Abstract
The Na+-translocating NADH:quinone oxidoreductase (NQR) is the entry site for electrons into the respiratory chain of Vibrio cholerae, the causative agent of cholera disease. NQR couples the electron transfer from NADH to ubiquinone to the translocation of sodium ions across the membrane. We investigated the pH dependence of electron transfer and generation of a transmembrane voltage (ΔΨ) by NQR reconstituted in liposomes with Na+ or Li+ as coupling cation. ΔΨ formation was followed with the voltage-sensitive dye oxonol. With Na+, ΔΨ was barely influenced by pH (6.5–8.5), while Q reduction activity exhibited a maximum at pH 7.5–8.0. With Li+, ΔΨ was generally lower, and the pH profile of electron transfer activity did not reveal a pronounced maximum. We conclude that the coupling efficiency of NQR is influenced by the nature of the transported cation, and by the concentration of protons. The 3D structure of NQR reveals a transmembrane channel in subunit NqrB. It is proposed that partial uncoupling of the NQR observed with the smaller Li+, or with Na+ at pH 7.5–8.0, is caused by the backflow of the coupling cation through the channel in NqrB.
Acknowledgments
This work was supported by contract research of the Baden-Württemberg Stiftung, Forschungsprogramm P-LS-Meth/4 (to J. S. and G.F.), by Deutsche Forschungsgemeinschaft grant FR 1321/3-1 (to J.S.), and a fellowship from the Carl Zeiss Foundation (to B.C.).
References
Apell, H.J. and Bersch, B. (1987). Oxonol VI as an optical indicator for membrane potentials in lipid vesicles. Biochim. Biophys. Acta. 903, 480–494.10.1016/0005-2736(87)90055-1Search in Google Scholar PubMed
Barquera, B., Hellwig, P., Zhou, W., Morgan, J.E., Häse, C.C., Gosink, K.K., Nilges, M., Bruesehoff, P.J., Roth A, Lancaster CR, et al. (2002). Purification and characterization of the recombinant Na+-translocating NADH:quinone oxidoreductase from Vibrio cholerae. Biochemistry 41, 3781–3789.10.1021/bi011873oSearch in Google Scholar PubMed
Bogachev, A.V., Murtazina, R.A., and Skulachev, V.P. (1997). The Na+/e− stoichiometry of the Na+-motive NADH:quinone oxidoreductase in Vibrio alginolyticus. FEBS Lett. 409, 475–477.10.1016/S0014-5793(97)00536-XSearch in Google Scholar
Bogachev, A.V., Bertsova, Y.V., Barquera, B., and Verkhovsky, M.I. (2001). Sodium-dependent steps in the redox reactions of the Na+-motive NADH:quinone oxidoreductase from Vibrio harveyi. Biochemistry 40, 7318–7323.10.1021/bi002545bSearch in Google Scholar PubMed
Casutt, M.S., Huber, T., Brunisholz, R., Tao, M., Fritz, G., and Steuber, J. (2010). Localization and function of the membrane-bound riboflavin in the Na+-translocating NADH:quinone oxidoreductase (Na+-NQR) from Vibrio cholerae. J. Biol. Chem. 285, 27088–27099.10.1074/jbc.M109.071126Search in Google Scholar PubMed PubMed Central
Dwivedi, M., Sukenik, S., Friedler, A., and Padan, E. (2016). The Ec-NhaA antiporter switches from antagonistic to synergistic antiport upon a single point mutation. Sci. Rep. 6, 23339.10.1038/srep23339Search in Google Scholar PubMed PubMed Central
Fritz, G. and Steuber, J. (2016). Sodium as coupling cation in respiratory energy conversion. Met. Ions Life Sci. 16, 349–390.10.1007/978-3-319-21756-7_11Search in Google Scholar PubMed
Glynn, I.M. and Karlish, S.J. (1990). Occluded cations in active transport. Annu. Rev. Biochem. 59, 171–205.10.1146/annurev.bi.59.070190.001131Search in Google Scholar PubMed
Gouaux, E. and MacKinnon, R. (2005). Principles of selective ion transport in channels and pumps. Science 310, 1461–1465.10.1126/science.1113666Search in Google Scholar PubMed
Griffitt, K.J. and Grimes, D.J. (2013). Abundance and distribution of Vibrio cholerae, V. parahaemolyticus, and V. vulnificus following a major freshwater intrusion into the Mississippi Sound. Microb. Ecol. 65, 578–583.10.1007/s00248-013-0203-6Search in Google Scholar PubMed
