Harris Derek F, Lukoyanov Dmitriy A, Shaw Sudipta, Compton Phil, Tokmina-Lukaszewska Monika, Bothner Brian, Kelleher Neil, Dean Dennis R, Hoffman Brian M, Seefeldt Lance C
Department of Chemistry and Biochemistry, Utah State University , Logan, Utah 84322, United States.
Departments of Chemistry and Molecular Biosciences, Northwestern University , Evanston, Illinois 60208, United States.
Biochemistry. 2018 Feb 6;57(5):701-710. doi: 10.1021/acs.biochem.7b01142. Epub 2018 Jan 17.
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.
在三种固氮酶形式（钼固氮酶、钒固氮酶和铁固氮酶）中，铁固氮酶相对于氢生成的氮还原比率最低。最近对钼固氮酶的研究表明，在活性位点铁钼辅因子上还原消除两个桥连的铁 - 氢 - 铁氢化物以产生氢气是氮还原机制的一个关键特征。铁固氮酶活性位点铁铁辅因子的氮还原机制尚不清楚。在这里，我们纯化了铁固氮酶系统的两种组成蛋白，电子传递铁蛋白（AnfH）和催化铁铁蛋白（AnfDGK），并确定了其氮还原机制。电感耦合等离子体发射光谱和质谱表明，铁铁蛋白组分不含大量的钼或钒，因此排除了这些金属对氮还原的需求。发现功能完全正常的铁固氮酶系统对氮还原（1个大气压）的比活性为181±5 nmol NH₃ min⁻¹ mg⁻¹铁铁蛋白，对质子还原（无氮时）的比活性为1085±41 nmol H₂ min⁻¹ mg⁻¹铁铁蛋白，对乙炔还原（0.3个大气压）的比活性为306±3 nmol C₂H₄ min⁻¹ mg⁻¹铁铁蛋白。在周转条件下，氮还原受到氢气的抑制，并且当存在氮气和重氢时，该酶催化生成HD。这些观察结果可以通过在铁铁辅因子上积累四个还原当量，即两个金属结合的氢化物和两个质子来解释，氮还原的激活是通过氢的还原消除发生的。
