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The structure of Na+-translocating of NADH:ubiquinone oxidoreductase of Vibrio cholerae: implications on coupling between electron transfer and Na+ transport

Julia Steuber, Georg Vohl, Valentin Muras, Charlotte Toulouse, Björn Claußen, Thomas Vorburger and Günter Fritz
From the journal Biological Chemistry

Abstract

The Na+-translocating NADH:ubiquinone oxidoreductase (Na+-NQR) of Vibrio cholerae is a respiratory complex that couples the exergonic oxidation of NADH to the transport of Na+ across the cytoplasmic membrane. It is composed of six different subunits, NqrA, NqrB, NqrC, NqrD, NqrE, and NqrF, which harbor FAD, FMN, riboflavin, quinone, and two FeS centers as redox co-factors. We recently determined the X-ray structure of the entire Na+-NQR complex at 3.5-Å resolution and complemented the analysis by high-resolution structures of NqrA, NqrC, and NqrF. The position of flavin and FeS co-factors both at the cytoplasmic and the periplasmic side revealed an electron transfer pathway from cytoplasmic subunit NqrF across the membrane to the periplasmic NqrC, and via NqrB back to the quinone reduction site on cytoplasmic NqrA. A so far unknown Fe site located in the midst of membrane-embedded subunits NqrD and NqrE shuttles the electrons over the membrane. Some distances observed between redox centers appear to be too large for effective electron transfer and require conformational changes that are most likely involved in Na+ transport. Based on the structure, we propose a mechanism where redox induced conformational changes critically couple electron transfer to Na+ translocation from the cytoplasm to the periplasm through a channel in subunit NqrB.


Corresponding author: Günter Fritz, Institute of Neuropathology, University of Freiburg, Breisacher Strasse 64, D-79106 Freiburg, Germany, e-mail:

Acknowledgments

This work was supported by contract research ‘Methoden in den Lebenswissenschaften’ of the Baden-Württemberg Stiftung P-LS-Meth/4 (to J.S. and G.F.) and by the Deutsche Forschungsgemeinschaft grant FR 1321/3-1 (to J.S.) and grant FR 1488/3-2 (to G.F.).

References

Baradaran, R., Berrisford, J.M., Minhas, G.S., and Sazanov, L.A. (2013). Crystal structure of the entire respiratory complex I. Nature 494, 443–448. Search in Google Scholar

Barquera, B., Hellwig, P., Zhou, W., Morgan, J.E., Häse, C.C., Gosink, K.K., Nilges, M., Bruesehoff, P.J., Roth, A., Lancaster, C.R., et al. (2002a). Purification and characterization of the recombinant Na+-translocating NADH:quinone oxidoreductase from Vibrio cholerae. Biochemistry 41, 3781–3789. Search in Google Scholar

Barquera, B., Zhou, W., Morgan, J.E., and Gennis, R.B. (2002b). Riboflavin is a component of the Na+-pumping NADH-quinone oxidoreductase from Vibrio cholerae. Proc. Natl. Acad. Sci. USA 99, 10322–10324. Search in Google Scholar

Batista, A.P., Marreiros, B.C., Louro, R.O., and Pereira, M.M. (2012). Study of ion translocation by respiratory complex I. A new insight using 23Na NMR spectroscopy. Biochim. Biophys. Acta 1817, 1810–1816. Search in Google Scholar

Batista, A.P., Marreiros, B.C. and Pereira, M.M. (2013). The antiporter-like subunit constituent of the universal adaptor of complex I, group 4 membrane-bound [NiFe]-hydrogenases and related complexes. Biol. Chem. 394, 659–666. Search in Google Scholar

Bertsova, Y.V., Fadeeva, M.S., Kostyrko, V.A., Serebryakova, M.V., Baykov, A.A., and Bogachev, A.V. (2013). Alternative pyrimidine biosynthesis protein ApbE is a flavin transferase catalyzing covalent attachment of FMN to a threonine residue in bacterial flavoproteins. J. Biol. Chem. 288, 14276–14286. Search in Google Scholar

Bertsova, Y.V., Kostyrko, V.A., Baykov, A.A., and Bogachev, A.V. (2014). Localization-controlled specificity of FAD:threonine flavin transferases in Klebsiella pneumoniae and its implications for the mechanism of Na+-translocating NADH:quinone oxidoreductase. Biochim. Biophys. Acta 1837, 1122–1129. Search in Google Scholar

Biegel, E. and Müller, V. (2010). Bacterial Na+-translocating ferredoxin:NAD+ oxidoreductase. Proc. Natl. Acad. Sci. USA 107, 18138–18142. Search in Google Scholar

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. Search 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. Search in Google Scholar

