NMR Reveals Double Occupancy of Quinone-type Ligands in the Catalytic Quinone Binding Site of the Na+-translocating NADH:Quinone Oxidoreductase from Vibrio cholerae

The sodium ion-translocating NADH:quinone oxidoreductase (Na⁺-NQR) from the pathogen Vibrio cholerae exploits the free energy liberated during oxidation of NADH with ubiquinone to pump sodium ions across the cytoplasmic membrane. The Na⁺-NQR consists of four membrane-bound subunits NqrBCDE and the peripheral NqrF and NqrA subunits. NqrA binds ubiquinone-8 as well as quinones with shorter prenyl chains (ubiquinone-1 and ubiquinone-2). Here we show that the quinone derivative 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone (DBMIB), a known inhibitor of the bc₁ and b₆f complexes found in mitochondria and chloroplasts, also inhibits quinone reduction by the Na⁺-NQR in a mixed inhibition mode. Tryptophan fluorescence quenching and saturation transfer difference NMR experiments in the presence of Na⁺-NQR inhibitor (DBMIB or 2-n-heptyl-4-hydroxyquinoline N-oxide) indicate that two quinone analog ligands are bound simultaneously by the NqrA subunit with very similar interaction constants as observed with the holoenzyme complex. We conclude that the catalytic site of quinone reduction is located on NqrA. The two ligands bind to an extended binding pocket in direct vicinity to each other as demonstrated by interligand Overhauser effects between ubiquinone-1 and DBMIB or 2-n-heptyl-4-hydroxyquinoline N-oxide, respectively. We propose that a similar spatially close arrangement of the native quinone substrates is also operational in vivo, enhancing the catalytic efficiency during the final electron transfer steps in the Na⁺-NQR.     

Localization and Function of the Membrane-bound Riboflavin in the Na+-translocating NADH:Quinone Oxidoreductase (Na+-NQR) from Vibrio cholerae

The sodium ion-translocating NADH:quinone oxidoreductase (Na⁺-NQR) from the human pathogen Vibrio cholerae is a respiratory membrane protein complex that couples the oxidation of NADH to the transport of Na⁺ across the bacterial membrane. The Na⁺-NQR comprises the six subunits NqrABCDEF, but the stoichiometry and arrangement of these subunits are unknown. Redox-active cofactors are FAD and a 2Fe-2S cluster on NqrF, covalently attached FMNs on NqrB and NqrC, and riboflavin and ubiquinone-8 with unknown localization in the complex. By analyzing the cofactor content and NADH oxidation activity of subcomplexes of the Na⁺-NQR lacking individual subunits, the riboflavin cofactor was unequivocally assigned to the membrane-bound NqrB subunit. Quantitative analysis of the N-terminal amino acids of the holo-complex revealed that NqrB is present in a single copy in the holo-complex. It is concluded that the hydrophobic NqrB harbors one riboflavin in addition to its covalently attached FMN. The catalytic role of two flavins in subunit NqrB during the reduction of ubiquinone to ubiquinol by the Na⁺-NQR is discussed.     

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

Na⁺-pumping NADH:ubiquinone oxidoreductase (Na⁺-NQR) is responsible for maintaining a sodium gradient across the inner bacterial membrane. This respiratory enzyme, which couples sodium pumping to the electron transfer between NADH and ubiquinone, is not present in eukaryotes and as such could be a target for antibiotics. In this paper it is shown that the site of ubiquinone reduction is conformationally coupled to the NqrB subunit, which also hosts the final cofactor in the electron transport chain, riboflavin. Previous work showed that mutations in conserved NqrB glycine residues 140 and 141 affect ubiquinone reduction and the proper functioning of the sodium pump. Surprisingly, these mutants did not affect the dissociation constant of ubiquinone or its analog HQNO (2-n-heptyl-4-hydroxyquinoline N-oxide) from Na⁺-NQR, which indicates that these residues do not participate directly in the ubiquinone binding site but probably control its accessibility. Indeed, redox-induced difference spectroscopy showed that these mutations prevented the conformational change involved in ubiquinone binding but did not modify the signals corresponding to bound ubiquinone. Moreover, data are presented that demonstrate the NqrA subunit is able to bind ubiquinone but with a low non-catalytically relevant affinity. It is also suggested that Na⁺-NQR contains a single catalytic ubiquinone binding site and a second site that can bind ubiquinone but is not active.     

