Department of Chemistry, Northwestern University, Evanston, Illinois 60208, United States.
Department of Chemistry and Biochemistry, Utah State University, Logan, Utah 84322, United States.
Inorg Chem. 2022 Apr 11;61(14):5459-5464. doi: 10.1021/acs.inorgchem.2c00180. Epub 2022 Mar 31.
The nitrogenase active-site cofactor must accumulate 4e/4H (E(4H) state) before N can bind and be reduced. Earlier studies demonstrated that this E(4H) state stores the reducing-equivalents as two hydrides, with the cofactor metal-ion core formally at its resting-state redox level. This led to the understanding that N binding is mechanistically coupled to reductive-elimination of the two hydrides that produce H. The state having acquired 2e/2H (E(2H)) correspondingly contains one hydride with a resting-state core redox level. How the cofactor accommodates addition of the first e/H (E(H) state) is unknown. The Fe-nitrogenase FeFe-cofactor was used to address this question because it is EPR-active in the E(H) state, unlike the FeMo-cofactor of Mo-nitrogenase, thus allowing characterization by EPR spectroscopy. The freeze-trapped E(H) state of Fe-nitrogenase shows an = 1/2 EPR spectrum with = [1.965, 1.928, 1.779]. This state is photoactive, and under 12 K cryogenic , 450 nm photolysis converts to a new and likewise photoactive = 1/2 state (denoted E(H)) with = [2.009, 1.950, 1.860], which results in a photostationary state, with E(H) relaxing to E(H) at temperatures above 145 K. An H/D kinetic isotope effect of 2.4 accompanies the 12 K E(H)/E(H)* photointerconversion. These observations indicate that the addition of the first e/H to the FeFe-cofactor of Fe-nitrogenase produces an Fe-bound hydride, not a sulfur-bound proton. As a result, the cluster metal-ion core is one-electron oxidized relative to the resting state. It is proposed that this behavior applies to all three nitrogenase isozymes.
固氮酶活性部位辅因子在 N 结合并被还原之前必须积累 4e/4H(E(4H) 态)。早期的研究表明,这种 E(4H) 态将还原当量存储为两个氢化物,辅因子金属离子核心在其静止状态的氧化还原水平处。这导致了这样的理解,即 N 结合与产生 H 的两个氢化物的还原消除机械偶联。相应地,获得 2e/2H(E(2H))的状态包含一个具有静止状态核心氧化还原水平的氢化物。辅因子如何适应第一个 e/H 的添加(E(H) 态)是未知的。使用 Fe-固氮酶 FeFe-辅因子来解决这个问题,因为它在 E(H) 态中具有 EPR 活性,而不同于 Mo-固氮酶的 FeMo-辅因子,因此允许通过 EPR 光谱学进行表征。Fe-固氮酶的冷冻捕获 E(H) 态显示出 g = 1/2 的 EPR 谱,g = [1.965,1.928,1.779]。该状态是光活性的,并且在 12 K 低温下,450nm 光解转化为新的同样光活性的 g = 1/2 状态(表示为 E(H)*),g = [2.009,1.950,1.860],这导致光稳定状态,E(H)*在 145 K 以上的温度下弛豫至 E(H)。12 K 下 E(H)/E(H)*光互变伴随 H/D 动力学同位素效应为 2.4。这些观察结果表明,向 Fe-固氮酶的 FeFe-辅因子添加第一个 e/H 会产生一个与 Fe 结合的氢化物,而不是与 S 结合的质子。结果,簇金属离子核心相对于静止状态被单电子氧化。据推测,这种行为适用于所有三种固氮酶同工酶。