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3.D.5 The Na+-translocating NADH:Quinone Dehydrogenase (Na-NDH or NQR) Family

The Na-NDH enzyme complex (also called the Na+-NQR complex) from Vibrio alginolyticus contains noncovalently bound FAD and two covalently bound FMNs as cofactors and has one iron-sulfur center of the Fe2S2 type. Electrons from NADH are donated to the quinone pool. The complex has six subunits which are not homologous to those of the proteins of the NDH family (TC #3.D.1). Five of these subunits are probably integral membrane proteins. The V. harveyi and V. cholerae enzyme complexes have also been purified and have similar properties. Homologous subunits are encoded within the genomes of Haemophilus influenzae and several other Gram-negative marine and blood-borne bacteria (Verkhovsky and Bogachev, 2010).  Additionally, two additional proteins, ApbE and NqrM (DUF539), are essential for activity. ApbE is responsible for covalent attachment of flavin mononucleotide (FMN) while NqrM is necessary for biogenesis (Kostyrko et al. 2016).

Juárez et al., 2009 defined the complete sequence of redox carriers in the electrons transfer pathway through the Na+-pumping NQR enzyme. Electrons flow from NADH to quinone through the FAD in subunit F, the 2Fe-2S center, the FMN in subunit C, the FMN in subunit B, and finally riboflavin. The reduction of the FMN(C) to its anionic flavosemiquinone state is the first Na+-dependent process, suggesting that reduction of this site is linked to Na+ uptake. During the reduction reaction, two FMNs are transformed to their anionic flavosemiquinone in a single kinetic step. Subsequently, FMN(C) is converted to the flavohydroquinone, accounting for the single anionic flavosemiquinone radical in the fully reduced enzyme. A model of the electron transfer steps in the catalytic cycle of Na+-NQR has been presented by Juárez et al., 2009 and Juárez et al., 2010.

Most bacteria that exhibit the Na-NDH system contain the six subunit-encoding genes in a single operon in the same gene order: nqrA-F. A is relatively hydrophilic; B, D and E are very hydrophobic; B and C contain the covalently bound flavins, linked by phosphodiester linkages, and F contains motifs for binding an FeS center, FAD and NADH (Häse and Barquera, 2001). The enzyme also contains riboflavin (1:1 stoichiometry) that is a neutral flavin semiquinone in the oxidized form of the enzyme and an anionic flavin semiquinone in the reduced form (Barquera et al., 2002). The e-/Na+ stoichiometry is 1:1. The protons required for ubiquinone reduction to ubiquinol are taken up from the cytoplasm. The large, peripheral NqrA subunit of Na+-NQR binds one molecule of ubiquinone-8 (Q(8)) (Casutt et al., 2011).  A mutation in NqrB (D397N) uncouples transport from electron transfer in the presence of K+ (Shea et al. 2015).

The membrane topologies of the six subunits of Na+-translocating NADH:quinone oxidoreductase (Na+-NQR) from Vibrio cholerae have been determined by a combination of topology prediction algorithms and the construction of C-terminal fusions (Duffy and Barquera, 2006). NqrA is localized to the cytoplasmic side of the membrane. NqrB has nine transmembrane helices and residue T236, the binding site for flavin mononucleotide (FMN), resides in the cytoplasm. NqrC consists of two transmembrane helices with the FMN binding site at residue T225 on the cytoplasmic side. NqrD and NqrE show almost mirror image topologies, each consisting of six transmembrane helices; the results for NqrD and NqrE are consistent with the topologies of Escherichia coli homologues YdgQ and YdgL, respectively. The NADH, flavin adenine dinucleotide, and Fe-S center binding sites of NqrF are localized to the cytoplasm. The determination of the topologies of these subunits provides insight into the locations of all redox cofactors localized to the cytoplasmic side of the membrane (Duffy and Barquera, 2006).