Bogachev, A.V., Bertsova, Y.V., Bloch, D.A., and Verkhovsky, M.I. (2006). Thermodynamic properties of the redox centers of Na+-translocating NADH:quinone oxidoreductase. Biochemistry 45, 3421–3428. Search in Google Scholar

Bogachev, A.V., Bertsova, Y.V., Aitio, O., Permi, P., Verkhovsky, M.I. (2007). Redox-dependent sodium binding by the Na+-translocating NADH:quinone oxidoreductase from Vibrio harveyi. Biochemistry 46, 10186–10191. Search in Google Scholar

Bogachev, A.V., Belevich, N.P., Bertsova, Y.V., and Verkhovsky, M.I. (2009a). Primary steps of the Na+-translocating NADH:ubiquinone oxidoreductase catalytic cycle resolved by the ultrafast freeze-quench approach. J. Biol. Chem. 284, 5533–5538. Search in Google Scholar

Bogachev, A.V., Bloch, D.A., Bertsova, Y.V., and Verkhovsky, M.I. (2009b). Redox properties of the prosthetic groups of Na+-translocating NADH:quinone oxidoreductase. 2. Study of the enzyme by optical spectroscopy. Biochemistry 48, 6299–6304. Search in Google Scholar

Bogachev, A. V., Kulik, L.V., Bloch, D.A., Bertsova, Y.V., Fadeeva, M.S., and Verkhovsky, M.I. (2009c). Redox properties of the prosthetic groups of Na+-translocating NADH:quinone oxidoreductase. 1. electron paramagnetic resonance study of the enzyme. Biochemistry 48, 6291–6298. Search in Google Scholar

Borshchevskiy, V., Round, E., Bertsova, Y., Polovinkin, V., Gushchin, I., A. Ishchenko, A., Kovalev, K., Mishin, A., Kachalova, G., Popov, A., et al. (2015). Structural and functional investigation of flavin binding center of the NqrC subunit of sodium-translocating NADH:quinone oxidoreductase from Vibrio harveyi. PLoS One 10, e0118548. Search in Google Scholar

Brown, K.A., Howell, E.E., and Kraut, J. (1993). Long-range structural effects in a second-site revertant of a mutant dihydrofolate reductase. Proc. Natl. Acad. Sci. USA 90, 11753–11756. Search in Google Scholar

Buckel, W. and Thauer, R.K. (2013). Energy conservation via electron bifurcating ferredoxin reduction and proton/Na+ translocating ferredoxin oxidation. Biochim. Biophys. Acta 1827, 94–113. Search in Google Scholar

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. Search in Google Scholar

Casutt, M.S., Nedielkov, R., Wendelspiess, S., Vossler, S., Gerken, U., M. Murai, M., Miyoshi, H., Moller, H.M., and Steuber, J. (2011). Localization of ubiquinone-8 in the Na+-pumping NADH:quinone oxidoreductase from Vibrio cholerae. J. Biol. Chem. 286, 40075–40082. Search in Google Scholar

Casutt, M.S., Schlosser, A., Buckel, W., and Steuber, J. (2012). The single NqrB and NqrC subunits in the Na+-translocating NADH:quinone oxidoreductase (Na+-NQR) from Vibrio cholerae each carry one covalently attached FMN. Biochim. Biophys. Acta 1817, 1817–1822. Search in Google Scholar

Chowdhury, N.P., Mowafy, A.M., Demmer, J.K., Upadhyay, V., Koelzer, S., Jayamani, E., Kahnt, J., Hornung, M., Demmer, U., Ermler, U., et al. (2014). Studies on the mechanism of electron bifurcation catalyzed by electron transferring flavoprotein (Etf) and butyryl-CoA dehydrogenase (Bcd) of Acidaminococcus fermentans. J. Biol. Chem. 289, 5145–5157. Search in Google Scholar

Cooley, J.W. (2013). Protein conformational changes involved in the cytochrome bc1 complex catalytic cycle. Biochim. Biophys. Acta 1827, 1340–1345. Search in Google Scholar

Darrouzet, E., Moser, C.C., Dutton, P.L., and Daldal, F. (2001). Large scale domain movement in cytochrome bc1: a new device for electron transfer in proteins. Trends Biochem. Sci. 26, 445–451. Search in Google Scholar

Duffy, E.B. and Barquera, B. (2006). Membrane topology mapping of the Na+-pumping NADH:quinone oxidoreductase from Vibrio cholerae by PhoA-green fluorescent protein fusion analysis. J. Bacteriol. 188, 8343–8351. Search in Google Scholar

