Recent progress in the Na(+)-translocating NADH-quinone reductase from the marine Vibrio alginolyticus

The respiratory chain of Gram-negative marine and halophilic bacteria has a Na(+)-dependent NADH-quinone reductase that functions as a primary Na(+) pump. The Na(+)-translocating NADH-quinone reductase (NQR) from the marine Vibrio alginolyticus is composed of six structural genes (nqrA to nqrF). The NqrF subunit has non-covalently bound FAD. There are conflicting results on the existence of other flavin cofactors. Recent studies revealed that the NqrB and NqrC subunits have a covalently bound flavin, possibly FMN, which is attached to a specified threonine residue. A novel antibiotic, korormicin, was found to specifically inhibit the NQR complex. From the homology search of the nqr operon, it was found that the Na(+)-pumping NQR complex is widely distributed among Gram-negative pathogenic bacteria.     

Insights into the mechanism of electron transfer and sodium translocation of the Na(+)-pumping NADH:quinone oxidoreductase

Na(+)-NQR is a unique energy-transducing complex, 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 that can provide energy for the cell. Na(+)-NQR is not homologous to any other respiratory protein but is closely related to the RNF complex. In this review we propose that sodium pumping in Na(+)-NQR is coupled to the redox reactions by a novel mechanism, which operates at multiple sites, is indirect and mediated by conformational changes of the protein. This article is part of a Special Issue entitled: 17th European Bioenergetics Conference (EBEC 2012).     

Sequencing and preliminary characterization of the Na+-translocating NADH:ubiquinone oxidoreductase from Vibrio harveyi

The Na(+)-translocating NADH: ubiquinone oxidoreductase (Na(+)-NQR) generates an electrochemical Na(+) potential driven by aerobic respiration. Previous studies on the enzyme from Vibrio alginolyticus have shown that the Na(+)-NQR has six subunits, and it is known to contain FAD and an FeS center as redox cofactors. In the current work, the enzyme from the marine bacterium Vibrio harveyi has been purified and characterized. In addition to FAD, a second flavin, tentatively identified as FMN, was discovered to be covalently attached to the NqrC subunit. The purified V. harveyi Na(+)-NQR was reconstituted into proteoliposomes. The generation of a transmembrane electric potential by the enzyme upon NADH:Q(1) oxidoreduction was strictly dependent on Na(+), resistant to the protonophore CCCP, and sensitive to the sodium ionophore ETH-157, showing that the enzyme operates as a primary electrogenic sodium pump. Interior alkalinization of the inside-out proteoliposomes due to the operation of the Na(+)-NQR was accelerated by CCCP, inhibited by valinomycin, and completely arrested by ETH-157. Hence, the protons required for ubiquinol formation must be taken up from the outside of the liposomes, which corresponds to the bacterial cytoplasm. The Na(+)-NQR operon from this bacterium was sequenced, and the sequence shows strong homology to the previously reported Na(+)-NQR operons from V. alginolyticus and Haemophilus influenzae. Homology studies show that a number of other bacteria, including a number of pathogenic species, also have an Na(+)-NQR operon.     

Purification and characterization of the recombinant Na(+)-translocating NADH:quinone oxidoreductase from Vibrio cholerae

The nqr operon from Vibrio cholerae, encoding the entire six-subunit, membrane-associated, Na(+)-translocating NADH:quinone oxidoreductase (Na(+)-NQR), was cloned under the regulation of the P(BAD) promoter. The enzyme was successfully expressed in V. cholerae. To facilitate molecular genetics studies of this sodium-pumping enzyme, a host strain of V. cholerae was constructed in which the genomic copy of the nqr operon was deleted. By using a vector containing a six-histidine tag on the carboxy terminus of the NqrF subunit, the last subunit in the operon, the recombinant enzyme was readily purified by affinity chromatography in a highly active form from detergent-solubilized membranes of V. cholerae. The recombinant enzyme has a high specific activity in the presence of sodium. NADH consumption was assessed at a turnover number of 720 electrons per second. When purified using dodecyl maltoside (DM), the isolated enzyme contains approximately one bound ubiquinone, whereas if the detergent LDAO is used instead, the quinone content of the isolated enzyme is negligible. Furthermore, the recombinant enzyme, purified with DM, has a relatively low rate of reaction with O(2) (10-20 s(-1)). In steady state turnover, the isolated, recombinant enzyme exhibits up to 5-fold stimulation by sodium and functions as a primary sodium pump, as reported previously for Na(+)()-NQR from other bacterial sources. When reconstituted into liposomes, the recombinant Na(+)-NQR generates a sodium gradient and a Delta Psi across the membrane. SDS-PAGE resolves all six subunits, two of which, NqrB and NqrC, contain covalently bound flavin. A redox titration of the enzyme, monitored by UV-visible spectroscopy, reveals three n = 2 redox centers and one n = 1 redox center, for which the presence of three flavins and a 2Fe-2S center can account. The V. cholerae Na(+)-NQR is well-suited for structural studies and for the use of molecular genetics techniques in addressing the mechanism by which NADH oxidation is coupled to the pumping of Na(+) across the membrane.     

