NADH-UBIQUINONE OXIDOREDUCTASE OF VIBRIO-ALGINOLYTICUS - PURIFICATION, PROPERTIES, AND RECONSTITUTION OF THE NA+ PUMP

The Na+-activated NADH:ubiquinone oxidoreductase of Vibrio alginolytic us was extracted from the membranes with lauryldimethylamine-N-oxide a nd 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 artific ial electron acceptor) and ubiquinone-l (Q) reductase activity. On fur ther fractionation of the enzyme, the Q-reductase activity essentially disappeared. Chemical analyses revealed the presence of FAD but not F MN, of non-heme iron and of acid-labile sulfur and tightly-bound ubiqu inone-8 in the purified Q-reductase preparation. The participation of an iron-sulfur cluster of the [2Fe-2S] type in the electron translocat ion was demonstrated by the appearance of a typical EPR signal for thi s prosthetic group after the reduction of Q-reductase with NADH. A str ong EPR signal typical for a radical observed upon reduction of the en zyme might arise from the formation of quinone radicals. In the absenc e of Na+, the path of the electrons apparently ends with the reduction of ubiquinone-l to the semiquinone derivative which in the presence o f O-2 becomes reoxidized with concomitant formation of superoxide radi cals. In the presence of Na+, these oxygen radicals are not formed and the semiquinone is further reduced to the quinol derivative. These re sults indicate that the Na+-dependent step in the electron transfer ca talyzed by NADH:ubiquinone oxidoreductase is the reduction of ubisemiq uinone to ubiquinol. After reconstitution of the purified Q-reductase into proteoliposomes, NADH oxidation by ubiquinone-l was coupled to Na + transport with an apparent stoichiometry of 0.5 Na+ per NADH oxidize d. The transport was stimulated by valinomycin (+K+) or by the uncoupl er carbonyl cyanide m-chlorophenylhydrazone (CCCP), The transport of N a+ is therefore a primary event and does not involve the intermediate formation of a proton gradient.