BBA - Bioenergetics (v.1857, #4)
Editorial Board (i).
Mimicking respiratory phosphorylation using purified enzymes by Christoph von Ballmoos; Olivier Biner; Tobias Nilsson; Peter Brzezinski (321-331).
The enzymes of oxidative phosphorylation is a striking example of the functional association of multiple enzyme complexes, working together to form ATP from cellular reducing equivalents. These complexes, such as cytochrome c oxidase or the ATP synthase, are typically investigated individually and therefore, their functional interplay is not well understood. Here, we present methodology that allows the co-reconstitution of purified terminal oxidases and ATP synthases in synthetic liposomes. The enzymes are functionally coupled via proton translocation where upon addition of reducing equivalents the oxidase creates and maintains a transmembrane electrochemical proton gradient that energizes the synthesis of ATP by the F1F0 ATP synthase. The method has been tested with the ATP synthases from Escherichia coli and spinach chloroplasts, and with the quinol and cytochrome c oxidases from E. coli and Rhodobacter sphaeroides, respectively. Unlike in experiments with the ATP synthase reconstituted alone, the setup allows in vitro ATP synthesis under steady state conditions, with rates up to 90 ATP × s− 1 × enzyme− 1. We have also used the novel system to study the phenomenon of “mild uncoupling” as observed in mitochondria upon addition of low concentrations of ionophores (e.g. FCCP, SF6847) and the recoupling effect of 6-ketocholestanol. While we could reproduce the described effects, our data with the in vitro system does not support the idea of a direct interaction between a mitochondrial protein and the uncoupling agents as proposed earlier.Display Omitted
Keywords: ATP synthesis; Respiratory chain; Liposomes; Mild uncoupling; Ionophore; Lateral proton diffusion;
On the ATP binding site of the ε subunit from bacterial F-type ATP synthases by Alexander Krah; Shoji Takada (332-340).
F-type ATP synthases are reversible machinery that not only synthesize adenosine triphosphate (ATP) using an electrochemical gradient across the membrane, but also can hydrolyze ATP to pump ions under certain conditions. To prevent wasteful ATP hydrolysis, subunit ε in bacterial ATP synthases changes its conformation from the non-inhibitory down- to the inhibitory up-state at a low cellular ATP concentration. Recently, a crystal structure of the ε subunit in complex with ATP was solved in a non-biologically relevant dimeric form. Here, to derive the functional ATP binding site motif, we carried out molecular dynamics simulations and free energy calculations. Our results suggest that the ATP binding site markedly differs from the experimental resolved one; we observe a reorientation of several residues, which bind to ATP in the crystal structure. In addition we find that an Mg2+ ion is coordinated by ATP, replacing interactions of the second chain in the crystal structure. Thus we demonstrate more generally the influence of crystallization effects on ligand binding sites and their respective binding modes. Furthermore, we propose a role for two highly conserved residues to control the ATP binding/unbinding event, which have not been considered before. Additionally our results provide the basis for the rational development of new biosensors based on subunit ε, as shown previously for novel sensors measuring the ATP concentration in cells.Display Omitted
Keywords: ATP binding site; ATPase inhibition; ε subunit; F-type ATPase; thermophilic Bacillus PS3;
Triplet–triplet energy transfer from chlorophylls to carotenoids in two antenna complexes from dinoflagellate Amphidinium carterae by Zuzana Kvíčalová; Jan Alster; Eckhard Hofmann; Petro Khoroshyy; Radek Litvín; David Bína; Tomáš Polívka; Jakub Pšenčík (341-349).
Room temperature transient absorption spectroscopy with nanosecond resolution was used to study quenching of the chlorophyll triplet states by carotenoids in two light-harvesting complexes of the dinoflagellate Amphidinium carterae: the water soluble peridinin–chlorophyll protein complex and intrinsic, membrane chlorophyll a–chlorophyll c 2–peridinin protein complex. The combined study of the two complexes facilitated interpretation of a rather complicated relaxation observed in the intrinsic complex. While a single carotenoid triplet state was resolved in the peridinin–chlorophyll protein complex, evidence of at least two different carotenoid triplets was obtained for the intrinsic light-harvesting complex. Most probably, each of these carotenoids protects different chlorophylls. In both complexes the quenching of the chlorophyll triplet states by carotenoids occurs with a very high efficiency (~ 100%), and with transfer times estimated to be in the order of 0.1 ns or even faster. The triplet–triplet energy transfer is thus much faster than formation of the chlorophyll triplet states by intersystem crossing. Since the triplet states of chlorophylls are formed during the whole lifetime of their singlet states, the apparent lifetimes of both states are the same, and observed to be equal to the carotenoid triplet state rise time (~ 5 ns).
