Mechanism of N Reduction Catalyzed by Fe-Nitrogenase Involves Reductive Elimination of H.

Of the three forms of nitrogenase (Mo-nitrogenase, V-nitrogenase, and Fe-nitrogenase), Fe-nitrogenase has the poorest ratio of N reduction relative to H 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 is a key feature in the N reduction mechanism. The N 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 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 reduction. The fully functioning Fe-nitrogenase system was found to have specific activities for N reduction (1 atm) of 181 ± 5 nmol NH min mg FeFe protein, for proton reduction (in the absence of N) of 1085 ± 41 nmol H min mg FeFe protein, and for acetylene reduction (0.3 atm) of 306 ± 3 nmol CH min mg FeFe protein. Under turnover conditions, N reduction is inhibited by H and the enzyme catalyzes the formation of HD when presented with N and D. 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 reduction occurring by reductive elimination of H.