Mutagenesis study of the 2Fe-2S center and the FAD binding site of the Na+-translocating NADH:Ubiquinone oxidoreductase from Vibrio cholerae
Mutagenesis study of the 2Fe-2S center and the FAD binding site of the Na+-translocating NADH:Ubiquinone oxidoreductase from Vibrio cholerae
复制标题
DOI：
10.1021/bi048689y
复制
发表时间：
2004-09-28
期刊：
影响因子：
2.9
通讯作者：
Gennis, RB
中科院分区：
文献类型：
--
作者：
Barquera, B;Nilges, MJ;Gennis, RB
许多海洋和病原细菌具有独特的钠易位 NADH：泛醌氧化还原酶 (Na+-NQR)，它在有氧呼吸过程中产生电化学 Na+ 梯度。 Na+-NQR 由六个亚基 (NqrA-F) 组成，并包含五种已知的氧化还原辅因子：两个共价结合的 FMN、一个非共价结合的 FAD、一种核黄素和一个 2Fe-2S 中心。在空气氧化的酶中观察到稳定的中性黄素-半醌自由基，而NADH-或连二亚硫酸盐还原的酶表现出稳定的阴离子黄素-半醌自由基。 NqrF 亚基与 2Fe-2S 簇和 FAD 的结合有关。 NqrF 中的四个保守半胱氨酸（C70、C76、C79 和 C111）与规范的 2Fe-2S 基序匹配，并且三个保守残基（R210、Y212、S246）预计是黄素结合域的一部分。在这项工作中，这两个基序已通过单个残基的定点诱变而改变，并被证实分别对于 2Fe-2S 簇和 FAD 的结合至关重要。氧化和还原形式的 FAD 缺陷突变体的 EPR 光谱分别表现出中性和阴离子黄酮半醌自由基信号，表明 NqrF 中的 FAD 不是任一自由基信号的来源。在 FAD 和 2Fe-2S 中心突变体中，中性和阴离子黄酮半醌 EPR 信号的线宽与野生型酶相比没有变化，表明这些中心都不在自由基附近或与自由基偶联。使用 NADH、Q-1 和人工电子受体铁氰化物对稳态周转的测量强烈支持电子传递途径模型，其中 NqrF 亚基中非共价结合的 FAD 是初始电子受体，然后电子流向 2Fe-2S 中心。
Many marine and pathogenic bacteria have a unique sodium-translocating NADH:ubiquinone oxidoreductase (Na+-NQR), which generates an electrochemical Na+ gradient during aerobic respiration. Na+-NQR consists of six subunits (NqrA-F) and contains five known redox cofactors: two covalently bound FMNs, one noncovalently bound FAD, one riboflavin, and one 2Fe-2S center. A stable neutral flavin-emiquinone radical is observed in the air-oxidized enzyme, while the NADH- or dithionite-reduced enzyme exhibits a stable anionic flavin-semiquinone radical. The NqrF subunit has been implicated in binding of both the 2Fe-2S cluster and the FAD. Four conserved cysteines (C70, C76, C79, and C111) in NqrF match the canonical 2Fe-2S motif, and three conserved residues (R210, Y212, S246) have been predicted to be part of a flavin binding domain. In this work, these two motifs have been altered by site-directed mutagenesis of individual residues and are confirmed to be essential for binding, respectively, the 2Fe-2S cluster and FAD. EPR spectra of the FAD-deficient mutants in the oxidized and reduced forms exhibit neutral and anionic flavo-semiquinone radical signals, respectively, demonstrating that the FAD in NqrF is not the source of either radical signal. In both the FAD and 2Fe-2S center mutants the line widths of the neutral and anionic flavo-semiquinone EPR signals are unchanged from the wild-type enzyme, indicating that neither of these centers is nearby or coupled to the radicals. Measurements of steady-state turnover using NADH, Q-1, and the artificial electron acceptor ferricyanide strongly support an electron transport pathway model in which the noncovalently bound FAD in the NqrF subunit is the initial electron acceptor and electrons then flow to the 2Fe-2S center.