Fadeeva, M.S., Nunez, C., Bertsova, Y.V., Espin, G., and Bogachev, A.V. (2008). Catalytic properties of Na+-translocating NADH:quinone oxidoreductases from Vibrio harveyi, Klebsiella pneumoniae, and Azotobacter vinelandii. FEMS Microbiol. Lett. 279, 116–123. Search in Google Scholar

Gemperli, A.C., Dimroth, P., and Steuber, J. (2002). The respiratory complex I (NDH I) from Klebsiella pneumoniae, a sodium pump. J. Biol. Chem. 277, 33811–33817. Search in Google Scholar

Häse, C.C., Fedorova, N.D., Galperin, M.Y., and Dibrov, P.A. (2001). Sodium ion cycle in bacterial pathogens: evidence from cross-genome comparisons. Microbiol. Mol. Biol. Rev. 65, 353–370. Search in Google Scholar

Hayashi, M. and Unemoto, T. (1987). Subunit component and their roles in the sodium-transport NADH: quinone reductase of a marine bacterium, Vibrio alginolyticus. Biochim. Biophys. Acta 890, 47–54. Search in Google Scholar

Hayashi, M., Hirai, K., and Unemoto, T. (1995). Sequencing and the alignment of structural genes in the nqr operon encoding the Na+-translocating NADH-quinone reductase from Vibrio alginolyticus. FEBS Lett. 363, 75–77. Search in Google Scholar

Hayashi, M., Shibata, N., Nakayama, Y., Yoshikawa, K., and Unemoto, T. (2002). Korormicin insensitivity in Vibrio alginolyticus is correlated with a single point mutation of Gly-140 in the NqrB subunit of the Na+-translocating NADH-quinone reductase. Arch. Biochem. Biophys. 401, 173–177. Search in Google Scholar

Hess, V., Schuchmann, K., and Müller, V. (2013). The ferredoxin:NAD+ oxidoreductase (Rnf) from the acetogen Acetobacterium woodii requires Na+ and is reversibly coupled to the membrane potential. J. Biol. Chem. 288, 31496–31502. Search in Google Scholar

Juarez, O. and Barquera, B. (2012). Insights into the mechanism of electron transfer and sodium translocation of the Na+-pumping NADH:quinone oxidoreductase. Biochim. Biophys. Acta 1817, 1823–1832. Search in Google Scholar

Juárez, O., Nilges, M.J., Gillespie, P., Cotton, J., and Barquera, B. (2008). Riboflavin is an active redox cofactor in the Na+-pumping NADH:quinone oxidoreductase (Na+-NQR) from Vibrio cholerae. J. Biol. Chem. 283, 33162–33167. Search in Google Scholar

Juarez, O., Morgan, J.E., and Barquera, B. (2009a). The electron transfer pathway of the Na+-pumping NADH:quinone oxidoreductase from Vibrio cholerae. J. Biol. Chem. 284, 8963–8972. Search in Google Scholar

Juarez, O., Athearn, K., Gillespie, P., and Barquera, B. (2009b). Acid residues in the transmembrane helices of the Na+-pumping NADH:quinone oxidoreductase from Vibrio cholerae involved in sodium translocation. Biochemistry 48, 9516–9524. Search in Google Scholar

Juarez, 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. Natl. Acad. Sci. USA 107, 12505–12510. Search in Google Scholar

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. Search in Google Scholar

Karplus, P.A. and Faber, H.R. (2004). Structural aspects of plant ferredoxin:NADP+ oxidoreductases. Photosynth. Res. 81, 303–315. Search in Google Scholar

Krebs, W., Steuber, J., Gemperli, A.C., and Dimroth, P. (1999). Na+ translocation by the NADH:ubiquinone oxidoreductase (complex I) from Klebsiella pneumoniae. Mol. Microbiol. 33, 590–598. Search in Google Scholar

Mitchell, P. (1961). Coupling of phosphorylation to electron and hydrogen transfer by a chemi-osmotic type of mechanism. Nature 191, 144–148. Search in Google Scholar

Mitchell, P. and Moyle, J. (1969). Estimation of membrane potential and pH difference across the cristae membrane of rat liver mitochondria. Eur. J. Biochem. 7, 471–484. Search in Google Scholar

Mulo, P. (2011). Chloroplast-targeted ferredoxin-NADP+ oxidoreductase (FNR): structure, function and location. Biochim. Biophys. Acta 1807, 927–934. Search in Google Scholar

Nakayama, Y., Yasui, M., Sugahara, K., Hayashi, M., and Unemoto, T. (2000). Covalently bound flavin in the NqrB and NqrC subunits of Na+-translocating NADH-quinone reductase from Vibrio alginolyticus. FEBS Lett. 474, 165–168. Search in Google Scholar