The Kinetic Reaction Mechanism of the Vibrio cholerae Sodium-dependent NADH Dehydrogenase

The sodium-dependent NADH dehydrogenase (Na⁺-NQR) is the main ion transporter in Vibrio cholerae. Its activity is linked to the operation of the respiratory chain and is essential for the development of the pathogenic phenotype. Previous studies have described different aspects of the enzyme, including the electron transfer pathways, sodium pumping structures, cofactor and subunit composition, among others. However, the mechanism of the enzyme remains to be completely elucidated. In this work, we have studied the kinetic mechanism of Na⁺-NQR with the use of steady state kinetics and stopped flow analysis. Na⁺-NQR follows a hexa-uni ping-pong mechanism, in which NADH acts as the first substrate, reacts with the enzyme, and the oxidized NAD leaves the catalytic site. In this conformation, the enzyme is able to capture two sodium ions and transport them to the external side of the membrane. In the last step, ubiquinone is bound and reduced, and ubiquinol is released. Our data also demonstrate that the catalytic cycle involves two redox states, the three- and five-electron reduced forms. A model that gathers all available information is proposed to explain the kinetic mechanism of Na⁺-NQR. This model provides a background to understand the current structural and functional information.     

Origin and Evolution of the Sodium -Pumping NADH: Ubiquinone Oxidoreductase

The sodium -pumping NADH: ubiquinone oxidoreductase (Na⁺-NQR) is the main ion pump and the primary entry site for electrons into the respiratory chain of many different types of pathogenic bacteria. This enzymatic complex creates a transmembrane gradient of sodium that is used by the cell to sustain ionic homeostasis, nutrient transport, ATP synthesis, flagellum rotation and other essential processes. Comparative genomics data demonstrate that the nqr operon, which encodes all Na⁺-NQR subunits, is found in a large variety of bacterial lineages with different habitats and metabolic strategies. Here we studied the distribution, origin and evolution of this enzymatic complex. The molecular phylogenetic analyses and the organizations of the nqr operon indicate that Na⁺-NQR evolved within the Chlorobi/Bacteroidetes group, after the duplication and subsequent neofunctionalization of the operon that encodes the homolog RNF complex. Subsequently, the nqr operon dispersed through multiple horizontal transfer events to other bacterial lineages such as Chlamydiae, Planctomyces and α, β, γ and δ -proteobacteria. Considering the biochemical properties of the Na⁺-NQR complex and its physiological role in different bacteria, we propose a detailed scenario to explain the molecular mechanisms that gave rise to its novel redox- dependent sodium -pumping activity. Our model postulates that the evolution of the Na⁺-NQR complex involved a functional divergence from its RNF homolog, following the duplication of the rnf operon, the loss of the rnfB gene and the recruitment of the reductase subunit of an aromatic monooxygenase.     

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).     

Sodium-translocating NADH:quinone oxidoreductase as a redox-driven ion pump

The Na⁺-translocating NADH:ubiquinone oxidoreductase (Na⁺-NQR) is a component of the respiratory chain of various bacteria. This enzyme is an analogous but not homologous counterpart of mitochondrial Complex I. Na⁺-NQR drives the same chemistry and also uses released energy to translocate ions across the membrane, but it pumps Na⁺ instead of H⁺. Most likely the mechanism of sodium pumping is quite different from that of proton pumping (for example, it could not accommodate the Grotthuss mechanism of ion movement); this is why the enzyme structure, subunits and prosthetic groups are completely special. This review summarizes modern knowledge on the structural and catalytic properties of bacterial Na⁺-translocating NADH:quinone oxidoreductases. The sequence of electron transfer through the enzyme cofactors and thermodynamic properties of those cofactors is discussed. The resolution of the intermediates of the catalytic cycle and localization of sodium-dependent steps are combined in a possible molecular mechanism of sodium transfer by the enzyme.     

Localization-controlled specificity of FAD:threonine flavin transferases in Klebsiella pneumoniae and its implications for the mechanism of Na-translocating NADH:quinone oxidoreductase

