Barney Brett M, Laryukhin Mikhail, Igarashi Robert Y, Lee Hong-In, Dos Santos Patricia C, Yang Tran-Chin, Hoffman Brian M, Dean Dennis R, Seefeldt Lance C
Department of Chemistry and Biochemistry, Utah State University, Logan, Utah 84322, USA.
Biochemistry. 2005 Jun 7;44(22):8030-7. doi: 10.1021/bi0504409.
A major challenge in understanding the mechanism of nitrogenase, the enzyme responsible for the biological fixation of N(2) to two ammonias, is to trap a nitrogenous substrate at the enzyme active site in a state that is amenable to further characterization. In the present work, a strategy is described that results in the trapping of the substrate hydrazine (H(2)N-NH(2)) as an adduct bound to the active site metal cluster of nitrogenase, and this bound adduct is characterized by EPR and ENDOR spectroscopies. Earlier work has been interpreted to indicate that nitrogenous (e.g., N(2) and hydrazine) as well as alkyne (e.g., acetylene) substrates can bind at a common FeS face of the FeMo-cofactor composed of Fe atoms 2, 3, 6, and 7. Substitution of alpha-70(Val) that resides over this FeS face by the smaller amino acid alanine was also previously shown to improve the affinity and reduction rate for hydrazine. We now show that when alpha-195(His), a putative proton donor near the active site, is substituted by glutamine in combination with substitution of alpha-70(Val) by alanine, and the resulting doubly substituted MoFe protein (alpha-70(Ala)/alpha-195(Gln)) is turned over with hydrazine as substrate, the FeMo-cofactor can be freeze-trapped in a S = (1)/(2) state in high yield ( approximately 70%). The presumed hydrazine-FeMo-cofactor adduct displays a rhombic EPR signal with g = [2.09, 2.01, 1.93]. The optimal pH for the population of this state was found to be 7.4. The EPR signal showed a Curie law temperature dependence similar to the resting state EPR signal. Mims pulsed ENDOR spectroscopy at 35 GHz using (15)N-labeled hydrazine reveals that the trapped intermediate incorporates a hydrazine-derived species bound to the FeMo-cofactor; in spectra taken at g(1) this species gives a single observed (15)N signal, A(g(1)) = 1.5 MHz.
理解固氮酶（负责将N₂生物固定为两个氨的酶）作用机制的一个主要挑战是在酶活性位点捕获处于易于进一步表征状态的含氮底物。在本工作中，描述了一种策略，该策略导致底物肼（H₂N-NH₂）作为加合物捕获在固氮酶的活性位点金属簇上，并且这种结合的加合物通过电子顺磁共振（EPR）和电子核双共振（ENDOR）光谱进行表征。早期的工作被解释为表明含氮（例如N₂和肼）以及炔烃（例如乙炔）底物可以在由铁原子2、3、6和7组成的FeMo辅因子的共同FeS面上结合。先前还表明，用较小的氨基酸丙氨酸取代位于该FeS面上的α-70（Val）可提高对肼的亲和力和还原速率。我们现在表明，当活性位点附近的假定质子供体α-195（His）被谷氨酰胺取代并与α-70（Val）被丙氨酸取代相结合，并且所得的双取代MoFe蛋白（α-70（Ala）/α-195（Gln））以肼作为底物进行周转时，FeMo辅因子可以高产率（约70%）冷冻捕获在S = 1/2状态。推测的肼 - FeMo辅因子加合物显示出g = [2.09, 2.01, 1.93]的菱形EPR信号。发现该状态的最佳pH值为7.4。EPR信号显示出与静止状态EPR信号相似的居里定律温度依赖性。使用¹⁵N标记的肼在35 GHz下进行的Mims脉冲ENDOR光谱显示，捕获的中间体包含与FeMo辅因子结合的肼衍生物种；在g(1)处拍摄的光谱中，该物种给出单个观察到的¹⁵N信号，A(g(1)) = 1.5 MHz。
