Mo-, V-, and Fe-Nitrogenases Use a Universal Eight-Electron Reductive-Elimination Mechanism To Achieve N Reduction.

Three genetically distinct, but structurally similar, isozymes of nitrogenase catalyze biological N reduction to 2NH: Mo-, V-, and Fe-nitrogenase, named respectively for the metal () in their active site metallocofactors (metal-ion composition, Fe). Studies of the Mo-enzyme have revealed key aspects of its mechanism for N binding and reduction. Central to this mechanism is accumulation of four electrons and protons on its active site metallocofactor, called FeMo-co, as metal bound hydrides to generate the key E(4H) ("Janus") state. N binding/reduction in this state is coupled to reductive elimination () of the two hydrides as H, the forward direction of a reductive-elimination/oxidative-addition () equilibrium. A recent study demonstrated that Fe-nitrogenase follows the same mechanism, as particularly evidenced by HD formation during turnover under N/D. Kinetic analysis revealed that Mo- and Fe-nitrogenases show similar rate constants for hydrogenase-like H formation by hydride protonolysis () but significant differences in the rate constant for H with N binding/reduction (). We now report that V-nitrogenase also exhibits HD formation during N/D turnover (and H inhibition of N reduction), thereby establishing the equilibrium as a universal mechanism for N binding and activation among the three nitrogenases. Kinetic analysis further reveals that differences in catalytic efficiencies do not stem from significant differences in the rate constant () for H production by the hydrogenase-like side reaction but directly arise from the differences in the rate constant () for the of H coupled to N binding/reduction, which decreases in the order Mo > V > Fe.