The Na(+)-translocating NADH:quinone oxidoreductase (Na+-NQR) from Vibrio cholerae is a respiratory flavo-FeS complex composed of the six subunits NqrA-F. The Na+-NQR was produced as His(6)-tagged protein by homologous expression in V. cholerae. The isolated complex contained near-stoichiometric amounts of non-covalently bound FAD and riboflavin, catalyzed NADH-driven Na+ transport, and was inhibited by 2-n-heptyl-4-hydroxyquinoline-N-oxide (Tao et al., 2008). EPR spectroscopy showed that Na+-NQR contained low amounts of a neutral flavosemiquinone. Reduction with NADH resulted in the formation of an anionic flavosemiquinone. Subsequent oxidation of the Na+-NQR with ubiquinone-1 or O2 led to the formation of a neutral flavosemiquinone.

Redox titration of the electronic spectra of the prosthetic groups of the Na+-translocating NADH:quinone oxidoreductase (Na+-NQR) from Vibrio harveyi at different pH values showed five redox transitions corresponding to the four flavin cofactors of the enzyme and one additional transition reflecting oxidoreduction of the [2Fe-2S] cluster (Bogachev et al., 2009). The pH dependence of the measured midpoint redox potentials showed that the two-electron reduction of the FAD located in the NqrF subunit was coupled with the uptake of only one H+. The one-electron reduction of the neutral semiquinone of riboflavin and the formation of anion flavosemiquinone from the oxidized FMN bound to the NqrB subunit were not coupled to proton uptake. The two sequential one-electron reductions of the FMN residue bound to the NqrC subunit showed pH-independent formation of the anion radical in the first step and the formation of fully reduced flavin coupled to the uptake of one H+ in the second step. All four flavins stayed in the anionic form in the fully reduced enzyme. None of the six redox transitions in Na+-NQR showed dependence of its midpoint redox potential on the concentration of sodium ions (Bogachev et al., 2009). 

Na+-NQR is widely distributed among marine and pathogenic bacteria. It converts the energy from the oxidation of NADH and the reduction of quinone into an electrochemical Na+-gradient. All six constituents of theNQR complex are homologous to the six proteins of the RNF complex (3.D.6). Juárez and Barquera 2012 proposed that sodium pumping is coupled to the redox reactions by a novel mechanism, which operates at multiple sites, is indirect, and is mediated by conformational changes in the proteins.

The Na+-NQR complex consists of six subunits, NqrA, B, C, D, E and F. Steuber et al. 2014 presented the crystal structure of the Na+-NQR complex of Vibrio cholerae at 3.5 Å resolution. The arrangement of cofactors both at the cytoplasmic and the periplasmic side of the complex, together with an iron centre in the membrane-embedded part, revealed an electron transfer pathway from the NADH-oxidizing cytoplasmic NqrF subunit across the membrane to the periplasmic NqrC and back to the quinone reduction site on NqrA, located in the cytoplasm. The sodium channel is localized in subunit NqrB, the largest membrane subunit of the Na+-NQR and is structurally related to urea and ammonia transporters. On the basis of the structure, a mechanism of redox-driven Na+ translocation was proposed where the change in redox state of the flavin mononucleotide cofactor in NqrB triggers the transport of Na+ through the channel (Steuber et al. 2014). 

The entire Na+-NQR complex has dimensions of 87 Å × 138 Å × 52 Å. The transmembrane part of Na+-NQR is formed by subunits NqrB, NqrD and NqrE, which are integral membrane proteins with small domains in the cytoplasm or periplasm. NqrA, NqrC and NqrF are large hydrophilic subunits with the latter two anchored via a single transmembrane helix in the cytoplasmic membrane. The hydrophilic subunits NqrA and NqrF protrude into the cytoplasm, whereas NqrC resides in the periplasm. NqrA, which lacks transmembrane helices, is tightly bound to NqrB, forming a complex with a large interaction surface. Subunits NqrD and NqrE form a central structural unit that interacts with NqrB on one side and the transmembrane helices of NqrC and NqrF on the opposed sid (Steuber et al. 2014).

The transport reaction catalyzed by the Na+-NDH complex is:

NADH + quinone + nNa+ (in) → NAD+ + quinol + nNa+ (out).

