Kinetics of the spectral changes during reduction of the Na+-motive NADH:quinone oxidoreductase from Vibrio harveyi
Kinetics of the spectral changes during reduction of the Na+-motive NADH:quinone oxidoreductase from Vibrio harveyi
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DOI：
10.1016/s0005-2728(02)00342-0
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发表时间：
2002-12-02
影响因子：
4.3
通讯作者：
Verkhovsky, MI
中科院分区：
文献类型：
--
作者：
Bogachev, AV;Bertsova, YV;Verkhovsky, MI
用电子顺磁共振波谱技术在哈氏弧菌的Na+转运型NADH：泛醌氧化还原酶(Na+-NQR)中观察到两个不同线宽的自由基信号。第一个自由基是在氧化酶中观察到的，并被指定为中性黄半喹酮。第二个自由基在还原的酶中观察到，并被指定为黄半喹酮的阴离子形式。由停流光谱学监测的NADH还原Na+-NQR的时间过程显示出三个不同的相，其光谱表明它们对应于三个不同的黄素物种的还原。第一阶段在有钠和无钠的情况下都很快，在NADH脱氢中心将FAD还原为FADH(2)。另外两个相的速率强烈依赖于钠浓度，这些相归因于两个共价结合的FMN的还原，结合光学和EPR数据表明，氧化酶中存在一个中性的FMN黄半喹酮，它被NADH还原为完全还原的黄素。另一个FMN部分最初被氧化，并被还原为阴离子黄半喹酮。因此，在Na+-NQR的催化循环中，两个离散的黄素物种的单电子跃迁被认为是钠相关的步骤。(C)2002 Elsevier Science B.V.保留所有权利。
Two radical signals with different line widths are seen in the Na+-translocating NADH:ubiquinone oxidoreductase (Na+-NQR) from Vibrio harveyi by EPR spectroscopy. The first radical is observed in the oxidized enzyme, and is assigned as a neutral flavosemiquinone. The second radical is observed in the reduced enzyme and is assigned to be the anionic form of flavosemiquinone. The time course of Na+-NQR reduction by NADH, as monitored by stopped-flow optical spectroscopy, shows three distinct phases, the spectra of which suggest that they correspond to the reduction of three different flavin species. The first phase is fast both in the presence and absence of sodium, and is assigned to reduction of FAD to FADH(2) at the NADH dehydrogenating site. The rates of the other two phases are strongly dependent on sodium concentration, and these phases are attributed to reduction of two covalently bound FMN's. Combination of the optical and EPR data suggests that a neutral FMN flavosemiquinone preexists in the oxidized enzyme, and that it is reduced to the fully reduced flavin by NADH. The other FMN moiety is initially oxidized, and is reduced to the anionic flavosemiquinone. One-electron transitions of two discrete flavin species are thus assigned as sodium-dependent steps in the catalytic cycle of Na+-NQR. (C) 2002 Elsevier Science B.V. All rights reserved.