The Mrp Na+/H+ Antiporter Increases the Activity of the Malate:Quinone Oxidoreductase of an Escherichia coli Respiratory Mutant (original) (raw)
FEBS Letters, 2001
The Na + /H + antiport activity encoded by the sevengene mrp operons of Bacillus subtilis and alkaliphilic Bacillus pseudofirmus OF4 were cloned into a low copy plasmid, were expressed in several Escherichia coli mutant strains and compared side-by-side with similarly cloned nhaA, a major secondary antiporter from E. coli. All three antiporter systems exhibited electron donor-dependent antiport in a fluorescencebased vesicle assay, with NhaA being the most active. In whole cells of the same antiporter-deficient strain from which the vesicles were made, E. coli KNabc, Mrp-mediated Na + exclusion was significantly more protonophore-resistant than that conferred by NhaA. The Mrp systems were also more efficacious than NhaA: in supporting anaerobic Na + resistance in wild type and a terminal oxidase mutant strain of E. coli (SBS2115); and in increasing non-fermentative growth of an NADH dehydrogenase-minus E. coli mutant (ANN0222). The results suggest the possibility that the Mrp systems may have both secondary and primary energization capacities.
Journal of Bacteriology, 2008
Mrp antiporters catalyze secondary Na+(Li+)/H+ antiport and/or K+/H+ antiport that is physiologically important in diverse bacteria. An additional capacity for anion flux has been observed for a few systems. Mrp is unique among antiporters in that it requires all six or seven hydrophobic gene products (MrpA to MrpG) of the mrp operon for full antiporter activity, but MrpE has been reported to be dispensable. Here, the membrane complexes formed by Mrp proteins were examined using a cloned mrp operon from alkaliphilic Bacillus pseudofirmus OF4. The operon was engineered so that the seven Mrp proteins could be detected in single samples. Membrane extracts of an antiporter-deficient Escherichia coli strain expressing this construct were analyzed by blue native-sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Mrp complexes of two sizes were identified containing all seven Mrp proteins. Studies of the single nonpolar mrp gene deletions in the construct showed that a subcomplex o...
Journal of Biological Chemistry, 2010
Mrp systems are widely distributed and structurally complex cation/proton antiporters. Antiport activity requires heterooligomeric complexes of all six or seven hydrophobic Mrp proteins (MrpA-MrpG). Here, a panel of site-directed mutants in conserved or proposed motif residues was made in the Mrp Na ؉ (Li ؉ )/H ؉ antiporter from an alkaliphilic Bacillus. The mutant operons were expressed in antiporter-deficient Escherichia coli KNabc and assessed for antiport properties, support of sodium resistance, membrane levels of each Mrp protein, and presence of monomeric and dimeric Mrp complexes. Antiport did not depend on a VFF motif or a conserved tyrosine pair, but a role for a conserved histidine in a potential quinone binding site of MrpA was supported. The importance of several acidic residues for antiport was confirmed, and the importance of additional residues was demonstrated (e.g. three lysine residues conserved across MrpA, MrpD, and membrane-bound respiratory Complex I subunits (NuoL/M/N)). The results extended indications that MrpE is required for normal membrane levels of other Mrp proteins and for complex formation. Moreover, mutations in several other Mrp proteins lead to greatly reduced membrane levels of MrpE. Thus, changes in either of the two Mrp modules, MrpA-MrpD and MrpE-MrpG, influence the other. Two mutants, MrpB-P37G and MrpC-Q70A, showed a normal phenotype but lacked the MrpA-MrpG monomeric complex while retaining the dimeric hetero-oligomeric complex. Finally, MrpG-P81A and MrpG-P81G mutants exhibited no antiport activity but supported sodium resistance and a low [Na ؉ ] in . Such mutants could be used to screen hypothesized but uncharacterized sodium efflux functions of Mrp apart from Na ؉ (Li ؉ )/H ؉ antiport.
