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.     

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.     

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.     

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.     

Membrane topology mapping of the Na+-pumping NADH: quinone oxidoreductase from Vibrio cholerae by PhoA-green fluorescent protein fusion analysis

The membrane topologies of the six subunits of Na⁺-translocating NADH:quinone oxidoreductase (Na⁺-NQR) from Vibrio cholerae were determined by a combination of topology prediction algorithms and the construction of C-terminal fusions. Fusion expression vectors contained either bacterial alkaline phosphatase (phoA) or green fluorescent protein (gfp) genes as reporters of periplasmic and cytoplasmic localization, respectively. A majority of the topology prediction algorithms did not predict any transmembrane helices for NqrA. A lack of PhoA activity when fused to the C terminus of NqrA and the observed fluorescence of the green fluorescent protein C-terminal fusion confirm that this subunit is localized to the cytoplasmic side of the membrane. Analysis of four PhoA fusions for NqrB indicates that this subunit has nine transmembrane helices and that residue T236, the binding site for flavin mononucleotide (FMN), resides in the cytoplasm. Three fusions confirm that the topology of NqrC consists of two transmembrane helices with the FMN binding site at residue T225 on the cytoplasmic side. Fusion analysis of NqrD and NqrE showed almost mirror image topologies, each consisting of six transmembrane helices; the results for NqrD and NqrE are consistent with the topologies of Escherichia coli homologs YdgQ and YdgL, respectively. The NADH, flavin adenine dinucleotide, and Fe-S center binding sites of NqrF were localized to the cytoplasm. The determination of the topologies of the subunits of Na⁺-NQR provides valuable insights into the location of cofactors and identifies targets for mutagenesis to characterize this enzyme in more detail. The finding that all the redox cofactors are localized to the cytoplasmic side of the membrane is discussed.     

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.     

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.     

NADH oxidation by the Na+-translocating NADH:quinone oxidoreductase from Vibrio cholerae: functional role of the NqrF subunit

The Na(+)-translocating NADH:quinone oxidoreductase from Vibrio cholerae is a six subunit enzyme containing four flavins and a single motif for the binding of a Fe-S cluster on its NqrF subunit. This study reports the production of a soluble variant of NqrF (NqrF') and its individual flavin and Fe-S-carrying domains using V. cholerae or Escherichia coli as expression hosts. NqrF' and the flavin domain each contain 1 mol of FAD/mol of enzyme and exhibit high NADH oxidation activity (20,000 micromol min(-1) mg(-1)). EPR, visible absorption, and circular dichroism spectroscopy indicate that the Fe-S cluster in NqrF' and its Fe-S domain is related to 2Fe ferredoxins of the vertebrate-type. The addition of NADH to NqrF' results in the formation of a neutral flavosemiquinone and a partial reduction of the Fe-S cluster. The NqrF subunit harbors the active site of NADH oxidation and acts as a converter between the hydride donor NADH and subsequent one-electron reaction steps in the Na(+)-translocating NADH:quinone oxidoreductase complex. The observed electron transfer NADH --> FAD --> [2Fe-2S] in NqrF requires positioning of the FAD and the Fe-S cluster in close proximity in accordance with a structural model of the subunit.     

Sodium-dependent steps in the redox reactions of the Na+-motive NADH:quinone oxidoreductase from Vibrio harveyi

The Na+-translocating NADH:ubiquinone oxidoreductase (Na+-NQR) from Vibrio harveyi was purified and studied by EPR and visible spectroscopy. Two EPR signals in the NADH-reduced enzyme were detected: one, a radical signal, and the other a line around g = 1.94, which is typical for a [2Fe-2S] cluster. An E(m) of -267 mV was found for the Fe-S cluster (n = 1), independent of sodium concentration. The spin concentration of the radical in the enzyme was approximately the same under a variety of redox conditions. The time course of Na+-NQR reduction by NADH indicated the presence of at least two different flavin species. Reduction of the first species (most likely, a FAD near the NADH dehydrogenase site) was very rapid in both the presence and absence of sodium. Reduction of the second flavin species (presumably, covalently bound FMN) was slower and strongly dependent on sodium concentration, with an apparent activation constant for Na+ of approximately 3.4 mM. This is very similar to the Km for Na+ in the steady-state quinone reductase reaction catalyzed by this enzyme. These data led us to conclude that the sodium-dependent step within the Na+-NQR is located between the noncovalently bound FAD and the covalently bound FMN.     