Oxidant-induced formation of a neutral flavosemiquinone in the Na+-translocating NADH:Quinone oxidoreductase (Na+-NQR) from Vibrio cholerae

The Na(+)-translocating NADH:quinone oxidoreductase (Na(+)-NQR) from the human pathogen 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 (0.78 mol/mol Na(+)-NQR) and riboflavin (0.70 mol/mol Na(+)-NQR), catalyzed NADH-driven Na(+) transport (40 nmol Na(+)min(-1) mg(-1)), and was inhibited by 2-n-heptyl-4-hydroxyquinoline-N-oxide. EPR spectroscopy showed that Na(+)-NQR as isolated contained very low amounts of a neutral flavosemiquinone (10(-3) mol/mol Na(+)-NQR). Reduction with NADH resulted in the formation of an anionic flavosemiquinone (0.10 mol/mol Na(+)-NQR). Subsequent oxidation of the Na(+)-NQR with ubiquinone-1 or O(2) led to the formation of a neutral flavosemiquinone (0.24 mol/mol Na(+)-NQR). We propose that the Na(+)-NQR is fully oxidized in its resting state, and discuss putative schemes of NADH-triggered redox transitions.     

Thermodynamic contribution to the regulation of electron transfer in the Na(+)-pumping NADH:quinone oxidoreductase from Vibrio cholerae

The Na(+)-pumping NADH:quinone oxidoreductase (Na(+)-NQR) is a fundamental enzyme of the oxidative phosphorylation metabolism and ionic homeostasis in several pathogenic and marine bacteria. To understand the mechanism that couples electron transfer with sodium translocation in Na(+)-NQR, the ion dependence of the redox potential of the individual cofactors was studied using a spectroelectrochemical approach. The redox potential of one of the FMN cofactors increased 90 mV in the presence of Na(+) or Li(+), compared to the redox potentials measured in the presence of other cations that are not transported by the enzyme, such as K(+), Rb(+), and NH(4)(+). This shift in redox potential of one FMN confirms the crucial role of the FMN anionic radicals in the Na(+) pumping mechanism and demonstrates that the control of the electron transfer rate has both kinetic (via conformational changes) and thermodynamic components.     

Sodium-Dependent Movement of Covalently Bound FMN Residue(s) in Na(+)-Translocating NADH:Quinone Oxidoreductase

Na(+)-translocating NADH:quinone oxidoreductase (Na(+)-NQR) is a component of respiratory electron-transport chain of various bacteria generating redox-driven transmembrane electrochemical Na(+) potential. We found that the change in Na(+) concentration in the reaction medium has no effect on the thermodynamic properties of prosthetic groups of Na(+)-NQR from Vibrio harveyi, as was revealed by the anaerobic equilibrium redox titration of the enzyme's EPR spectra. On the other hand, the change in Na(+) concentration strongly alters the EPR spectral properties of the radical pair formed by the two anionic semiquinones of FMN residues bound to the NqrB and NqrC subunits (FMN(NqrB) and FMN(NqrC)). Using data obtained by pulse X- and Q-band EPR as well as by pulse ENDOR and ELDOR spectroscopy, the interspin distance between FMN(NqrB) and FMN(NqrC) was found to be 15.3 ? in the absence and 20.4 ? in the presence of Na(+), respectively. Thus, the distance between the covalently bound FMN residues can vary by about 5 ? upon changes in Na(+) concentration. Using these results, we propose a scheme of the sodium potential generation by Na(+)-NQR based on the redox- and sodium-dependent conformational changes in the enzyme.     

