NADH:ubiquinone oxidoreductase of Vibrio alginolyticus: purification, properties, and reconstitution of the Na+ pump.
NADH:ubiquinone oxidoreductase of Vibrio alginolyticus: purification, properties, and reconstitution of the Na+ pump.
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NADH：溶藻弧菌的泛醌氧化还原酶：钠泵的纯化、特性和重建。
DOI：
10.1021/bi953032l
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发表时间：
1996
期刊：
影响因子：
2.9
通讯作者：
P. Dimroth
中科院分区：
文献类型：
--
作者：
X. D. Pfenninger;S. Albracht;R. van Belzen;P. Dimroth
用N-氧化月桂基二甲胺从膜中提取溶藻弧菌的Na+激活的NADH：泛醌氧化还原酶，并通过两个连续的阴离子交换柱纯化。该制备物在SDS-PAGE后产生四条主要染色带和几条次要染色带，保留了NADH-脱氢酶活性（以甲萘醌作为人工电子受体）和泛醌-1（Q）还原酶活性。在进一步分馏的酶，Q-还原酶活性基本上消失。化学分析表明，在纯化的Q-还原酶制剂中存在FAD但不存在FMN、非血红素铁和酸不稳定硫以及紧密结合的泛醌-8。参与的[2Fe-2S]型的铁-硫簇的电子易位证明了一个典型的EPR信号的外观后，Q-还原酶与NADH还原的辅基。一个强大的EPR信号典型的自由基观察后，还原酶可能会产生醌自由基的形成。在不存在Na+的情况下，电子的路径显然结束于泛醌-1还原成半醌衍生物，该半醌衍生物在O2的存在下变得再氧化，伴随着形成超氧自由基。在Na+存在下，这些氧自由基不会形成，半醌进一步还原为醌醇衍生物。这些结果表明，在由NADH：泛醌氧化还原酶催化的电子转移中的Na+依赖步骤是泛半醌还原为泛醇。纯化的Q-还原酶重组成脂蛋白体后，辅酶Q-1的NADH氧化与Na+转运偶联，表观化学计量为0.5 Na+/氧化的NADH。缬氨霉素（+ K+）或解偶联剂羰基氰间氯苯腙（CCCP）刺激转运。因此，Na+的运输是一个主要事件，不涉及质子梯度的中间形成。
The Na+-activated NADH:ubiquinone oxidoreductase of Vibrio alginolyticus was extracted from the membranes with lauryldimethylamine-N-oxide and purified by two successive anion exchange columns. This preparation, yielding four major and several minor stained bands after SDS-PAGE, retained the NADH-dehydrogenase activity (with menadione as an artificial electron acceptor) and ubiquinone-1 (Q) reductase activity. On further fractionation of the enzyme, the Q-reductase activity essentially disappeared. Chemical analyses revealed the presence of FAD but not FMN, of non-heme iron and of acid-labile sulfur and tightly-bound ubiquinone-8 in the purified Q-reductase preparation. The participation of an iron-sulfur cluster of the [2Fe-2S] type in the electron translocation was demonstrated by the appearance of a typical EPR signal for this prosthetic group after the reduction of Q-reductase with NADH. A strong EPR signal typical for a radical observed upon reduction of the enzyme might arise from the formation of quinone radicals. In the absence of Na+, the path of the electrons apparently ends with the reduction of ubiquinone-1 to the semiquinone derivative which in the presence of O2 becomes reoxidized with concomitant formation of superoxide radicals. In the presence of Na+, these oxygen radicals are not formed and the semiquinone is further reduced to the quinol derivative. These results indicate that the Na+-dependent step in the electron transfer catalyzed by NADH:ubiquinone oxidoreductase is the reduction of ubisemiquinone to ubiquinol. After reconstitution of the purified Q-reductase into proteoliposomes, NADH oxidation by ubiquinone-1 was coupled to Na+ transport with an apparent stoichiometry of 0.5 Na+ per NADH oxidized. The transport was stimulated by valinomycin (+ K+) or by the uncoupler carbonyl cyanide m-chlorophenylhydrazone (CCCP). The transport of Na+ is therefore a primary event and does not involve the intermediate formation of a proton gradient.