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).     

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.     

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 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.     

Na+ is translocated at NADH:quinone oxidoreductase segment in the respiratory chain of Vibrio alginolyticus

The coupling site of the Na+ pump to the respiratory chain of Vibrio alginolyticus was examined using membrane fractions prepared from the wild type, Na+ pump-deficient mutants, and spontaneous revertant. NADH oxidase of the wild type and revertant specifically required NA+ for maximum activity, whereas Na+ was not essential for the NADH oxidase of mutants. Similar to the Na+ pump in whole cells, the Na+-dependent NADH oxidase in membranes had a pH optimum in the alkaline region. A respiratory inhibitor, 2-heptyl-4-hydroxyquinoline-N-oxide (HQNO), inhibited the Na+-dependent NADH oxidase but had little effect on the NA+-independent activity of mutant membranes. NADH:quinone oxidoreductase was found to be the Na+-dependent HQNO-sensitive site of the NADH oxidase. In the wild type cells, HQNO was also found to cause a strong inhibition of the Na+ pump with little effect on the overall H+ extrusion by respiration. The inhibition of the Na+ pump by HQNO was overcome by oxidized, but not reduced, N,N,N',N'-tetra-methyl-p-phenylenediamine (TMPD). In the presence of oxidised TMPD, the electron flow NADH to oxygen seemed to bypass the HQNO-sensitive site and energize the Na+ pump. From these results, it was concluded that the Na+ pump is coupled to the respiratory chain at the step of NADH:quinone oxidoreductase.     

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.     

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).     

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).     

Redox-dependent sodium binding by the Na(+)-translocating NADH:quinone oxidoreductase from Vibrio harveyi

Relaxation characteristics of the 23Na nuclei magnetization were used to determine the sodium-binding properties of the Na+-translocating NADH:quinone oxidoreductase from Vibrio harveyi (NQR). The dissociation constant of Na+ for the oxidized enzyme was found to be 24 mM and for the reduced enzyme about 30 microM. Such large (3 orders in magnitude) redox dependence of the NQR affinity to sodium ions shows that the molecular machinery was designed to use the drop in redox energy for creating an electrochemical sodium gradient. Redox titration of NQR monitored by changes in line width of the 23Na NMR signal at 2 mM Na+ showed that the enzyme affinity to sodium ions follows the Nernst law for a one-electron carrier with Em about -300 mV (vs SHE). The data indicate that energy conservation by NQR involves a mechanism modulating ion affinity by the redox state of an enzyme redox cofactor.     

Kinetics of the spectral changes during reduction of the Na+-motive NADH:quinone oxidoreductase from Vibrio harveyi

Two radical signals with different line widths are seen in the Na+-translocating NADH:ubiquinone oxidoreductase (Na+-NQR) from Vibrio harveyi by EPR spectroscopy. The first radical is observed in the oxidized enzyme, and is assigned as a neutral flavosemiquinone. The second radical is observed in the reduced enzyme and is assigned to be the anionic form of flavosemiquinone. The time course of Na+-NQR reduction by NADH, as monitored by stopped-flow optical spectroscopy, shows three distinct phases, the spectra of which suggest that they correspond to the reduction of three different flavin species. The first phase is fast both in the presence and absence of sodium, and is assigned to reduction of FAD to FADH2 at the NADH dehydrogenating site. The rates of the other two phases are strongly dependent on sodium concentration, and these phases are attributed to reduction of two covalently bound FMN's. Combination of the optical and EPR data suggests that a neutral FMN flavosemiquinone preexists in the oxidized enzyme, and that it is reduced to the fully reduced flavin by NADH. The other FMN moiety is initially oxidized, and is reduced to the anionic flavosemiquinone. One-electron transitions of two discrete flavin species are thus assigned as sodium-dependent steps in the catalytic cycle of Na+-NQR.