Journal of Bacteriology, 2007
Monovalent cation proton antiporter-3 (Mrp) family antiporters are widely distributed and physiologically important in prokaryotes. Unlike other antiporters, they require six or seven hydrophobic gene products for full activity. Standard fluorescence-based assays of Mrp antiport in membrane vesicles from Escherichia coli transformants have not yielded strong enough signals for characterization of antiport kinetics. Here, an optimized assay protocol for vesicles of antiporter-deficient E. coli EP432 transformants produced higher levels of secondary Na ؉ (Li ؉ )/H ؉ antiport than previously reported. Assays were conducted on Mrps from alkaliphilic Bacillus pseudofirmus OF4 and Bacillus subtilis and the homologous antiporter of Staphylococcus aureus (Mnh), all of which exhibited Na ؉ (Li ؉ )/H ؉ antiport. A second paralogue of S. aureus (Mnh2) did not. K ؉ , Ca 2؉ , and Mg 2؉ did not support significant antiport by any of the test antiporters. All three Na ؉ (Li ؉ )/H ؉ Mrp antiporters had alkaline pH optima and apparent K m values for Na ؉ that are among the lowest reported for bacterial Na ؉ /H ؉ antiporters. Using a fluorescent probe of the transmembrane electrical potential (⌬⌿), Mrp Na ؉ /H ؉ antiport was shown to be ⌬⌿ consuming, from which it is inferred to be electrogenic. These assays also showed that membranes from E. coli EP432 expressing Mrp antiporters generated higher ⌬⌿ levels than control membranes, as did membranes from E. coli EP432 expressing plasmid-borne NhaA, the well-characterized electrogenic E. coli antiporter. Assays of respiratory chain components in membranes from Mrp and control E. coli transformants led to a hypothesis explaining how activity of secondary, ⌬⌿-consuming antiporters can elicit increased capacity for ⌬⌿ generation in a bacterial host.
Journal of Biological Chemistry, 2004
The NADH:ubiquinone oxidoreductase (complex I) couples the transfer of electrons from NADH to ubiquinone with the translocation of protons across the membrane. Recently, it was demonstrated that complex I from Klebsiella pneumoniae translocates sodium ions instead of protons. Experimental evidence suggested that complex I from the close relative Escherichia coli works as a primary sodium pump as well. However, data obtained with whole cells showed the presence of an NADH-induced electrochemical proton gradient. In addition, Fourier transform IR spectroscopy demonstrated that the redox reaction of the E. coli complex I is coupled to a protonation of amino acids. To resolve this contradiction we measured the properties of isolated E. coli complex I reconstituted in phospholipids. We found that the NADH:ubiquinone oxidoreductase activity did not depend on the sodium concentration. The redox reaction of the complex in proteoliposomes caused a membrane potential due to an electrochemical proton gradient as measured with fluorescent probes. The signals were sensitive to the protonophore carbonyl cyanide m-chlorophenylhydrazone (CCCP), the inhibitors piericidin A, dicyclohexylcarbodi-imide (DCCD), and amiloride derivatives, but were insensitive to the sodium ionophore ETH-157. Furthermore, monensin acting as a Na ؉ /H ؉ exchanger prevented the generation of a proton gradient. Thus, our data demonstrated that the E. coli complex I is a primary electrogenic proton pump. However, the magnitude of the pH gradient depended on the sodium concentration. The capability of complex I for secondary Na ؉ /H ؉ antiport is discussed.
Journal of Bacteriology, 2005
Sha (also known as Mrp/Mnh/Pha) is a Na+/H+ antiporter encoded by a cluster of six or seven genes that probably form a multisubunit transport complex. The Sha system is important for the homeostasis of H+, Na+, and other monovalent cations and plays a critical role in various functions, including alkaliphily, sporulation, and symbiosis. Here, we characterized the sha homologue genes from the opportunistic pathogen Pseudomonas aeruginosa, which exist as a cluster of six genes (PA1054 to PA1059). The gene cluster PA1054 to PA1059, but not the cluster with a deletion of PA1054, complemented a growth defect in the presence of 0.2 M NaCl and a defect in Na+/H+ antiport activity of the Escherichia coli TO114 mutant lacking the three major Na+/H+ antiporters, indicating that genes PA1054 to PA1059 are responsible for Na+/H+ antiport activity. We disrupted PA1054 (a shaA homologue gene) and determined its effect on Na+ tolerance during growth, Na+ efflux, and pathogenicity in mice. Disrupti...
