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.

中文翻译：

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Fe-固氮酶催化N ₂还原的机理涉及H _2的还原消除

在三种形式的固氮酶（Mo-固氮酶，V-固氮酶和Fe-固氮酶）中，Fe-固氮酶相对于H ₂析出的N ₂还原率最差。Mo固氮酶的最新研究表明，在活性位点FeMo辅因子上消除两个桥接的Fe–H–Fe氢化物以产生H ₂是N ₂还原机制的关键特征。Fe固氮酶活性位点FeFe辅因子的N ₂还原机理尚不清楚。在这里，我们纯化了铁氮酶系统的两个组成蛋白，电子传递铁蛋白（AnfH）和催化铁蛋白FeFe（AnfDGK），并确定了其N _2的作用机理。减少。电感耦合等离子体发射光谱法和质谱法表明，FeFe蛋白成分不包含大量的Mo或V，因此排除了这些金属对N ₂还原的需求。已发现功能齐全的Fe固氮酶系统具有181±5 nmol NH ₃ min ^–1 mg ^–1 FeFe蛋白的N ₂还原（1 atm）的特定活性，对于1085的质子还原（无N ₂）具有特定的活性。±41 nmol H ₂ min ^–1 mg ^–1 FeFe蛋白，乙炔还原（0.3 atm）为306±3 nmol C ₂ H ₄ min ^–1mg ^–1 FeFe蛋白。在周转条件下，N _2的还原被H ₂抑制，当与N ₂和D ₂一起存在时，该酶催化HD的形成。这些观察结果通过FeFe-辅因子在FeFe-辅因子上作为两个金属结合的氢化物和两个质子的四个还原当量的积累而得以解释，其中N ₂还原的活化通过还原性消除H _2发生。