3.D.5 The Na+-translocating NADH:Quinone Dehydrogenase (Na-NDH) 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).
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).
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).
The transport reaction catalyzed by the Na-NDH complex is:
NADH + quinone + nNa+ (in) → NAD+ + quinol + nNa+ (out).