Physiological consequences of expression of the Na+/H+ antiporter sod2 in Escherichia coli
1998
Sod2 is the sodium-proton antiporter on the plasma membrane of the fission yeast Schizosaccharomyces pombe. It is vitally important for sodium export and pH homeostasis in this organism. Recently, the sod2 gene has been cloned and sequenced. However, initial attempts to express sod2 in Escherichia coli using the T7 promoter failed. In the present work we examined physiological consequences of expression of sod2 in E. coli. To alleviate problems caused by expression of sod2 we: (i) used sodium-free media at all steps; (ii) used the moderate tac promoter for expression and; (iii) used E. coli strain MH1 which has impaired sodium exchange. The effect of sod2 expression on E. coli varied depending on the E. coli genotype. When sod2 was expressed in BL21 cells which have normal N a + /H + antiporters, the result was a Li + sensitive phenotype. LiCl completely arrested or prevented growth of BL21 E. coli transformed with the sod2 gene. The effect on growth was pronounced in media of low external pH. Sod2 was then expressed in E. coli MH1 which is devoid of endogenous Na + /H + antiporters. These cells became more resistant to external LiCl, but only in Na + containing media. In the absence of external Na + , the presence of sod2 reduced growth. The results are explained in a model which demonstrates the physiological consequences of interference by expression of a foreign electroneutral Na + /H + antiporter in conjunction with different housekeeping systems of E. coli host cells.
Biochemistry, 2009
Vibrio cholerae and many other marine and pathogenic bacteria posses a unique respiratory complex, the Na +-pumping NADH: quinone oxidoreductase (Na +-NQR)1, which pumps Na + across the cell membrane using the energy released by the redox reaction between NADH and ubiquinone. In order to function as a selective sodium pump, Na +-NQR must contain structures that: 1) allow the sodium ion to pass through the hydrophobic core of the membrane, and 2) provide cation specificity to the translocation system. In other sodium transporting proteins, the structures that carry out these roles frequently include aspartate and glutamate residues. The negative charge of these residues facilitates binding and translocation of sodium. In this study we have analyzed mutants of acid residues located in the transmembrane helices of subunits B, D and E of Na +-NQR. The results are consistent with the participation of seven of these residues in the translocation process of sodium. Mutations at NqrB-D397, NqrD-D133 and NqrE-E95 produced a decrease of approximately ten times or more in the apparent affinity of the enzyme for sodium (Km app), which suggests that these residues may form part of a sodium-binding site. Mutation at other residues, including NqrB-E28, NqrB-E144, NqrB-E346 and NqrD-D88, had a large effect on the quinone reductase activity of the enzyme and its sodium sensitivity, but less effect on the apparent sodium affinity, consistent with a possible role in sodium conductance pathways. The sodium pumping NADH:quinone oxidoreductase (Na +-NQR) is a unique prokaryotic respiratory enzyme capable of sustaining a sodium gradient across the plasma membrane, using the free energy released in the coupled oxidation of NADH and reduction of ubiquinone (1-3). Na +-NQR is composed of six subunits (NqrA-F) and contains five cofactors involved in the internal electron transfer: a non-covalently bound FAD and a 2Fe-2S center located in NqrF (4-8), two covalently-bound FMN's in NqrB and NqrC (9-11), which have been shown to give rise to two anionic flavosemiquinone radicals, observed in the partially and fully reduced forms of the enzyme (12), and a non-covalently bound riboflavin molecule that is found as a stable neutral flavosemiquinone radical in the oxidized state of the enzyme (13,14). This is notable because it is the only known instance in which riboflavin is present as a bona fide redox cofactor in any enzyme.
Biochimica et Biophysica Acta (BBA) - Bioenergetics, 2012
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).
Biochemistry, 2005
The ND4L subunit is the smallest mitochondrial DNA-encoded subunit of the protontranslocating NADH-quinone oxidoreductase (complex I). In an attempt to study the functional and structural roles of the NuoK subunit (the Escherichia coli homologue of ND4L) of the bacterial NADH-quinone oxidoreductase (NDH-1), we have performed a series of site-specific mutations on the nuoK gene of the NDH-1 operon by using the homologous recombination technique. The amino acid residues we targeted included two highly conserved glutamic acids that are presumably located in the middle of the membrane and several arginine residues that are predicted to be on the cytosolic side. All point mutants examined had fully assembled NDH-1 as detected by blue-native gel electrophoresis and immunostaining. Mutations of nearly perfectly conserved Glu-36 lead to almost null activities of coupled electron transfer with a concomitant loss of generation of electrochemical gradient. A significant diminution of the coupled activities was also observed with mutations of another highly conserved residue, Glu-72. These results may suggest that both membrane-embedded acidic residues are important for the coupling mechanism of NDH-1. Furthermore, a severe impairment of the coupled activities occurred when two vicinal arginine residues on a cytosolic loop were simultaneously mutated. Possible roles of these arginine residues and other conserved residues in the NuoK subunit for NDH-1 function were discussed. 10.
