Abstract
The enzyme nitrogenase catalyzes the reduction of N(2) to ammonia but also that of protons to H(2). These reactions compete at the mechanistically central 'Janus' intermediate, denoted E(4)(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(2) with H(2) loss, yielding an E(4)(2N2H) state that goes on to generate two NH(3) molecules. Thus, E(4)(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(4) states at the expense of less reduced states, quenching HP at those states. The ratio of rate constants for E(4)(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 N(2) pressure their tendency to productively react with N(2) to produce two NH(3) molecules and an accompanying H(2), rather than diverting electrons to the side reaction, HP production of H(2), 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 H(2) production side reaction, is not caused by a higher k(HP) but by a significantly lower k (re).