References associated with 3.D.5 family:

Barquera, B., W. Zhou, J.E. Morgan, and R.B. Gennis. (2002). Riboflavin is a component of the Na+-pumping NADH-quinone oxidoreductase from Vibrio cholerae. Proc. Natl. Acad. Sci. USA 99: 10322-10324. 12122213
Bogachev, A.V., D.A. Bloch, Y.V. Bertsova, and M.I. Verkhovsky. (2009). Redox properties of the prosthetic groups of Na+-translocating NADH:quinone oxidoreductase. 2. Study of the enzyme by optical spectroscopy. Biochemistry 48: 6299-6304. 19496622
Casutt, M.S., R. Nedielkov, S. Wendelspiess, S. Vossler, U. Gerken, M. Murai, H. Miyoshi, H.M. Möller, and J. Steuber. (2011). Localization of ubiquinone-8 in the Na+-pumping NADH:quinone oxidoreductase from Vibrio cholerae. J. Biol. Chem. 286: 40075-40082. 21885438
Dimroth, P. (1997). Primary sodium ion translocating enzymes. Biochim. Biophys. Acta 1318: 11-51. 9030254
Duffy, E.B. and B. Barquera. (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. 17041063
Häse, C.C. and B. Barquera. (2001). Role of sodium bioenergetics in Vibrio cholerae. Biochim. Biophys. Acta 1505: 169-178. 11248198
Hayashi, M., K. Hirai, and T. Unemoto. (1995). Sequencing and the alignment of structural genes in the nqr operon encoding the Na+-translocating NADH-quinone reductase from Vibrio alginolyticus. FEBS Letts. 363: 75-77. 7729558
Juárez, O. and B. Barquera. (2012). Insights into the mechanism of electron transfer and sodium translocation of the Na+-pumping NADH:quinone oxidoreductase. Biochim. Biophys. Acta. 1817: 1823-1832. 22465856
Juárez, O., J.E. Morgan, and B. Barquera. (2009). The Electron Transfer Pathway of the Na+-pumping NADH:Quinone Oxidoreductase from Vibrio cholerae. J. Biol. Chem. 284: 8963-8972. 19155212
Juárez, O., J.E. Morgan, M.J. Nilges, and B. Barquera. (2010). Energy transducing redox steps of the Na+-pumping NADH:quinone oxidoreductase from Vibrio cholerae. Proc. Natl. Acad. Sci. USA 107: 12505-12510. 20616050
Kostyrko, V.A., Y.V. Bertsova, M.V. Serebryakova, A.A. Baykov, and A.V. Bogachev. (2016). NqrM (DUF539) Protein Is Required for Maturation of Bacterial Na+-Translocating NADH:Quinone Oxidoreductase. J. Bacteriol. 198: 655-663. 26644436
Shea, M.E., K.G. Mezic, O. Juárez, and B. Barquera. (2015). A mutation in Na+-NQR uncouples electron flow from Na+ translocation in the presence of K+. Biochemistry 54: 490-496. 25486106
Steuber, J., G. Vohl, M.S. Casutt, T. Vorburger, K. Diederichs, and G. Fritz. (2014). Structure of the V. cholerae Na+-pumping NADH:quinone oxidoreductase. Nature 516: 62-67. 25471880
Tao, M., M.S. Casutt, G. Fritz, and J. Steuber. (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. 18454933
Toulouse, C., B. Claussen, V. Muras, G. Fritz, and J. Steuber. (2016). Strong pH dependence of coupling efficiency of the Na+ - translocating NADH:quinone oxidoreductase (Na+ -NQR) of Vibrio cholerae. Biol Chem. [Epub: Ahead of Print] 27639271
Verkhovsky MI. and Bogachev AV. (2010). Sodium-translocating NADH:quinone oxidoreductase as a redox-driven ion pump. Biochim Biophys Acta. 1797(6-7):738-46. 20056102
Yagi, T., T. Yano, S. Di Bernardo, and A. Matsuno-Yagi. (1998). Procaryotic complex I (NDH-1), an overview. Biochim. Biophys. Acta 1364: 125-133. 9593856