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

The Na(+)-pumping NADH:quinone oxidoreductase (Na(+)-NQR) is the main entrance for electrons into the respiratory chain of many marine and pathogenic bacteria. The enzyme accepts electrons from NADH and donates them to ubiquinone, and the free energy released by this redox reaction is used to create an electrochemical gradient of sodium across the cell membrane. Here we report the role of glycine 140 and glycine 141 of the NqrB subunit in the functional binding of ubiquinone. Mutations at these residues altered the affinity of the enzyme for ubiquinol. Moreover, mutations in residue NqrB-G140 almost completely abolished the electron transfer to ubiquinone. Thus, NqrB-G140 and -G141 are critical for the binding and reaction of Na(+)-NQR with its electron acceptor, ubiquinone.     

Localization of ubiquinone-8 in the Na+-pumping NADH:quinone oxidoreductase from Vibrio cholerae

Na(+) is the second major coupling ion at membranes after protons, and many pathogenic bacteria use the sodium-motive force to their advantage. A prominent example is Vibrio cholerae, which relies on the Na(+)-pumping NADH:quinone oxidoreductase (Na(+)-NQR) as the first complex in its respiratory chain. The Na(+)-NQR is a multisubunit, membrane-embedded NADH dehydrogenase that oxidizes NADH and reduces quinone to quinol. Existing models describing redox-driven Na(+) translocation by the Na(+)-NQR are based on the assumption that the pump contains four flavins and one FeS cluster. Here we show that the large, peripheral NqrA subunit of the Na(+)-NQR binds one molecule of ubiquinone-8. Investigations of the dynamic interaction of NqrA with quinones by surface plasmon resonance and saturation transfer difference NMR reveal a high affinity, which is determined by the methoxy groups at the C-2 and C-3 positions of the quinone headgroup. Using photoactivatable quinone derivatives, it is demonstrated that ubiquinone-8 bound to NqrA occupies a functional site. A novel scheme of electron transfer in Na(+)-NQR is proposed that is initiated by NADH oxidation on subunit NqrF and leads to quinol formation on subunit NqrA.     

Quinone reduction by the Na+-translocating NADH dehydrogenase promotes extracellular superoxide production in Vibrio cholerae

The pathogenicity of Vibrio cholerae is influenced by sodium ions which are actively extruded from the cell by the Na(+)-translocating NADH:quinone oxidoreductase (Na(+)-NQR). To study the function of the Na(+)-NQR in the respiratory chain of V. cholerae, we examined the formation of organic radicals and superoxide in a wild-type strain and a mutant strain lacking the Na(+)-NQR. Upon reduction with NADH, an organic radical was detected in native membranes by electron paramagnetic resonance spectroscopy which was assigned to ubisemiquinones generated by the Na(+)-NQR. The radical concentration increased from 0.2 mM at 0.08 mM Na(+) to 0.4 mM at 14.7 mM Na(+), indicating that the concentration of the coupling cation influences the redox state of the quinone pool in V. cholerae membranes. During respiration, V. cholerae cells produced extracellular superoxide with a specific activity of 10.2 nmol min(-1) mg(-1) in the wild type compared to 3.1 nmol min(-1) mg(-1) in the NQR deletion strain. Raising the Na(+) concentration from 0.1 to 5 mM increased the rate of superoxide formation in the wild-type V. cholerae strain by at least 70%. Rates of respiratory H(2)O(2) formation by wild-type V. cholerae cells (30.9 nmol min(-1) mg(-1)) were threefold higher than rates observed with the mutant strain lacking the Na(+)-NQR (9.7 nmol min(-1) mg(-1)). Our study shows that environmental Na(+) could stimulate ubisemiquinone formation by the Na(+)-NQR and hereby enhance the production of reactive oxygen species formed during the autoxidation of reduced quinones.     

Na(+) translocation by bacterial NADH:quinone oxidoreductases: an extension to the complex-I family of primary redox pumps

The current knowledge on the Na(+)-translocating NADH:ubiquinone oxidoreductase of the Na(+)-NQR type from Vibrio alginolyticus, and on Na(+) transport by the electrogenic NADH:Q oxidoreductases from Escherichia coli and Klebsiella pneumoniae (complex I, or NDH-I) is summarized. A general mode of redox-linked Na(+) transport by NADH:Q oxidoreductases is proposed that is based on the electrostatic attraction of a positively charged Na(+) towards a negatively charged, enzyme-bound ubisemiquinone anion in a medium of low dielectricity. A structural model of the [2Fe-2S]- and FAD-carrying NqrF subunit of the Na(+)-NQR from V. alginolyticus based on ferredoxin and ferredoxin:NADP(+) oxidoreductase suggests that a direct participation of the Fe/S center in Na(+) transport is rather unlikely. A ubisemiquinone-dependent mechanism of Na(+) translocation is proposed that results in the transport of two Na(+) ions per two electrons transferred. Whereas this stoichiometry of the pump is in accordance with in vivo determinations of Na(+) transport by the respiratory chain of V. alginolyticus, higher (Na(+) or H(+)) transport stoichiometries are expected for complex I, suggesting the presence of a second coupling site.     

