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 N-2 reduction relative to H-2 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 H-2 is a key feature in the N-2 reduction mechanism. The N-2 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 N-2 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 N-2 reduction. The fully functioning Fe-nitrogenase system was found to have specific activities for N-2 reduction (1 atm) of 181 +/- 5 nmol NH3 min(-1) mg(-1) FeFe protein, for proton reduction (in the absence of N-2) of 1085 +/- 41 nmol H-2 Ing(-1) FeFe protein, and for acetylene reduction (0.3 atm) of 306 +/- 3 nmol C2H4 min(-1) mg(-1) FeFe protein. Under turnover conditions, N-2 reduction is inhibited by H-2 and the enzyme catalyzes the formation of HD when presented with N-2 and D-2. 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 N-2 reduction occurring by reductive elimination of H-2.