Keywords: Dinoflagellate; Chlorophyll; Carotenoid; Triplet state;
cAMP regulates the functional activity, coupling efficiency and structural organization of mammalian FOF1 ATP synthase by Domenico De Rasmo; Loris Micelli; Arcangela Santeramo; Anna Signorile; Paolo Lattanzio; Sergio Papa (350-358).
The present study shows that in isolated mitochondria and myoblast cultures depletion of cAMP, induced by sAC inhibition, depresses both ATP synthesis and hydrolysis by the FOF1 ATP synthase (complex V) of the oxidative phosphorylation system (OXPHOS). These effects are accompanied by the decrease of the respiratory membrane potential, decreased level of FOF1 connecting subunits and depressed oligomerization of the complex. All these effects of sAC inhibition are prevented by the addition of the membrane-permeant 8-Br-cAMP. These results show, for the first time, that cAMP promotes ATP production by complex V and prevents, at the same time, its detour to a mitochondrial membrane leak conductance, which is involved in cell death.
Keywords: Mitochondria; FOF1 ATP synthase; Intramitochondrial cAMP; sAC;
Subunit Asa1 spans all the peripheral stalk of the mitochondrial ATP synthase of the chlorophycean alga Polytomella sp. by Lilia Colina-Tenorio; Héctor Miranda-Astudillo; Araceli Cano-Estrada; Miriam Vázquez-Acevedo; Pierre Cardol; Claire Remacle; Diego González-Halphen (359-369).
Mitochondrial F1FO-ATP synthase of chlorophycean algae is dimeric. It contains eight orthodox subunits (alpha, beta, gamma, delta, epsilon, OSCP, a and c) and nine atypical subunits (Asa1 to 9). These subunits build the peripheral stalk of the enzyme and stabilize its dimeric structure. The location of the 66.1 kDa subunit Asa1 has been debated. On one hand, it was found in a transient subcomplex that contained membrane-bound subunits Asa1/Asa3/Asa5/Asa8/a (Atp6)/c (Atp9). On the other hand, Asa1 was proposed to form the bulky structure of the peripheral stalk that contacts the OSCP subunit in the F1 sector. Here, we overexpressed and purified the recombinant proteins Asa1 and OSCP and explored their interactions in vitro, using immunochemical techniques and affinity chromatography. Asa1 and OSCP interact strongly, and the carboxy-terminal half of OSCP seems to be instrumental for this association. In addition, the algal ATP synthase was partially dissociated at relatively high detergent concentrations, and an Asa1/Asa3/Asa5/Asa8/a/c 10 subcomplex was identified. Furthermore, Far-Western analysis suggests an Asa1–Asa8 interaction. Based on these results, a model is proposed in which Asa1 spans the whole peripheral arm of the enzyme, from a region close to the matrix-exposed side of the mitochondrial inner membrane to the F1 region where OSCP is located. 3D models show elongated, helix-rich structures for chlorophycean Asa1 subunits. Asa1 subunit probably plays a scaffolding role in the peripheral stalk analogous to the one of subunit b in orthodox mitochondrial enzymes.
Keywords: F1FO-ATP synthase peripheral stalk; Dimeric mitochondrial complex V; Chlorophycean algae; Chlamydomonas reinhardtii; Polytomella sp.; Asa subunits;
Efficient light-harvesting using non-carbonyl carotenoids: Energy transfer dynamics in the VCP complex from Nannochloropsis oceanica by Gürkan Keşan; Radek Litvín; David Bína; Milan Durchan; Václav Šlouf; Tomáš Polívka (370-379).