The Klebsiella pneumoniae genome contains genes for two putative flavin transferase enzymes (ApbE1 and ApbE2) that add FMN to protein Thr residues. ApbE1, but not ApbE2, has a periplasm-addressing signal sequence. The genome also contains genes for three target proteins with the Dxx(s/t)gAT flavinylation motif: two subunits of Na⁺-translocating NADH:quinone oxidoreductase (Na⁺-NQR), and a 99.5 kDa protein, KPK_2907, with a previously unknown function. We show here that KPK_2907 is an active cytoplasmically-localized fumarate reductase. K. pneumoniae cells with an inactivated kpk_2907 gene lack cytoplasmic fumarate reductase activity, while retaining this activity in the membrane fraction. Complementation of the mutant strain with a kpk_2907-containing plasmid resulted in a complete recovery of cytoplasmic fumarate reductase activity. KPK_2907 produced in Escherichia coli cells contains 1 mol/mol each of covalently bound FMN, noncovalently bound FMN and noncovalently bound FAD. Lesion in the ApbE1 gene in K. pneumoniae resulted in inactive Na⁺-NQR, but cytoplasmic fumarate reductase activity remained unchanged. On the contrary, lesion in the ApbE2 gene abolished the fumarate reductase but not the Na⁺-NQR activity. Both activities could be restored by transformation of the ApbE1- or ApbE2-deficient K. pneumoniae strains with plasmids containing the Vibrio cholerae apbE gene with or without the periplasm-directing signal sequence, respectively. Our data thus indicate that ApbE1 and ApbE2 bind FMN to Na⁺-NQR and fumarate reductase, respectively, and that, contrary to the presently accepted view, the FMN residues are on the periplasmic side of Na⁺-NQR. A new, “electron loop” mechanism is proposed for Na⁺-NQR, involving an electroneutral Na⁺/electron symport. This article is part of a Special Issue entitled: 18th European Bioenergetic Conference.     

Cys377 residue in NqrF subunit confers Ag<Superscript>+</Superscript> sensitivity of Na<Superscript>+</Superscript>-translocating NADH:quinone oxidoreductase from <Emphasis Type="Italic">Vibrio harveyi</Emphasis>

The Na⁺-translocating NADH:quinone oxidoreductase (Na⁺-NQR) is a component of the respiratory chain of various bacteria that generates a redox-driven transmembrane electrochemical Na⁺ potential. The Na⁺-NQR activity is known to be specifically inhibited by low concentrations of silver ions. Replacement of the conserved Cys377 residue with alanine in the NqrF subunit of Na⁺-NQR from Vibrio harveyi resulted in resistance of the enzyme to Ag⁺ and to other heavy metal ions. Analysis of the catalytic activity also showed that the rate of electron input into the mutant Na⁺-NQR decreased by about 14-fold in comparison to the wild type enzyme, whereas all other properties of _NqrFC377A Na⁺-NQR including its stability remained unaffected.     

The sodium pumping NADH:quinone oxidoreductase (Na+-NQR), a unique redox-driven ion pump

The Na⁺-translocating NADH:quinone oxidoreductase (Na⁺-NQR) is a unique Na⁺ pumping respiratory complex found only in prokaryotes, that plays a key role in the metabolism of marine and pathogenic bacteria, including Vibrio cholerae and other human pathogens. Na⁺-NQR is the main entrance for reducing equivalents into the respiratory chain of these bacteria, catalyzing the oxidation of NADH and the reduction of quinone, the free energy of this redox reaction drives the selective translocation of Na⁺ across the cell membrane, which energizes key cellular processes. In this review we summarize the unique properties of Na⁺-NQR in terms of its redox cofactor composition, electron transfer reactions and a possible mechanism of coupling and pumping.     

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 1.7 mol 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).     

Respiratory Na+ pump and Na+-dependent energetics inVibrio alginolyticus

The marine bacteriumVibrio alginolyticus was found to possess the respiratory Na⁺ pump that generates an electrochemical potential of Na⁺, which plays a central role in bioenergetics ofV. alginolyticus, as a direct result of respiration. Mutants defective in the Na⁺ pump revealed that one of the two kinds of NADH: quinone oxidoreductase requires Na⁺ for activity and functions as the Na⁺ pump. The Na⁺ pump composed of three subunits was purified and reconstituted into liposomes. Generation of membrane potential by the reconstituted proteoliposomes required Na⁺. The respiratory Na⁺ pump coupled to the NADH: quinone oxidoreductase was found in wide varieties of Gramnegative marine bacteria belonging to the generaAlcaligenes, Alteromonas, andVibrio, and showed a striking similarity in the mode of electron transfer and enzymic properties. Na⁺ extrusion seemed to be coupled to a dismutation reaction, which leads to the formation of quinol and quinone from semi-quinone radical.     

