Mo-, V-, and Fe-Nitrogenases Use a Universal Eight-Electron Reductive-Elimination Mechanism To Achieve N2Reduction

Three genetically distinct, but structurally similar, isozymes of nitrogenase catalyze biological N2reduction to 2NH3: Mo-, V-, and Fe-nitrogenase, named respectively for the metal (M) in their active site metallocofactors (metal-ion composition, MFe7). Studies of the Mo-enzyme have revealed key aspects of its mechanism for N2binding 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 E4(4H) (“Janus”) state. N2binding/reduction in this state is coupled to reductive elimination (re) of the two hydrides as H2, the forward direction of a reductive-elimination/oxidative-addition (re/oa) equilibrium. A recent study demonstrated that Fe-nitrogenase follows the same re/oamechanism, as particularly evidenced by HD formation during turnover under N2/D2. Kinetic analysis revealed that Mo- and Fe-nitrogenases show similar rate constants for hydrogenase-like H2formation by hydride protonolysis (kHP) but significant differences in the rate constant for H2rewith N2binding/reduction (kre). We now report that V-nitrogenase also exhibits HD formation during N2/D2turnover (and H2inhibition of N2reduction), thereby establishing the re/oaequilibrium as a universal mechanism for N2binding 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 (kHP) for H2production by the hydrogenase-like side reaction but directly arise from the differences in the rate constant (kre) for the reof H2coupled to N2binding/reduction, which decreases in the order Mo > V > Fe.