Violaxanthin–chlorophyll a protein (VCP) from Nannochloropsis oceanica is a Chl a-only member of the LHC family of light-harvesting proteins. VCP binds carotenoids violaxanthin (Vio), vaucheriaxanthin (Vau), and vaucheriaxanthin-ester (Vau-ester). Here we report on energy transfer pathways in the VCP complex. The overall carotenoid-to-Chla energy transfer has efficiency over 90%. Based on their energy transfer properties, the carotenoids in VCP can be divided into two groups; blue carotenoids with the lowest energy absorption band around 480 nm and red carotenoids with absorption extended up to 530 nm. Both carotenoid groups transfer energy efficiently from their S2 states, reaching efficiencies of ~ 70% (blue) and ~ 60% (red). The S1 pathway, however, is efficient only for the red carotenoid pool for which two S1 routes characterized by 0.33 and 2.4 ps time constants were identified. For the blue carotenoids the S1-mediated pathway is represented only by a minor route likely involving a hot S1 state. The relaxed S1 state of blue carotenoids decays to the ground state within 21 ps. Presence of a fraction of non-transferring red carotenoids with the S1 lifetime of 13 ps indicates some specific carotenoid-protein interaction that must shorten the intrinsic S1 lifetime of Vio and/or Vau whose S1 lifetimes in methanol are 26 and 29 ps, respectively. The VCP complex from N. oceanica is the first example of a light-harvesting complex binding only non-carbonyl carotenoids with carotenoid-to-chlorophyll energy transfer efficiency over 90%.
Keywords: Carotenoids; Energy transfer; Light-harvesting complex; Violaxanthin; Femtosecond spectroscopy;
Electron transfer between the QmoABC membrane complex and adenosine 5′-phosphosulfate reductase by Américo G. Duarte; André A. Santos; Inês A.C. Pereira (380-386).
The dissimilatory adenosine 5′-phosphosulfate reductase (AprAB) is a key enzyme in the sulfate reduction pathway that catalyzes the reversible two electron reduction of adenosine 5′-phosphosulfate (APS) to sulfite and adenosine monophosphate (AMP). The physiological electron donor for AprAB is proposed to be the QmoABC membrane complex, coupling the quinone-pool to sulfate reduction. However, direct electron transfer between these two proteins has never been observed. In this work we demonstrate for the first time direct electron transfer between the Desulfovibrio desulfuricans ATCC 27774 QmoABC complex and AprAB. Cyclic voltammetry conducted with the modified Qmo electrode and AprAB in the electrolyte solution presented the Qmo electrochemical signature with two additional well-defined one electron redox processes, attributed to the AprAB FAD redox behavior. Moreover, experiments performed under catalytic conditions using the QmoABC modified electrode, with AprAB and APS in solution, show a catalytic current peak develop in the cathodic wave, attributed to substrate reduction, and which is not observed in the absence of QmoABC. Substrate dependence conducted with different electrode preparations (with and without immobilized Qmo) demonstrated that the QmoABC complex is essential for efficient electron delivery to AprAB, in order to sustain catalysis. These results confirm the role of Qmo in electron transfer to AprAB.Display Omitted
Keywords: Dissimilatory sulfur metabolism; Respiratory membrane complex; Electron-transfer; Electrochemistry; Quinone-pool;
Structural and functional characterization of phosphomimetic mutants of cytochrome c at threonine 28 and serine 47 by Alejandra Guerra-Castellano; Irene Díaz-Moreno; Adrián Velázquez-Campoy; Miguel A. De la Rosa; Antonio Díaz-Quintana (387-395).
Protein function is frequently modulated by post-translational modifications of specific residues. Cytochrome c, in particular, is phosphorylated in vivo at threonine 28 and serine 47. However, the effect of such modifications on the physiological functions of cytochrome c – namely, the transfer of electrons in the respiratory electron transport chain and the triggering of programmed cell death – is still unknown. Here we replace each of these two residues by aspartate, in order to mimic phosphorylation, and report the structural and functional changes in the resulting cytochrome c variants. We find that the T28D mutant causes a 30-mV decrease on the midpoint redox potential and lowers the affinity for the distal site of Arabidopsis thaliana cytochrome c 1 in complex III. Both the T28D and S47D variants display a higher efficiency as electron donors for the cytochrome c oxidase activity of complex IV. In both protein mutants, the peroxidase activity is significantly higher, which is related to the ability of cytochrome c to leave the mitochondria and reach the cytoplasm. We also find that both mutations at serine 47 (S47D and S47A) impair the ability of cytoplasmic cytochrome c to activate the caspases cascade, which is essential for triggering programmed cell death.