Regulation of expression of Na<Superscript>+</Superscript>-translocating NADH:quinone oxidoreductase genes in <Emphasis Type="Italic">Vibrio harveyi</Emphasis> and <Emphasis Type="Italic">Klebsiella pneumoniae</Emphasis>

The expression of genes encoding sodium-translocating NADH:quinone oxidoreductase (Na⁺-NQR) was studied in the marine bacterium Vibrio harveyi and in the enterobacterium Klebsiella pneumoniae. It has been shown that such parameters as NaCl concentration, pH value, and presence of an uncoupler in the growth media do not influence significantly the level of nqr expression. However, nqr expression depends on the growth substrates used by these bacteria. Na⁺-NQR is highly repressed in V. harveyi during anaerobic growth, and nqr expression is modulated by electron acceptors and values of their redox potentials. The latter effect was shown to be independent of the ArcAB regulatory system. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users. Accession number: EF394942 (Vibrio harveyi arcB gene, partial cds).     

Solubilization and reconsitution of renal brush border Na+−H+ exchanger

Summary In order to permit future characterization and possible isolation of the Na⁺–H⁺ exchanger from the apical membrane of proximal tubular cells, studies were performed to solubilize and reconstitute this transporter. Rabbit brush border membranes were prepared by a magnesium aggregation method, solubilized with the detergent octyl glucoside, and reconstituted into artificial phospholipid vesicles. In the presence of a pH gradient (pH_in 6.0, pH_out 8.0), the uptake of 1mm ²²Na⁺ into the proteoliposomes was five- to sevenfold higher than into liposomes. Amiloride (2mm) inhibited proton gradient-stimulated uptake of sodium by 50%. As compared to proton gradient conditions, the uptake of sodium was lower in the absence of a pH gradient but was significantly higher when the outside and inside pH was 6.0 than 8.0. TheK _a for sodium in reconstituted proteoliposomes studied under pH gradient conditions was 4mm. The uptake of sodium in proteoliposomes prepared from heat-denatured membrane proteins was significantly decreased. These studies demonstrate that proteoliposomes prepared from octyl glucoside-solubilized brush border membrane proteins and asolectin exhibit proton gradient-stimulated, amiloride-inhibitable, electroneutral uptake of sodium. The ability to solubilize and reconstitute the Na⁺–H⁺ exchanger from the apical membrane of the proximal tubule will be of value in isolating and characterizing this transporter.     

Molecular dynamics modeling of the Vibrio cholera Na+-translocating NADH:quinone oxidoreductase NqrB–NqrD subunit interface

The Na⁺-translocating NADH:quinone oxidoreductase (Na⁺-NQR) is the major Na⁺ pump in aerobic pathogens such as Vibrio cholerae. The interface between two of the NQR subunits, NqrB and NqrD, has been proposed to harbor a binding site for inhibitors of Na⁺-NQR. While the mechanisms underlying Na⁺-NQR function and inhibition remain underinvestigated, their clarification would facilitate the design of compounds suitable for clinical use against pathogens containing Na⁺-NQR. An in silico model of the NqrB–D interface suitable for use in molecular dynamics simulations was successfully constructed. A combination of algorithmic and manual methods was used to reconstruct portions of the two subunits unresolved in the published crystal structure and validate the resulting structure. Hardware and software optimizations that improved the efficiency of the simulation were considered and tested. The geometry of the reconstructed complex compared favorably to the published V. cholerae Na⁺-NQR crystal structure. Results from one 1 µs, three 150 ns and two 50 ns molecular dynamics simulations illustrated the stability of the system and defined the limitations of this model. When placed in a lipid bilayer under periodic boundary conditions, the reconstructed complex was completely stable for at least 1 µs. However, the NqrB–D interface underwent a non-physiological transition after 350 ns.

     

Reconstitution of cAMP-Dependent protein kinase regulated renal Na+−H+ exchanger

Summary Studies were performed to determine if the Na⁺–H⁺ exchanger, solubilized from renal brush border membranes from the rabbit and assayed in reconstituted artificial proteoliposomes, could be regulated by cAMP-dependent protein kinase. Octyl glucoside solubilized renal apical membrane proteins from the rabbit kidney were phosphorylated by incubation with ATP and highly purified catalytic subunit of cAMP-dependent kinase.²²Na⁺ uptake was determined subsequently after reconstitution of the proteins into proteoliposomes. cAMP-dependent protein kinase resulted in sustained protein phosphorylation and a concentration-dependent decrease in the amiloride-sensitive component of pH gradient-stimulated sodium uptake. The inhibitory effect of cAMP-dependent protein kinase demonstrated an absolute requirement for ATP and was blocked by the specific protein inhibitor of this kinase. cAMP-dependent protein kinase also inhibited²²Na⁺ uptake in the absence of a pH gradient (pH_in 6.0. pH_out 6.0) and the inhibitory effect was blocked by the specific inhibitor of the kinase. Solubilized membrane proteins exhibited little endogenous protein kinase or protein phosphatase activity.These studies indicate that Na⁺–H⁺ exchange activity of proteoliposomes reconstituted with proteins from renal brush border membranes is inhibited by phosphorylation of selected proteins by cAMP-dependent protein kinase. These findings also indicate that the regulatory components of the Na⁺–H⁺ exchanger remain active during the process of solubilization and reconstitution of renal apical membrane proteins.     

