NADH:Ubiquinone Oxidoreductase of Vibrio alginolyticus: Purification, Properties, and Reconstitution of the Na+ Pump

Abstract

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.