Barney Brett M, Lukoyanov Dmitriy, Igarashi Robert Y, Laryukhin Mikhail, Yang Tran-Chin, Dean Dennis R, Hoffman Brian M, Seefeldt Lance C
Department of Chemistry and Biochemistry, Utah State University, Logan, Utah 84322, USA.
Biochemistry. 2009 Sep 29;48(38):9094-102. doi: 10.1021/bi901092z.
Nitrogenase reduces dinitrogen (N2) by six electrons and six protons at an active-site metallocluster called FeMo cofactor, to yield two ammonia molecules. Insights into the mechanism of substrate reduction by nitrogenase have come from recent successes in trapping and characterizing intermediates generated during the reduction of protons as well as nitrogenous and alkyne substrates by MoFe proteins with amino acid substitutions. Here, we describe an intermediate generated at a high concentration during reduction of the natural nitrogenase substrate, N2, by wild-type MoFe protein, providing evidence that it contains N2 bound to the active-site FeMo cofactor. When MoFe protein was frozen at 77 K during steady-state turnover with N2, the S = 3/2 EPR signal (g = [4.3, 3.64, 2.00]) arising from the resting state of FeMo cofactor was observed to convert to a rhombic, S = 1/2, signal (g = [2.08, 1.99, 1.97]). The intensity of the N2-dependent EPR signal increased with increasing N2 partial pressure, reaching a maximum intensity of approximately 20% of that of the original FeMo cofactor signal at > or = 0.2 atm N2. An almost complete loss of resting FeMo cofactor signal in this sample implies that the remainder of the enzyme has been reduced to an EPR-silent intermediate state. The N2-dependent EPR signal intensity also varied with the ratio of Fe protein to MoFe protein (electron flux through nitrogenase), with the maximum signal intensity observed with a ratio of 2:1 (1:1 Fe protein:FeMo cofactor) or higher. The pH optimum for the signal was 7.1. The N2-dependent EPR signal intensity exhibited a linear dependence on the square root of the EPR microwave power in contrast to the nonlinear response of signal intensity observed for hydrazine-, diazene-, and methyldiazene-trapped states. 15N ENDOR spectroscopic analysis of MoFe protein captured during turnover with 15N2 revealed a 15N nuclear spin coupled to the FeMo cofactor with a hyperfine tensor A = [0.9, 1.4, 0.45] MHz establishing that an N2-derived species was trapped on the FeMo cofactor. The observation of a single type of 15N-coupled nucleus from the field dependence, along with the absence of an associated exchangeable 1H ENDOR signal, is consistent with an N2 molecule bound end-on to the FeMo cofactor.
固氮酶在一个名为铁钼辅因子的活性位点金属簇上，通过六个电子和六个质子将氮气（N₂）还原，生成两个氨分子。对固氮酶底物还原机制的深入了解，源于最近通过对带有氨基酸替代的钼铁蛋白还原质子以及含氮和炔烃底物过程中产生的中间体进行捕获和表征所取得的成功。在此，我们描述了野生型钼铁蛋白在还原天然固氮酶底物N₂过程中高浓度生成的一种中间体，这为其包含与活性位点铁钼辅因子结合的N₂提供了证据。当钼铁蛋白在与N₂进行稳态周转时于77 K冷冻，观察到源于铁钼辅因子静止状态的S = 3/2电子顺磁共振（EPR）信号（g = [4.3, 3.64, 2.00]）转变为菱形的S = 1/2信号（g = [2.08, 1.99, 1.97]）。依赖于N₂的EPR信号强度随N₂分压增加而增强，在N₂分压≥0.2 atm时达到最大强度，约为原始铁钼辅因子信号强度的20%。该样品中静止的铁钼辅因子信号几乎完全消失，这意味着酶的其余部分已被还原为一种EPR沉默的中间状态。依赖于N₂的EPR信号强度也随铁蛋白与钼铁蛋白的比例（通过固氮酶的电子通量）而变化，在比例为2:1（1:1铁蛋白:铁钼辅因子）或更高时观察到最大信号强度。该信号的最适pH为7.1。与肼、二氮烯和甲基二氮烯捕获状态下观察到的信号强度非线性响应不同，依赖于N₂的EPR信号强度与EPR微波功率的平方根呈线性关系。用¹⁵N₂进行周转过程中捕获到的钼铁蛋白的¹⁵N电子核双共振（ENDOR）光谱分析显示，一个¹⁵N核自旋与铁钼辅因子耦合，超精细张量A = [0.9, 1.4, 0.45] MHz，这表明一种源自N₂的物种被困在铁钼辅因子上。从场依赖性观察到单一类型的¹⁵N耦合核，以及不存在相关的可交换¹H ENDOR信号，这与一个N₂分子以端对端方式结合到铁钼辅因子上是一致的。