Downregulation of Na(+)-NQR complex is essential for Vibrio alginolyticus in resistance to balofloxacin

Increasingly isolated frequency of antibiotic-resistant V. alginolyticus strains in clinic and aquaculture has been reported, but the mechanisms of V. alginolyticus antibiotic resistance are largely absent. In the present study, native/SDS-PAGE based proteomics, which may provide information on protein-protein interaction, was utilized to investigate differential proteins of V. alginolyticus in resistance to balofloxacin. Ten proteins were altered, in which V12G01_04671, V12G01_00457, V12G01_15927, V12G01_15240, NqrA (spot 26), and NqrF (spot 30) were downregulated, while V12G01_22043, TolC, V12G01_15130, V12G01_19297 were upregulated. Importantly, the two components of Na(+)-NQR complex, NqrA and NqrF, were vertically lined and was further investigated. Western blotting assay indicated that downregulation of the two proteins contrasted sharply with upregulation of a control protein TolC, which was consistent with the result obtained from 2-DE gel analysis. Furthermore, overexpression of NqrA, NqrF and TolC resulted in decrease and elevation of bacterial survival ability in medium with balofloxacin, respectively. These results indicate that downregulation of Na(+)-NQR complex is essential for V. alginolyticus resistance to balofloxacin. This is the first report on the role of Na(+)-NQR complex in antibiotic resistance. This finding highlights the way to an understanding of antibiotic-resistant mechanisms in content of metabolic regulation.     

The role and specificity of the catalytic and regulatory cation-binding sites of the Na+-pumping NADH:quinone oxidoreductase from Vibrio cholerae

The Na(+)-translocating NADH:quinone oxidoreductase is the entry site for electrons into the respiratory chain and the main sodium pump in Vibrio cholerae and many other pathogenic bacteria. In this work, we have employed steady-state and transient kinetics, together with equilibrium binding measurements to define the number of cation-binding sites and characterize their roles in the enzyme. Our results show that sodium and lithium ions stimulate enzyme activity, and that Na(+)-NQR enables pumping of Li(+), as well as Na(+) across the membrane. We also confirm that the enzyme is not able to translocate other monovalent cations, such as potassium or rubidium. Although potassium is not used as a substrate, Na(+)-NQR contains a regulatory site for this ion, which acts as a nonessential activator, increasing the activity and affinity for sodium. Rubidium can bind to the same site as potassium, but instead of being activated, enzyme turnover is inhibited. Activity measurements in the presence of both sodium and lithium indicate that the enzyme contains at least two functional sodium-binding sites. We also show that the binding sites are not exclusively responsible for ion selectivity, and other steps downstream in the mechanism also play a role. Finally, equilibrium-binding measurements with (22)Na(+) show that, in both its oxidized and reduced states, Na(+)-NQR binds three sodium ions, and that the affinity for sodium is the same for both of these states.     

The single NqrB and NqrC subunits in the Na(+)-translocating NADH: Quinone oxidoreductase (Na(+)-NQR) from Vibrio cholerae each carry one covalently attached FMN

The Na(+)-translocating NADH:quinone oxidoreductase (Na(+)-NQR) is the prototype of a novel class of flavoproteins carrying a riboflavin phosphate bound to serine or threonine by a phosphodiester bond to the ribityl side chain. This membrane-bound, respiratory complex also contains one non-covalently bound FAD, one non-covalently bound riboflavin, ubiquinone-8 and a [2Fe-2S] cluster. Here, we report the quantitative analysis of the full set of flavin cofactors in the Na(+)-NQR and characterize the mode of linkage of the riboflavin phosphate to the membrane-bound NqrB and NqrC subunits. Release of the flavin by β-elimination and analysis of the cofactor demonstrates that the phosphate group is attached at the 5'-position of the ribityl as in authentic FMN and that the Na(+)-NQR contains approximately 1.7mol covalently bound FMN per mol non-covalently bound FAD. Therefore, each of the single NqrB and NqrC subunits in the Na(+)-NQR carries a single FMN. Elimination of the phosphodiester bond yields a dehydro-2-aminobutyrate residue, which is modified with β-mercaptoethanol by Michael addition. Proteolytic digestion followed by mass determination of peptide fragments reveals exclusive modification of threonine residues, which carry FMN in the native enzyme. The described reactions allow quantification and localization of the covalently attached FMNs in the Na(+)-NQR and in related proteins belonging to the Rhodobacter nitrogen fixation (RNF) family of enzymes. This article is part of a Special Issue entitled: 17th European Bioenergetics Conference (EBEC 2012).     