Reconstitution of a bacterial Na+/H+ antiporter
Journal of Biological Chemistry
Membrane proteins from alkalophilic Bacillus fir-mu8 RAB were extracted with octylglucoside, reconstituted into liposomes made from alkalophile lipids. The proteoliposomes were loaded with 2aNa+. Imposition of a valinomycin-mediated potassium diffusion potential, positive out, resulted in very rapid efflux of radioactive Na+ against its electrochemical gradient. That the Na+ efflux was mediated by the electrogenic Na+/H+ antiporter is indicated by the following characteristics that had been established for the porter in previous studies: dependence upon an electrical potential; pH sensitivity, with activity dependent upon an alkaline pH; inhibition by Li+; and an apparent concentration dependence upon Na+ that correlated well with measurements in cells and membrane vesicles. Na+/H+ antiport activities have been reported in a very large variety of cells, and have been credited with a myriad of physiological roles, ranging from Na+ extrusion and pH homeostasis in prokaryotes to pH-mediated signalling events associated with differentiation and transformation in some eukaryotic systems (1). In general, the exchange of Na+ for H+ in eukaryotes is electroneutral, amiloride-sensitive, and involves the uptake of Na+. In prokaryotes, Na+ is usually extruded, and the activity is most often electrogenic with net positive charge of H+ moving inward (1). In view of the widespread interest in Na+/H+ antiporters, it is important to achieve purification of the carrier molecules to confirm and extend the characterizations derived from in vivo and membrane studies. Purification, in turn, requires the availability of a specific ligand or a functional reconstitution so that the activities can be assayed. The reconstitution of a eukaryotic, Na+/H+ antiport has been achieved (2). We report here the reconstitution of a Na+/H+ antiporter that was extracted from the membrane of an obligately alkalophilic bacterium. Such bacteria possess high activity of an electrogenic antiporter that is apparently necessary for the maintenance of a relatively acidified cytoplasm during growth of the organisms at very alkaline pH (1, 3-5). The crucial role of the antiporter as well as its high activity has made the alkalophiles an attractive system in which to study and attempt to isolate the Na+/H+ antiporter. The assay used in the current effort was developed during studies of the antiporter in vivo, i.e. an assay in which "Na+ efflux from starved cells was initiated by generation of a valinomycin-mediated potassium diffusion potential (outside positive) (5). The Na+/H+ antiporter from Bacillus firmus RAB and Bacillus alcalophilus catalyzed rapid, potential-dependent **Na+ efflux at pH 9.0 that was competitively inhibited by Li+. Neither antiporter was active at pH 7.0 (3,5). These characteristics have now been demonstrated with proteoliposomes prepared from B. firmus RAB membrane lipids and octylglucoside extracts of the membranes.
Nature Communications
Respiratory complex I plays a central role in cellular energy metabolism coupling NADH oxidation to proton translocation. In humans its dysfunction is associated with degenerative diseases. Here we report the structure of the electron input part of Aquifex aeolicus complex I at up to 1.8 Å resolution with bound substrates in the reduced and oxidized states. The redox states differ by the flip of a peptide bond close to the NADH binding site. The orientation of this peptide bond is determined by the reduction state of the nearby [Fe-S] cluster N1a. Fixation of the peptide bond by site-directed mutagenesis led to an inactivation of electron transfer and a decreased reactive oxygen species (ROS) production. We suggest the redoxgated peptide flip to represent a previously unrecognized molecular switch synchronizing NADH oxidation in response to the redox state of the complex as part of an intramolecular feedback mechanism to prevent ROS production.
The aerobic respiratory chain of Escherichia coli: from genes to supercomplexes
Microbiology, 2012
In spite of the large number of reports on the aerobic respiratory chain of Escherichia coli, from gene transcription regulation to enzyme kinetics and structural studies, an integrative perspective of this pathway is yet to be produced. Here, a multi-level analysis of the aerobic respiratory chain of E. coli was performed to find correlations between gene transcription, enzyme activity, growth dynamics, and supercomplex formation and composition. The transcription level of all genes encoding the aerobic respiratory chain of E. coli varied significantly in response to bacterial growth. Coordinated expression patterns were observed between the genes encoding NADH : quinone oxidoreductase and complex I (NDH-1), alternative NADH : quinone oxidoreductase (NDH-2) and cytochrome bdI, and also between sdhA and appC, encoding succinate dehydrogenase and cytochrome bdII, respectively. In general, the rates of the respiratory chain activities increased from mid-exponential to late-stationary phase, with no significant further variation occurring until the mid-stationary phase. Multi-level correlations between gene transcription, enzyme activity and growth dynamics were also found in this study. The previously reported NADH dehydrogenase and formate : oxygen oxidoreductase supercomplexes of E. coli were already assembled at mid-exponential phase and remained throughout growth. A new succinate oxidase supercomplex composed of succinate dehydrogenase and cytochrome bdII was identified, in agreement with the suggestion provided by the coordinated transcription of sdhA and appC.