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. Search in Google Scholar

Neehaul, Y., Juarez, 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. Search in Google Scholar

Nicholls, D.G. and Ferguson, S.J. (2013). Bioenergetics 4. (London: Academic Press). Search in Google Scholar

Page, C.C., Moser, C.C., Chen, X., and Dutton, P.L. (1999). Natural engineering principles of electron tunnelling in biological oxidation-reduction. Nature 402, 47–52. Search in Google Scholar

Petrek, M., Otyepka, M., Banas, P., Kosinova, P., Koca, J., and Damborsky, J. (2006). CAVER: a new tool to explore routes from protein clefts, pockets and cavities. BMC Bioinformatics 7, 316. Search in Google Scholar

Pfenninger-Li, X.D. and Dimroth, P. (1995). The Na+-translocating NADH:ubiquinone oxidoreductase from the marine bacterium Vibrio alginolyticus contains FAD but not FMN. FEBS Lett. 369, 173–176. Search in Google Scholar

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. Search in Google Scholar

Reyes-Prieto, A., Barquera, B., and Juarez, O. (2014). Origin and evolution of the sodium-pumping NADH:ubiquinone oxidoreductase. PLoS One 9, e96696. Search in Google Scholar

Sääf, A., Johansson, M., Wallin, E., and von Heijne, G. (1999). Divergent evolution of membrane protein topology: the Escherichia coli RnfA and RnfE homologues. Proc. Natl. Acad. Sci. USA 96, 8540–8544. Search in Google Scholar

Shea, M.E., Juarez, 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. Search in Google Scholar

Song, F., Hurtado del Pozo, C., Rosario, R., Zou, Y.S., Ananthakrishnan, R., Xu, X., Patel, P.R., Benoit, V.M., Yan, S.F., Li, H., et al. (2014). RAGE regulates the metabolic and inflammatory response to high-fat feeding in mice. Diabetes 63, 1948–1965. Search in Google Scholar

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. Search in Google Scholar

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. Search in Google Scholar

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. Search in Google Scholar

Terwilliger, T.C., Klei, H., Adams, P.D., Moriarty, N.W., and Cohn, J.D. (2006). Automated ligand fitting by core-fragment fitting and extension into density. Acta Crystallogr. D 62, 915–922. Search in Google Scholar

Türk, K., Puhar, A., Neese, F., Bill, E., Fritz, G., and Steuber, J. (2004). NADH oxidation by the Na+-translocating NADH:quinone oxidoreductase from Vibrio cholerae: functional role of the NqrF subunit. J. Biol. Chem. 279, 21349–21355. Search in Google Scholar

Unemoto, T. and Hayashi, M. (1977). Na+-dependent activation of NADH oxidase in membrane fractions from halophilic Vibrio alginolyticus and V. costicolus. J. Biochem. 82, 1389–1395. Search in Google Scholar

Unemoto, T. and Hayashi, M. (1979). NADH:quinone oxidoreductase as a site of Na+-dependent activation in the respiratory chain of marine Vibrio alginolyticus. J. Biochem. 85, 1461–1467. Search in Google Scholar

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. Search in Google Scholar

Vinothkumar, K.R., Zhu, J., and Hirst, J. (2014). Architecture of mammalian respiratory complex I. Nature 515, 80–84. Search in Google Scholar

Xie, W., Nangle, L.A., Zhang, W., Schimmel, P., and Yang, X.L. (2007). Long-range structural effects of a Charcot-Marie-Tooth disease-causing mutation in human glycyl-tRNA synthetase. Proc. Natl. Acad. Sci. USA 104, 9976–9981. Search in Google Scholar

Zhang, T., Liu, L.A., Lewis, D.F., and Wei, D.Q. (2011). Long-range effects of a peripheral mutation on the enzymatic activity of cytochrome P450 1A2. J. Chem. Inf. Model. 51, 1336–1346. Search in Google Scholar

Zhou, W., Bertsova, Y.V., Feng, B., Tsatsos, P., Verkhovskaya, M.L., Gennis, R.B., Bogachev, A.V., and Barquera, B. (1999). Sequencing and preliminary characterization of the Na+-translocating NADH:ubiquinone oxidoreductase from Vibrio harveyi. Biochemistry 38, 16246–16252. Search in Google Scholar

Zickermann, V., Wirth, C., Nasiri, H., Siegmund, K., Schwalbe, H., Hunte, C., and Brandt, U. (2015). Structural biology. Mechanistic insight from the crystal structure of mitochondrial complex I. Science 347, 44–49. Search in Google Scholar

Received: 2015-2-17
Accepted: 2015-6-29
Published Online: 2015-7-4
Published in Print: 2015-9-1

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