Mutagenesis study of the 2Fe-2S center and the FAD binding site of the Na(+)-translocating NADH:ubiquinone oxidoreductase from Vibrio cholerae

Many marine and pathogenic bacteria have a unique sodium-translocating NADH:ubiquinone oxidoreductase (Na(+)-NQR), which generates an electrochemical Na(+) gradient during aerobic respiration. Na(+)-NQR consists of six subunits (NqrA-F) and contains five known redox cofactors: two covalently bound FMNs, one noncovalently bound FAD, one riboflavin, and one 2Fe-2S center. A stable neutral flavin-semiquinone radical is observed in the air-oxidized enzyme, while the NADH- or dithionite-reduced enzyme exhibits a stable anionic flavin-semiquinone radical. The NqrF subunit has been implicated in binding of both the 2Fe-2S cluster and the FAD. Four conserved cysteines (C70, C76, C79, and C111) in NqrF match the canonical 2Fe-2S motif, and three conserved residues (R210, Y212, S246) have been predicted to be part of a flavin binding domain. In this work, these two motifs have been altered by site-directed mutagenesis of individual residues and are confirmed to be essential for binding, respectively, the 2Fe-2S cluster and FAD. EPR spectra of the FAD-deficient mutants in the oxidized and reduced forms exhibit neutral and anionic flavo-semiquinone radical signals, respectively, demonstrating that the FAD in NqrF is not the source of either radical signal. In both the FAD and 2Fe-2S center mutants the line widths of the neutral and anionic flavo-semiquinone EPR signals are unchanged from the wild-type enzyme, indicating that neither of these centers is nearby or coupled to the radicals. Measurements of steady-state turnover using NADH, Q-1, and the artificial electron acceptor ferricyanide strongly support an electron transport pathway model in which the noncovalently bound FAD in the NqrF subunit is the initial electron acceptor and electrons then flow to the 2Fe-2S center.     

The 9.8 kDa subunit of complex I,related to bacterial Na(+)-translocating NADH dehydrogenases,is required for enzyme assembly and function in Neurospora crassa

A nuclear gene encoding a 9.8 kDa subunit of complex I, the homologue of mammalian MWFE protein, was identified in the genome of Neurospora crassa. The gene was cloned and inactivated in vivo by the generation of repeat-induced point mutations. Fungal mutant strains lacking the 9.8 kDa polypeptide were subsequently isolated. Analyses of mitochondrial proteins from mutant nuo9.8 indicate that the membrane and peripheral arms of complex I fail to assemble. Respiration of mutant mitochondria on matrix NADH is rotenone-insensitive, confirming that the 9.8 kDa protein is required for the assembly and activity of complex I. We found a similarity between the MWFE homologues and the C-terminal part of the nqrA subunit of bacterial Na(+)-translocating NADH:quinone oxidoreductases (Na(+)-NQR), suggesting a link between proton-pumping and sodium-pumping NADH dehydrogenases.     

Consensus-degenerate hybrid oligonucleotide primers for amplification of priming glycosyltransferase genes of the exopolysaccharide locus in strains of the Lactobacillus casei group