The Na-translocating NADH:quinone oxidoreductase (Na-NQR) from Vibrio cholerae enhances insertion of FeS in overproduced NqrF subunit

The Na⁺-translocating NADH:quinone oxidoreductase (Na⁺-NQR) from Vibrio cholerae is a membrane-bound, respiratory Na⁺ pump. Its NqrF subunit contains one FAD and a [2Fe–2S] cluster and catalyzes the initial oxidation of NADH. A soluble variant of NqrF lacking its hydrophobic, N-terminal helix (NqrF′) was produced in V. cholerae wild type and nqr deletion strain. Under identical conditions of growth and induction, the yield of NqrF′ increased by 30% in the presence of the Na⁺-NQR. FAD-containing NqrF′ species with or without the FeS cluster were observed, indicating that assembly of the FeS center, but not insertion of the flavin cofactor, was limited during overproduction in V. cholerae. A comparison of these distinct NqrF′ species with regard to specific NADH dehydrogenase activity, pH dependence of activity and thermal inactivation showed that NqrF′ lacking the [2Fe–2S] cluster was less stable, partially unfolded, and therefore prone to proteolytic degradation in V. cholerae. We conclude that the overall yield of NqrF′ critically depends on the amount of fully assembled, FeS-containing NqrF′ in the V. cholerae host cells. The Na⁺-NQR is proposed to increase the stability of NqrF′ by stimulating the maturation of FeS centers.     

NADH oxidation drives respiratory Na+ transport in mitochondria from Yarrowia lipolytica

It is generally assumed that respiratory complexes exclusively use protons to energize the inner mitochondrial membrane. Here we show that oxidation of NADH by submitochondrial particles (SMPs) from the yeast Yarrowia lipolytica is coupled to protonophore-resistant Na⁺ uptake, indicating that a redox-driven, primary Na⁺ pump is operative in the inner mitochondrial membrane. By purification and reconstitution into proteoliposomes, a respiratory NADH dehydrogenase was identified which coupled NADH-dependent reduction of ubiquinone (1.4 μmol min⁻¹ mg⁻¹) to Na⁺ translocation (2.0 μmol min⁻¹ mg⁻¹). NADH-driven Na⁺ transport was sensitive towards rotenone, a specific inhibitor of complex I. We conclude that mitochondria from Y. lipolytica contain a NADH-driven Na⁺ pump and propose that it represents the complex I of the respiratory chain. Our study indicates that energy conversion by mitochondria does not exclusively rely on the proton motive force but may benefit from the electrochemical Na⁺ gradient established by complex I. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

The side chain of ubiquinone plays a critical role in Na+ translocation by the NADH-ubiquinone oxidoreductase (Na+-NQR) from Vibrio cholerae

The Na⁺-pumping NADH-ubiquinone (UQ) oxidoreductase (Na⁺-NQR) is an essential bacterial respiratory enzyme that generates a Na⁺ gradient across the cell membrane. However, the mechanism that couples the redox reactions to Na⁺ translocation remains unknown. To address this, we examined the relation between reduction of UQ and Na⁺ translocation using a series of synthetic UQs with Vibrio cholerae Na⁺-NQR reconstituted into liposomes. UQ₀ that has no side chain and UQ_CH3 and UQ_C2H5, which have methyl and ethyl side chains, respectively, were catalytically reduced by Na⁺-NQR, but their reduction generated no membrane potential, indicating that the overall electron transfer and Na⁺ translocation are not coupled. While these UQs were partly reduced by electron leak from the cofactor(s) located upstream of riboflavin, this complete loss of Na⁺ translocation cannot be explained by the electron leak. Lengthening the UQ side chain to n-propyl (C₃H₇) or longer significantly restored Na⁺ translocation. It has been considered that Na⁺ translocation is completed when riboflavin, a terminal redox cofactor residing within the membrane, is reduced. In this view, the role of UQ is simply to accept electrons from the reduced riboflavin to regenerate the stable neutral riboflavin radical and reset the catalytic cycle. However, the present study revealed that the final UQ reduction via reduced riboflavin makes an important contribution to Na⁺ translocation through a critical role of its side chain. Based on the results, we discuss the critical role of the UQ side chain in Na⁺ translocation.