding an E₄(2N2H) state that goes on to generate two NH₃ molecules. Thus, E₄(4H) represents the key branch point for these competing reactions. Here, we present a steady-state kinetic analysis that precisely describes this competition. The analysis demonstrates that steady-state, high-electron flux turnover overwhelmingly populates the E₄ states at the expense of less reduced states, quenching HP at those states. The ratio of rate constants for E₄(4H) hydride protonolysis ( k_HP) versus reductive elimination ( k_re) provides a sensitive measure of competition between these two processes and thus is a central parameter of nitrogenase catalysis. Analysis of measurements with the three nitrogenase variants (Mo-nitrogenase, V-nitrogenase, and Fe-nitrogenase) reveals that at a fixed N₂ pressure their tendency to productively react with N₂ to produce two NH₃ molecules and an accompanying H₂, rather than diverting electrons to the side reaction, HP production of H₂, decreases with their ratio of rate constants, k _re/ k_HP: Mo-nitrogenase, 5.1 atm^-1; V-nitrogenase, 2 atm^-1; and Fe-nitrogenase, 0.77 atm^-1 (namely, in a 1:0.39:0.15 ratio). Moreover, the lower catalytic effectiveness of the alternative nitrogenases, with more H₂ production side reaction, is not caused by a higher k_HP but by a significantly lower k _re.