Häse, C.C. and Barquera, B. (2001). Role of sodium bioenergetics in Vibrio cholerae. Biochim. Biophys. Acta. 1505, 169–178.10.1016/S0005-2728(00)00286-3Search in Google Scholar
Juárez, O., Athearn, K., Gillespie, P., and Barquera, B. (2009a). Acid Residues in the Transmembrane helices of the Na+-pumping NADH:quinone oxidoreductase from Vibrio cholerae involved in sodium translocation. Biochemistry 48, 9516–9524.10.1021/bi900845ySearch in Google Scholar PubMed PubMed Central
Juárez, O., Morgan, J.E., and Barquera, B. (2009b). The electron transfer pathway of the Na+-pumping NADH:quinone oxidoreductase from Vibrio cholerae. J. Biol. Chem. 284, 8963–8972.10.1074/jbc.M809395200Search in Google Scholar PubMed PubMed Central
Juárez, O., Morgan, J.E., Nilges, M.J., and Barquera, B. (2010). Energy transducing redox steps of the Na+-pumping NADH:quinone oxidoreductase from Vibrio cholerae. Proc. Nat. Acad. Sci. USA 107, 12505–12510.10.1073/pnas.1002866107Search in Google Scholar PubMed PubMed Central
Juárez, O., Shea, M.E., Makhatadze, G.I., and Barquera, B. (2011). The role and specificity of the catalytic and regulatory cation-binding sites of the Na+-pumping NADH:quinone oxidoreductase from Vibrio cholerae. J. Biol. Chem. 286, 26383–26390.10.1074/jbc.M111.257873Search in Google Scholar PubMed PubMed Central
Juárez, O., Neehaul, Y., Turk, E., Chahboun, N., DeMicco, J.M., Hellwig, P., and Barquera, B. (2012). The role of glycine residues 140 and 141 of subunit B in the functional ubiquinone binding site of the Na+-pumping NADH:quinone oxidoreductase from Vibrio cholerae. J. Biol. Chem. 287, 25678–25685.10.1074/jbc.M112.366088Search in Google Scholar PubMed PubMed Central
Kaim, G., Wehrle, F., Gerike, U., and Dimroth, P. (1997). Molecular basis for the coupling ion selectivity of F1F0 ATP synthases: probing the liganding groups for Na+ and Li+ in the c subunit of the ATP synthase from Propionigenium modestum. Biochemistry 36, 9185–9194.10.1021/bi970831qSearch in Google Scholar PubMed
Kojima, S., Yamamoto, K., Kawagishi, I., and Homma, M. (1999). The polar flagellar motor of Vibrio cholerae is driven by an Na+ motive force. J. Bacteriol. 181, 1927–1930.10.1128/JB.181.6.1927-1930.1999Search in Google Scholar PubMed PubMed Central
Lin, P.C., Türk, K., Häse, C.C., Fritz, G., and Steuber, J. (2007). Quinone reduction by the Na+-translocating NADH dehydrogenase promotes extracellular superoxide production in Vibrio cholerae. J. Bacteriol. 189, 3902–3908.10.1128/JB.01651-06Search in Google Scholar PubMed PubMed Central
Muras, V., Claussen, B., Karuppasamy, M., Schaffitzel, C., and Steuber, J. (2014). Continuous fluorescence-based measurement of redox-driven sodium ion translocation. Anal. Biochem. 459, 53–55.10.1016/j.ab.2014.05.012Search in Google Scholar PubMed
Nedielkov, R., Steffen, W., Steuber, J., and Möller, H.M. (2013). NMR reveals double occupancy of quinone-type ligands in the catalytic quinone binding site of the Na+-translocating NADH:quinone oxidoreductase from Vibrio cholerae. J. Biol. Chem. 288, 30597–30606.10.1074/jbc.M112.435750Search in Google Scholar PubMed PubMed Central
Neehaul, Y., Juárez, O., Barquera, B., and Hellwig, P. (2013). Infrared spectroscopic evidence of a redox-dependent conformational change involving ion binding residue NqrB-D397 in the Na+ -pumping NADH:quinone oxidoreductase from Vibrio cholerae. Biochemistry 52, 3085–3093.10.1021/bi4000386Search in Google Scholar PubMed
Pfenninger-Li, X.D., Albracht, S.P., van Belzen, R., and Dimroth, P. (1996). NADH:ubiquinone oxidoreductase of Vibrio alginolyticus: purification, properties, and reconstitution of the Na+ pump. Biochemistry 35, 6233–6242.10.1021/bi953032lSearch in Google Scholar PubMed