A nhaD Na + /H + antiporter and a pcd homologues are among the Rhodothermus marinus complex I genes
Biochimica Et Biophysica Acta-bioenergetics, 2005
The NADH:menaquinone oxidoreductase (Nqo) is one of the enzymes present in the respiratory chain of the thermohalophilic bacterium Rhodothermus marinus. The genes coding for the R. marinus Nqo subunits were isolated and sequenced, clustering in two operons [nqo 1 to nqo 7 (nqo A ) and nqo 10 to nqo 14 (nqo B )] and two independent genes (nqo 8 and nqo 9 ). Unexpectedly, two genes encoding homologues of a NhaD Na + /H + antiporter (NhaD) and of a pterin-4a-carbinolamine dehydratase (PCD) were identified within nqo B , flanked by nqo 13 and nqo 14 . Eight conserved motives to harbour iron -sulphur centres are identified in the deduced primary structures, as well as two consensus sequences to bind nucleotides, in this case NADH and FMN. Moreover, the open-reading-frames of the putative NhaD and PCD were shown to be co-transcribed with the other complex I genes encoded by nqo B . The possible role of these two genes in R. marinus complex I is discussed. D
Energy transducing redox steps of the Na + -pumping NADH:quinone oxidoreductase from Vibrio cholerae
Proceedings of the National Academy of Sciences, 2010
Na + -NQR is a unique respiratory enzyme that couples the free energy of electron transfer reactions to electrogenic pumping of sodium across the cell membrane. This enzyme is found in many marine and pathogenic bacteria where it plays an analogous role to the H + -pumping complex I. It has generally been assumed that the sodium pump of Na + -NQR operates on the basis of thermodynamic coupling between reduction of a single redox cofactor and the binding of sodium at a nearby site. In this study, we have defined the coupling to sodium translocation of individual steps in the redox reaction of Na + -NQR. Sodium uptake takes place in the reaction step in which an electron moves from the 2Fe-2S center to FMN C , while the translocation of sodium across the membrane dielectric (and probably its release into the external medium) occurs when an electron moves from FMN B to riboflavin. This argues against a single-site coupling model because the redox steps that drive these two parts of the...
Molecular Microbiology, 2013
Reactive oxygen species (ROS) production by respiratory Complex I from Escherichia coli was studied in bacterial membrane fragments and in the isolated and purified enzyme, either solubilized or incorporated in proteoliposomes. We found that the replacement of a single amino acid residue in close proximity to the nicotinamide adenine dinucleotide (NADH)-binding catalytic site (E95 in the NuoF subunit) dramatically increases the reactivity of Complex I towards dioxygen (O2). In the E95Q variant short-chain ubiquinones exhibit strong artificial one-electron reduction at the catalytic site, also leading to a stronger increase in ROS production. Two mechanisms can contribute to the observed kinetic effects: (a) a change in the reactivity of flavin mononucleotide (FMN) towards dioxygen at the catalytic site, and (b) a change in the population of the ROS-generating state. We propose the existence of two (closed and open) states of the NAD + -bound enzyme as one feature of the substratebinding site of Complex I. The analysis of the kinetic model of ROS production allowed us to propose that the population of Complex I with reduced FMN is always low in the wild-type enzyme even at low ambient redox potentials, minimizing the rate of reaction with O2 in contrast to E95Q variant.
Respiratory complex I fromEscherichia colidoes not transport Na+in the absence of its NuoL subunit
FEBS Letters, 2014
We investigated H + and Na + transport by complex I from Escherichia coli devoid of the NuoL subunit, which is probably part of the ion translocating machinery. We observed that complex I devoid of the NuoL subunit still translocates H + , although to a smaller extension than the complete version of complex I, but does not transport Na +. Our results unequivocally reinforce the observation that E. coli complex I transports Na + in the opposite direction to that of the H + and show that NuoL subunit is involved in the translocation of both ions by complex I.
Sodium-translocating NADH:quinone oxidoreductase as a redox-driven ion pump
Biochimica et Biophysica Acta (BBA) - Bioenergetics, 2010
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.