Keywords: Caspase activity; Cytochrome c; Electron transport chain; Liposomes binding; Peroxidase activity; Phosphorylation;
Deletion of the gene family of small chlorophyll-binding proteins (ScpABCDE) offsets C/N homeostasis in Synechocystis PCC 6803 by Tania Tibiletti; Miguel A. Hernández-Prieto; Hans C.P. Matthijs; Krishna K. Niyogi; Christiane Funk (396-407).
In the family of chlorophyll binding proteins, single helix small CAB-like proteins (SCPs) are found in all organisms performing oxygenic photosynthesis. Here, we investigated the function of these stress-inducible proteins in the cyanobacterium Synechocystis sp. PCC 6803. We compared physiological, proteome and transcriptome traits of a Photosystem I (PSI) deletion strain, which constitutively induces SCPs, and a PSI-less/ScpABCDE− without SCPs. The SCP mutant cells were larger in size, showed irregular thylakoid structure and differed in cell-surface morphology. Deletion of scp genes strongly affected the carbon (C) and nitrogen (N) balance, resulting in accumulation of carbohydrates and a decrease in N-rich compounds (proteins and chlorophyll). Data from transcriptomic and metabolomic experiments revealed a role of SCPs in the control of chlorophyll biosynthesis. Additionally, SCPs diminished formation of reactive oxygen species, thereby preventing damage within Photosystem II. We conclude that the lack of SCP-function to remove free chlorophyll under stress conditions has a large impact on the metabolism of the entire cell.
Keywords: Small chlorophyll-proteins; High light protection; Pleiotropic effects; C/N metabolism; Cyanobacterium (Synechocystis sp. strain PCC 6803);
Temperature dependent LH1 → RC energy transfer in purple bacteria Tch. tepidum with shiftable LH1-Qy band: A natural system to investigate thermally activated energy transfer in photosynthesis by Fei Ma; Long-Jiang Yu; Zheng-Yu Wang-Otomo; Rienk van Grondelle (408-414).
The native LH1-RC complex of the purple bacterium Thermochromatium (Tch.) tepidum has an ultra-red LH1-Qy absorption at 915 nm, which can shift to 893 and 882 nm by means of chemical modifications. These unique complexes are a good natural system to investigate the thermally activated energy transfer process, with the donor energies different while the other factors (such as the acceptor energy, special pair at 890 nm, and the distance/relative orientation between the donor and acceptor) remain the same. The native B915-RC, B893-RC and B882-RC complexes, as well as the LH1-RC complex of Rhodobacter (Rba.) sphaeroides were studied by temperature-dependent time-resolved absorption spectroscopy. The energy transfer time constants, k ET − 1, are 65, 45, 46 and 45 ps at room temperature while 225, 58, 85, 33 ps at 77 K for the B915-RC, B893-RC, B882-RC and Rba. sphaeroides LH1-RC, respectively. The dependences of k ET on temperature have different trends. The reorganization energies are determined to be 70, 290, 200 and 45 cm− 1, respectively, by fitting k ET vs temperature using Marcus equation. The activation energies are 200, 60, 115 and 20 cm− 1, respectively. The influences of the structure (the arrangement of the 32 BChl a molecules) on k ET are discussed based on these results, to reveal how the B915-RC complex accomplishes its energy transfer function with a large uphill energy of 290 cm− 1.
Keywords: Thermochromatium (Tch.) tepidum; Time-resolved absorption spectroscopy; LH1 → RC energy transfer; Thermally activated energy transfer;
Speract, a sea urchin egg peptide that regulates sperm motility, also stimulates sperm mitochondrial metabolism by Juan García-Rincón; Alberto Darszon; Carmen Beltrán (415-426).
