NADH:Ubiquinone Oxidoreductase of Vibrio alginolyticus: Purification, Properties, and Reconstitution of the Na+ Pump
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- 作者：Xiao Dan Pfenninger-Li ; Simon P. J. Albracht ; Ronald van Belzen ; and Peter Dimroth
- 刊名：Biochemistry
- 出版年：1996
- 出版时间：May 21, 1996
- 年：1996
- 卷：35
- 期：20
- 页码：6233 - 6242
- 全文大小：487K
- 年卷期：v.35,no.20(May 21, 1996)
- ISSN：1520-4995
文摘
The Na+-activated NADH:ubiquinoneoxidoreductase of Vibrio alginolyticus wasextractedfrom the membranes with lauryldimethylamine-N-oxide andpurified by two successive anion exchangecolumns. This preparation, yielding four major and several minorstained bands after SDS-PAGE, retainedthe NADH-dehydrogenase activity (with menadione as an artificialelectron acceptor) and ubiquinone-1(Q) reductase activity. On further fractionation of the enzyme,the Q-reductase activity essentiallydisappeared. Chemical analyses revealed the presence of FAD butnot FMN, of non-heme iron and ofacid-labile sulfur and tightly-bound ubiquinone-8 in the purifiedQ-reductase preparation. The participationof an iron-sulfur cluster of the [2Fe-2S] type in the electrontranslocation was demonstrated by theappearance of a typical EPR signal for this prosthetic group after thereduction of Q-reductase with NADH.A strong EPR signal typical for a radical observed upon reductionof the enzyme might arise from theformation of quinone radicals. In the absence ofNa+, the path of the electrons apparently ends withthereduction of ubiquinone-1 to the semiquinone derivative which in thepresence of O2 becomes reoxidizedwith concomitant formation of superoxide radicals. In the presenceof Na+, these oxygen radicals are notformed and the semiquinone is further reduced to the quinol derivative.These results indicate that theNa+-dependent step in the electron transfer catalyzed byNADH:ubiquinone oxidoreductase is the reductionof ubisemiquinone to ubiquinol. After reconstitution of thepurified Q-reductase into proteoliposomes,NADH oxidation by ubiquinone-1 was coupled to Na+transport with an apparent stoichiometry of 0.5Na+ per NADH oxidized. The transport was stimulatedby valinomycin (+K+) or by the uncouplercarbonylcyanide m-chlorophenylhydrazone (CCCP). The transportof Na+ is therefore a primary event and doesnot involve the intermediate formation of a protongradient.