Shani, M., Goldschleger, R., and Karlish, S.J. (1987). Rb+ occlusion in renal (Na++K+)-ATPase characterized with a simple manual assay. Biochim. Biophys. Acta. 904, 13–21.10.1016/0005-2736(87)90081-2Search in Google Scholar PubMed
Shea, M.E., Juárez, O., Cho, J., and Barquera, B. (2013). Aspartic acid 397 in subunit B of the Na+-pumping NADH:quinone oxidoreductase from Vibrio cholerae forms part of a sodium-binding site, is involved in cation selectivity, and affects cation-binding site cooperativity. J. Biol. Chem. 288, 31241–31249.10.1074/jbc.M113.510776Search in Google Scholar PubMed PubMed Central
Shea, M.E., Mezic, K.G., Juárez, O., and Barquera, B. (2015). A mutation in Na+ -NQR uncouples electron flow from Na+ translocation in the presence of K+. Biochemistry 54, 490–496.10.1021/bi501266eSearch in Google Scholar PubMed PubMed Central
Smith, P.K., Krohn, R.I., Hermanson, G.T., Mallia, A.K., Gartner, F.H., Provenzano, M.D., Fujimoto, E.K., Goeke, N.M., Olson BJ, Klenk DC. (1985). Measurement of protein using bicinchoninic acid. Anal. Biochem. 150, 76–85.10.1016/0003-2697(85)90442-7Search in Google Scholar PubMed
Steuber, J., Halang, P., Vorburger, T., Steffen, W., Vohl, G., and Fritz, G. (2014a). Central role of the Na+-translocating NADH:quinone oxidoreductase (Na+-NQR) in sodium bioenergetics of Vibrio cholerae. Biol. Chem. 395, 1389–1399.10.1515/hsz-2014-0204Search in Google Scholar PubMed
Steuber, J., Vohl, G., Casutt, M.S., Vorburger, T., Diederichs, K., and Fritz, G. (2014b). Structure of the V. cholerae Na+-pumping NADH:quinone oxidoreductase. Nature 516, 62–67.10.1038/nature14003Search in Google Scholar PubMed
Strickland, M., Juárez, O., Neehaul, Y., Cook, D.A., Barquera, B., and Hellwig, P. (2014). The conformational changes induced by ubiquinone binding in the Na+-pumping NADH:ubiquinone oxidoreductase (Na+-NQR) are kinetically controlled by conserved glycines 140 and 141 of the NqrB subunit. J. Biol. Chem. 289, 23723–23733.10.1074/jbc.M114.574640Search in Google Scholar PubMed PubMed Central
Tao, M., Casutt, M.S., Fritz, G., and Steuber, J. (2008). Oxidant-induced formation of a neutral flavosemiquinone in the Na+-translocating NADH:Quinone oxidoreductase (Na+-NQR) from Vibrio cholerae. Biochim. Biophys. Acta. 1777, 696–702.10.1016/j.bbabio.2008.04.006Search in Google Scholar PubMed
Tuz, K., Mezic, K.G., Xu, T., Barquera, B., and Juárez, O. (2015). The kinetic reaction mechanism of the Vibrio cholerae sodium-dependent NADH Dehydrogenase. J. Biol. Chem. 290, 20009–20021.10.1074/jbc.M115.658773Search in Google Scholar PubMed PubMed Central
Verkhovsky, M.I. and Bogachev, A.V. (2010). Sodium-translocating NADH:quinone oxidoreductase as a redox-driven ion pump. Biochim. Biophys. Acta. 1797, 738–746.10.1016/j.bbabio.2009.12.020Search in Google Scholar PubMed
Verkhovsky, M.I., Bogachev, A.V., Pivtsov, A.V., Bertsova, Y.V., Fedin, M.V., Bloch, D.A., and Kulik, L.V. (2012). Sodium-Dependent movement of covalently bound FMN residue(s) in Na+-translocating NADH:quinone oxidoreductase. Biochemistry 51, 5414–5421.10.1021/bi300322nSearch in Google Scholar PubMed
Vimont, S. and Berche, P. (2000). NhaA, an Na+/H+ antiporter involved in environmental survival of Vibrio cholerae. J. Bacteriol. 182, 2937–2944.10.1128/JB.182.10.2937-2944.2000Search in Google Scholar PubMed PubMed Central
Vorburger, T., Nedielkov, R., Brosig, A., Bok, E., Schunke, E., Steffen, W., Mayer, S., Götz, F., Möller, H.M., Steuber, J. (2016). Role of the Na+ -translocating NADH:quinone oxidoreductase in voltage generation and Na+ extrusion in Vibrio cholerae. Biochim. Biophys. Acta. 1857, 473–482.10.1016/j.bbabio.2015.12.010Search in Google Scholar PubMed
©2017 Walter de Gruyter GmbH, Berlin/Boston