Sea urchin sperm have only one mitochondrion, that in addition to being the main source of energy, may modulate intracellular Ca2 + concentration ([Ca2 +]i) to regulate their motility and possibly the acrosome reaction. Speract is a decapeptide from the outer jelly layer of the Strongylocentrotus purpuratus egg that upon binding to its receptor in the sperm, stimulates sperm motility, respiration and ion fluxes, among other physiological events. Altering the sea urchin sperm mitochondrial function with specific inhibitors of this organelle, increases [Ca2 +]i in an external Ca2 + concentration ([Ca2 +]ext)-dependent manner (Ardón, et al., 2009. BBActa 1787: 15), suggesting that the mitochondrion is involved in sperm [Ca2 +]i homeostasis. To further understand the interrelationship between the mitochondrion and the speract responses, we measured mitochondrial membrane potential (ΔΨ) and NADH levels. We found that the stimulation of sperm with speract depolarizes the mitochondrion and increases the levels of NADH. Surprisingly, these responses are independent of external Ca2 + and are due to the increase in intracellular pH (pHi) induced by speract. Our findings indicate that speract, by regulating pHi, in addition to [Ca2 +]i, may finely modulate mitochondrial metabolism to control motility and ensure that sperm reach the egg and fertilize it.
Keywords: Intracellular pH; Mitochondrial membrane potential; NADH; Carnitine palmitoyl transferase-I; Speract; Sperm;
Contribution of bacteriochlorophyll conformation to the distribution of site-energies in the FMO protein by Stuart A. MacGowan; Mathias O. Senge (427-442).
The structural data for the Fenna–Matthews–Olson (FMO) protein indicate that the bacteriochlorophylls (BChls) display a significant degree of conformational heterogeneity of their peripheral substituents and the protein-induced nonplanar skeletal deformations of the tetrapyrrole macrocycle. As electronic properties of chromophores are altered by such differences, a conformational effect may influence the site-energies of specific pigments and thus play a role in mediating the excitation energy transfer dynamics, but this has not yet been established. The difficulty of assessing this question is shown to be partly the result of the inability of the sequential truncation approach usually employed to account for interactions between the conformations of the macrocycle and its substituents and an alternative approach is suggested. By assigning the BChl atoms to meaningful atom groups and performing all possible permutations of partial optimizations in a full-factorial design, where each group is either frozen in the crystal geometry or optimized in vacuo, followed by excited state calculations on each resulting structure (PM6//ZIndo/S), the specific effects of the conformations of each BChl component as well as mutual interactions between the molecular fragments on the site-energy can be delineated. This factorial relaxation procedure gives different estimates of the macrocycle conformational perturbation than the approach of sequentially truncating the BChl periphery. The results were evaluated in the context of published site-energies for the FMO pigments from three species to identify how conformational effects contribute to their distribution and instances of cross-species conservation and functional divergence of the BChl nonplanarity conformational contribution are described.Display Omitted
Keywords: Chlorophylls; Factorial design; Fenna–Matthews–Olson protein; Light-harvesting; Nonplanar porphyrins; Partial optimizations; Photosynthesis; Semi-empirical quantum chemistry;
Reduced cardiolipin content decreases respiratory chain capacities and increases ATP synthesis yield in the human HepaRG cells by Laure Peyta; Kathleen Jarnouen; Michelle Pinault; Cyrille Guimaraes; Jean-Paul Pais de Barros; Stephan Chevalier; Jean-François Dumas; François Maillot; Grant M. Hatch; Pascal Loyer; Stephane Servais (443-453).
Cardiolipin (CL) is a unique mitochondrial phospholipid potentially affecting many aspects of mitochondrial function/processes, i.e. energy production through oxidative phosphorylation. Most data focusing on implication of CL content and mitochondrial bioenergetics were performed in yeast or in cellular models of Barth syndrome. Previous work reported that increase in CL content leads to decrease in liver mitochondrial ATP synthesis yield. Therefore the aim of this study was to determine the effects of moderate decrease in CL content on mitochondrial bioenergetics in human hepatocytes. For this purpose, we generated a cardiolipin synthase knockdown (shCLS) in HepaRG hepatoma cells showing bioenergetics features similar to primary human hepatocytes. shCLS cells exhibited a 55% reduction in CLS gene and a 40% decrease in protein expression resulting in a 45% lower content in CL compared to control (shCTL) cells. Oxygen consumption was significantly reduced in shCLS cells compared to shCTL regardless of substrate used and energy state analyzed. Mitochondrial low molecular weight supercomplex content was higher in shCLS cells (+ 60%) compared to shCTL. Significant fragmentation of the mitochondrial network was observed in shCLS cells compared to shCTL cells. Surprisingly, mitochondrial ATP synthesis was unchanged in shCLS compared to shCTL cells but exhibited a higher ATP:O ratio (+ 46%) in shCLS cells. Our results suggest that lowered respiratory chain activity induced by moderate reduction in CL content may be due to both destabilization of supercomplexes and mitochondrial network fragmentation. In addition, CL content may regulate mitochondrial ATP synthesis yield.
