Mo-, V-, and Fe-Nitrogenases Use a Universal Eight-Electron Reductive-Elimination Mechanism To Achieve N2 Reduction.

Three genetically distinct, but structurally similar, isozymes of nitrogenase catalyze biological N2 reduction 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 N2 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 E4(4H) ("Janus") state. N2 binding/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/oa mechanism, 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 H2 formation by hydride protonolysis (kHP) but significant differences in the rate constant for H2 re with N2 binding/reduction (kre). We now report that V-nitrogenase also exhibits HD formation during N2/D2 turnover (and H2 inhibition of N2 reduction), thereby establishing the re/oa equilibrium as a universal mechanism for N2 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 (kHP) for H2 production by the hydrogenase-like side reaction but directly arise from the differences in the rate constant (kre) for the re of H2 coupled to N2 binding/reduction, which decreases in the order Mo > V > Fe.

Mechanism of N2 Reduction Catalyzed by Fe-Nitrogenase Involves Reductive Elimination of H2.

Of the three forms of nitrogenase (Mo-nitrogenase, V-nitrogenase, and Fe-nitrogenase), Fe-nitrogenase has the poorest ratio of N2 reduction relative to H2 evolution. Recent work on the Mo-nitrogenase has revealed that reductive elimination of two bridging Fe-H-Fe hydrides on the active site FeMo-cofactor to yield H2 is a key feature in the N2 reduction mechanism. The N2 reduction mechanism for the Fe-nitrogenase active site FeFe-cofactor was unknown. Here, we have purified both component proteins of the Fe-nitrogenase system, the electron-delivery Fe protein (AnfH) plus the catalytic FeFe protein (AnfDGK), and established its mechanism of N2 reduction. Inductively coupled plasma optical emission spectroscopy and mass spectrometry show that the FeFe protein component does not contain significant amounts of Mo or V, thus ruling out a requirement of these metals for N2 reduction. The fully functioning Fe-nitrogenase system was found to have specific activities for N2 reduction (1 atm) of 181 ± 5 nmol NH3 min-1 mg-1 FeFe protein, for proton reduction (in the absence of N2) of 1085 ± 41 nmol H2 min-1 mg-1 FeFe protein, and for acetylene reduction (0.3 atm) of 306 ± 3 nmol C2H4 min-1 mg-1 FeFe protein. Under turnover conditions, N2 reduction is inhibited by H2 and the enzyme catalyzes the formation of HD when presented with N2 and D2. These observations are explained by the accumulation of four reducing equivalents as two metal-bound hydrides and two protons at the FeFe-cofactor, with activation for N2 reduction occurring by reductive elimination of H2.