Kinetic Understanding of N 2 Reduction versus H 2 Evolution at the E 4 (4H) Janus State in the Three Nitrogenases

The enzyme nitrogenase catalyzes the reduction of N to ammonia but also that of protons to H . These reactions compete at the mechanistically central 'Janus' intermediate, denoted E (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 N with H loss, yielding an E (2N2H) state that goes on to generate two NH molecules. Thus, E (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 E states at the expense of less reduced states, quenching HP at those states. The ratio of rate constants for E (4H) hydride protonolysis ( k ) versus reductive elimination ( k ) 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 N pressure their tendency to productively react with N to produce two NH molecules and an accompanying H , rather than diverting electrons to the side reaction, HP production of H , decreases with their ratio of rate constants, k / k : Mo-nitrogenase, 5.1 atm ; V-nitrogenase, 2 atm ; and Fe-nitrogenase, 0.77 atm (namely, in a 1:0.39:0.15 ratio). Moreover, the lower catalytic effectiveness of the alternative nitrogenases, with more H production side reaction, is not caused by a higher k but by a significantly lower k .