The NADH: ubiquinone oxidoreductase of Vibrio Alginolyticus: purification, properties and reconstitution of the Na+ pump. Pfenniger-Li, X.D.; Albracht, S.P.J.; van Belzen, R.; Dimroth, P. Published in: Biochemistry DOI:10.1021/bi953032lLink to publication Citation for published version (APA):Pfenniger-Li, X. D., Albracht, S. P. J., van Belzen, R., & Dimroth, P. (1996). The NADH: ubiquinone oxidoreductase of Vibrio Alginolyticus: purification, properties and reconstitution of the Na+ pump. Biochemistry, 35, 6233-6242. DOI: 10.1021/bi953032l General rights It is not permitted to download or to forward/distribute the text or part of it without the consent of the author(s) and/or copyright holder(s), other than for strictly personal, individual use, unless the work is under an open content license (like Creative Commons). Disclaimer/Complaints regulationsIf you believe that digital publication of certain material infringes any of your rights or (privacy) interests, please let the Library know, stating your reasons. In case of a legitimate complaint, the Library will make the material inaccessible and/or remove it from the website. Please Ask the Library: http://uba.uva.nl/en/contact, or a letter to: Library of the University of Amsterdam, Secretariat, Singel 425, 1012 WP Amsterdam, The Netherlands. You will be contacted as soon as possible. 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 O 2 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 + tra...

The iron-sulfur clusters 2 and ubisemiquinone radicals of NADH: ubiquinone oxidoreductase are involved in energy coupling in submitochondrial particles van Belzen, R.; Kotlyar, A.B.; Moon, N.; Dunham, W.R.; Albracht, S.P.J. General rightsIt is not permitted to download or to forward/distribute the text or part of it without the consent of the author(s) and/or copyright holder(s), other than for strictly personal, individual use, unless the work is under an open content license (like Creative Commons). Disclaimer/Complaints regulationsIf you believe that digital publication of certain material infringes any of your rights or (privacy) interests, please let the Library know, stating your reasons. In case of a legitimate complaint, the Library will make the material inaccessible and/or remove it from the website. Please Ask the Library: http://uba.uva.nl/en/contact, or a letter to: Library of the University of Amsterdam, Secretariat, Singel 425, 1012 WP Amsterdam, The Netherlands. You will be contacted as soon as possible. [163][164][165][166][167][168][169][170][171], are the result of a spin-spin interaction of 2.8 mT. Investigation of the radical signals present in coupled SMP indicated that more than 90% of the radicals can be ascribed to two types of semiquinones which are bound to Complex I (Q I -radicals) or ubiquinol:cytochrome c oxidoreductase (Complex III; Q III -radicals). The presence of Q III -radicals, but not that of Q I -radicals, was completely abolished by uncoupler. Part of the Q I -radicals weakly interact with the clusters 2 of Complex I. This uncoupler-sensitive interaction can amount to a splitting of the radical EPR signal of at most 1 mT, considerably weaker than the 2.8 mT splitting of the g z lines of the clusters 2. We propose that the 2.8 mT splitting of these g z lines results from an energy-induced spin-spin interaction between the two clusters 2 within the TYKY subunit of Complex I. The two clusters 2 show no interaction during electron transfer in uncoupled SMP or in fully-reduced anaerobic-coupled SMP. The results point to a direct role of the Fe-S clusters 2 and the Q I -radicals in the mechanism of coupled electron transfer catalyzed by Complex I.

No abstract

The nuoI gene that encodes a ferredoxin-like subunit of the Rhodobacter capsulatus Complex I (a subunit equivalent to the bovine TYKY subunit) was mutated by homologous recombination. Both a nuoI-deleted mutant (∆nuoI mutant) and a point mutant in which Cys74 was replaced by a serine (C74S mutant) proved to be completely deficient in Complex I activity. These strains were unable to grow under anaerobic photosynthetic conditions. Their cytoplasmic membranes were also characterized by the absence of specific EPR signals assigned to FeS clusters N1 and N2. Immunochemical analysis of the mutant membranes with subunit-specific antibodies showed that the peripheral subunits were not assembled. Trans-complementation of the mutant strains by a native nuoI gene restored the wild-type phenotypes. In the C74S mutant, a limited amount of NuoI subunit still bound to the membraneous domain of Complex I, which is an indication that NuoI directly interacts with this domain. All these results clearly show that NuoI plays a critical role in the connection between the membraneous domain and the peripheral domain of Complex I.