A primer design strategy named CODEHOP (consensus-degenerate hybrid oligonucleotide primer) for amplification of distantly related sequences was used to detect the priming glycosyltransferase (GT) gene in strains of the Lactobacillus casei group. Each hybrid primer consisted of a short 3' degenerate core based on four highly conserved amino acids and a longer 5' consensus clamp region based on six sequences of the priming GT gene products from exopolysaccharide (EPS)-producing bacteria. The hybrid primers were used to detect the priming GT gene of 44 commercial isolates and reference strains of Lactobacillus rhamnosus, L. casei, Lactobacillus zeae, and Streptococcus thermophilus. The priming GT gene was detected in the genome of both non-EPS-producing (EPS(-)) and EPS-producing (EPS(+)) strains of L. rhamnosus. The sequences of the cloned PCR products were similar to those of the priming GT gene of various gram-negative and gram-positive EPS(+) bacteria. Specific primers designed from the L. rhamnosus RW-9595M GT gene were used to sequence the end of the priming GT gene in selected EPS(+) strains of L. rhamnosus. Phylogenetic analysis revealed that Lactobacillus spp. form a distinctive group apart from other lactic acid bacteria for which GT genes have been characterized to date. Moreover, the sequences show a divergence existing among strains of L. rhamnosus with respect to the terminal region of the priming GT gene. Thus, the PCR approach with consensus-degenerate hybrid primers designed with CODEHOP is a practical approach for the detection of similar genes containing conserved motifs in different bacterial genomes.     

Catalytic properties of Na+-translocating NADH:quinone oxidoreductases from Vibrio harveyi, Klebsiella pneumoniae, and Azotobacter vinelandii.

The catalytic properties of sodium-translocating NADH:quinone oxidoreductases (Na+-NQRs) from the marine bacterium Vibrio harveyi, the enterobacterium Klebsiella pneumoniae, and the soil microorganism Azotobacter vinelandii have been comparatively analyzed. It is shown that these enzymes drastically differ in their affinity to sodium ions. The enzymes also possess different sensitivity to inhibitors. Na+-NQR from A. vinelandii is not sensitive to low 2-n-heptyl-4-hydroxyquinoline N-oxide (HQNO) concentrations, while Na+-NQR from K. pneumoniae is fully resistant to either Ag+ or N-ethylmaleimide. All the Na+-NQR-type enzymes are sensitive to diphenyliodonium, which is shown to modify the noncovalently bound FAD of the enzyme.     

Thermodynamic properties of the redox centers of Na(+)-translocating NADH:quinone oxidoreductase

Redox titration of all optically detectable prosthetic groups of Na(+)-translocating NADH:quinone oxidoreductase (Na(+)-NQR) at pH 7.5 showed that the functionally active enzyme possesses only three titratable flavin cofactors, one noncovalently bound FAD and two covalently bound FMN residues. All three flavins undergo different redox transitions during the function of the enzyme. The noncovalently bound FAD works as a "classical" two-electron carrier with a midpoint potential (E(m)) of -200 mV. Each of the FMN residues is capable of only one-electron reduction: one from neutral flavosemiquinone to fully reduced flavin (E(m) = 20 mV) and the other from oxidized flavin to flavosemiquinone anion (E(m) = -150 mV). The lacking second half of the redox transitions for the FMNs cannot be reached under our experimental conditions and is most likely not employed in the catalytic cycle. Besides the flavins, a [2Fe-2S] cluster was shown to function in the enzyme as a one-electron carrier with an E(m) of -270 mV. The midpoint potentials of all the redox transitions determined in the enzyme were found to be independent of Na(+) concentration. Even the components that exhibit very strong retardation in the rate of their reduction by NADH at low sodium concentrations experienced no change in the E(m) values when the concentration of the coupling ion was changed 1000 times. On the basis of these data, plausible mechanisms for the translocation of transmembrane sodium ions by Na(+)-NQR are discussed.     

The past and present of sodium energetics: may the sodium-motive force be with you

All living cells routinely expel Na(+) ions, maintaining lower concentration of Na(+) in the cytoplasm than in the surrounding milieu. In the vast majority of bacteria, as well as in mitochondria and chloroplasts, export of Na(+) occurs at the expense of the proton-motive force. Some bacteria, however, possess primary generators of the transmembrane electrochemical gradient of Na(+) (sodium-motive force). These primary Na(+) pumps have been traditionally seen as adaptations to high external pH or to high temperature. Subsequent studies revealed, however, the mechanisms for primary sodium pumping in a variety of non-extremophiles, such as marine bacteria and certain bacterial pathogens. Further, many alkaliphiles and hyperthermophiles were shown to rely on H(+), not Na(+), as the coupling ion. We review here the recent progress in understanding the role of sodium-motive force, including (i) the conclusion on evolutionary primacy of the sodium-motive force as energy intermediate, (ii) the mechanisms, evolutionary advantages and limitations of switching from Na(+) to H(+) as the coupling ion, and (iii) the possible reasons why certain pathogenic bacteria still rely on the sodium-motive force.