Trapping a hydrazine reduction intermediate on the nitrogenase active site
Trapping a hydrazine reduction intermediate on the nitrogenase active site
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DOI：
10.1021/bi0504409
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发表时间：
2005-06-07
期刊：
影响因子：
2.9
通讯作者：
Seefeldt, LC
中科院分区：
文献类型：
--
作者：
Barney, BM;Laryukhin, M;Seefeldt, LC
在理解固氮酶（负责将N-2生物固定到两个氨的酶）的机理中的主要挑战是将含氮底物捕获在酶活性位点处，使其处于适合于进一步表征的状态。在本工作中，描述了一种策略，该策略导致捕获底物肼（H2 N-NH 2）作为与固氮酶活性位点金属簇结合的加合物，并且通过EPR和ENDOR光谱对这种结合的加合物进行了表征。早期的工作已经被解释为表明含氮的（例如，N-2和肼）以及炔（例如，乙炔）基质可以结合在由Fe原子2、3、6和7组成的FeMo-辅因子的共同FeS面上。先前还显示，用较小的氨基酸丙氨酸取代位于该FeS表面上的α-70（瓦尔）可改善对肼的亲和力和还原速率。我们现在表明，当α-195（His）（活性位点附近的假定质子供体）被谷氨酰胺取代，同时α-70（瓦尔）被丙氨酸取代时，（α-70（Ala）/α-195（Gln））以肼为底物翻转，FeMo-辅因子可以在S = 1/2中被冷冻捕获，高收益率（接近70%）。推测的肼-FeMo-辅因子加合物显示菱形EPR信号，g = [2.09，2.01，1.93]。发现这种状态的种群的最佳pH为7.4。EPR信号表现出居里定律的温度依赖性类似于静息态EPR信号。使用N-15标记的肼在35 GHz下的Mims脉冲ENDOR光谱显示，捕获的中间体结合了与FeMo辅因子结合的肼衍生物质;在g（1）处拍摄的光谱中，该物质给出了单个观察到的N-15信号，A（g（1））= 1.5 MHz。
A major challenge in understanding the rnechanism 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 (H2N-NH2) 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 (similar to 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 N-15-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 N-15 signal, A(g(1)) = 1.5 MHz.