Localization of a substrate binding site on the FeMo-cofactor in nitrogenase:: Trapping propargyl alcohol with an α-70-substituted MoFe protein
Localization of a substrate binding site on the FeMo-cofactor in nitrogenase:: Trapping propargyl alcohol with an α-70-substituted MoFe protein
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DOI：
10.1021/bi034595x
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发表时间：
2003-08-05
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：
Benton, PMC;Laryukhin, M;Seefeldt, LC
用 Ala 或 Gly 取代 MoFe 蛋白 α-70(Val) 残基扩大了固氮酶的底物范围，从而可以还原较大的炔烃，包括炔丙醇 (HCdropCCH(2)OH)。在此，我们报告了 α-70(Val-->Ala) MoFe 蛋白的表征，其中炔丙醇被捕获在活性位点。 α-70(Ala) 变体 MoFe 蛋白在炔丙醇还原过程中迅速冻结，导致静息态 FeMo 辅因子 EPR 信号（S = 3/2 和 g = [4.41, 3.60, 2.00]）转换为新状态（S = 1/2 和 g = [2.123, 1.998, 1.986]）。新状态的 EPR 信号强度随着炔丙醇浓度的增加而增加，与单一底物的结合一致。炔丙醇状态的 EPR 信号显示出与经典 FeMo 辅因子 EPR 信号明显不同的温度和微波功率依赖性，这与自旋差异一致。新状态类似于抑制剂CO（“lo CO”状态）与野生型MoFe蛋白中的FeMo辅因子结合所诱导的状态。具有捕获的 C-13 标记的炔丙醇的 α-70(Ala) MoFe 蛋白的 C-13 ENDOR 谱显示出三个分辨率良好的 C-13 双峰，中心位于 C-13 Larmor 频率，具有类似于 3.2、类似于 1.4 和类似于 0.7 MHz 的各向同性超精细耦合，表明醇（或片段）与辅因子配位。本文给出的结果将炔丙醇的结合位点定位于 FeMo 辅因子的一个 [4Fe-4S] 面，并表明 α-70(Val) 残基在控制 FeMo 辅因子反应性中的作用。
Substitution of the MoFe protein alpha-70(Val) residue with Ala or Gly expands the substrate range of nitrogenase, allowing the reduction of larger alkynes, including propargyl alcohol (HCdropCCH(2)OH). Herein, we report characterization of the alpha-70(Val-->Ala) MoFe protein with propargyl alcohol trapped at the active site. The alpha-70(Ala) variant MoFe protein was rapidly frozen during reduction of propargyl alcohol, resulting in the conversion of the resting-state FeMo-cofactor EPR signal (S = 3/2 and g = [4.41, 3.60, 2.00]) to a new state (S = 1/2 and g = [2.123, 1.998, 1.986]). This EPR signal of the new state increased in intensity with increasing propargyl alcohol concentration, consistent with the binding of a single substrate. The EPR signal of the propargyl alcohol state showed temperature and microwave power dependencies markedly different from those of the classic FeMo-cofactor EPR signal, consistent with the difference in spin. The new state is analogous to that induced by the binding of the inhibitor CO ("lo CO" state) to FeMo-cofactor in the wild-type MoFe protein. The C-13 ENDOR spectrum of the alpha-70(Ala) MoFe protein with trapped C-13-labeled propargyl alcohol exhibited three well-resolved C-13 doublets centered at the C-13 Larmor frequency with isotropic hyperfine couplings of similar to3.2, similar to1.4, and similar to0.7 MHz, indicating that the alcohol (or a fragment) is coordinated to the cofactor. The results presented here localize the binding site of propargyl alcohol to one [4Fe-4S] face of FeMo-cofactor and indicate roles for the alpha-70(Val) residue in controlling FeMo-cofactor reactivity.