Keywords: Cardiolipin synthase; Mitochondrial network; Supercomplexes; Oxidative phosphorylation; Hepatocytes;
Mechanism of inhibition of NiFe hydrogenase by nitric oxide by Pierre Ceccaldi; Emilien Etienne; Sébastien Dementin; Bruno Guigliarelli; Christophe Léger; Bénédicte Burlat (454-461).
Hydrogenases reversibly catalyze the oxidation of molecular hydrogen and are inhibited by several small molecules including O2, CO and NO. In the present work, we investigate the mechanism of inhibition by NO of the oxygen-sensitive NiFe hydrogenase from Desulfovibrio fructosovorans by coupling site-directed mutagenesis, protein film voltammetry (PFV) and EPR spectroscopy. We show that micromolar NO strongly inhibits NiFe hydrogenase and that the mechanism of inhibition is complex, with NO targeting several metallic sites in the protein. NO reacts readily at the NiFe active site according to a two-step mechanism. The first and faster step is the reversible binding of NO to the active site followed by a slower and irreversible transformation at the active site. NO also induces irreversible damage of the iron–sulfur centers chain. We give direct evidence of preferential nitrosylation of the medial [3Fe–4S] to form dinitrosyl–iron complexes.
Keywords: Hydrogenase; Nitric oxide; Inhibition mechanism; EPR spectroscopy; Protein film voltammetry; Metalloenzyme;
Excitation energy transfer between Light-harvesting complex II and Photosystem I in reconstituted membranes by Parveen Akhtar; Mónika Lingvay; Teréz Kiss; Róbert Deák; Attila Bóta; Bettina Ughy; Győző Garab; Petar H. Lambrev (462-472).
Light-harvesting complex II (LHCII), the major peripheral antenna of Photosystem II in plants, participates in several concerted mechanisms for regulation of the excitation energy and electron fluxes in thylakoid membranes. In part, these include interaction of LHCII with Photosystem I (PSI) enhancing the latter's absorption cross-section – for example in the well-known state 1 – state 2 transitions or as a long-term acclimation to high light. In this work we examined the capability of LHCII to deliver excitations to PSI in reconstituted membranes in vitro. Proteoliposomes with native plant thylakoid membrane lipids and different stoichiometric ratios of LHCII:PSI were reconstituted and studied by steady-state and time-resolved fluorescence spectroscopy. Fluorescence emission from LHCII was strongly decreased in PSI–LHCII membranes due to trapping of excitations by PSI. Kinetic modelling of the time-resolved fluorescence data revealed the existence of separate pools of LHCII distinguished by the time scale of energy transfer. A strongly coupled pool, equivalent to one LHCII trimer per PSI, transferred excitations to PSI with near-unity efficiency on a time scale of less than 10 ps but extra LHCIIs also contributed significantly to the effective antenna size of PSI, which could be increased by up to 47% in membranes containing 3 LHCII trimers per PSI. The results demonstrate a remarkable competence of LHCII to increase the absorption cross-section of PSI, given the opportunity that the two types of complexes interact in the membrane.Display Omitted
Keywords: Artificial membranes; Light harvesting; Proteoliposomes; State transitions; Time-resolved fluorescence; Thylakoid membranes;
Role of the Na+-translocating NADH:quinone oxidoreductase in voltage generation and Na+ extrusion in Vibrio cholerae by Thomas Vorburger; Ruslan Nedielkov; Alexander Brosig; Eva Bok; Emina Schunke; Wojtek Steffen; Sonja Mayer; Friedrich Götz; Heiko M. Möller; Julia Steuber (473-482).
For Vibrio cholerae, the coordinated import and export of Na+ is crucial for adaptation to habitats with different osmolarities. We investigated the Na+-extruding branch of the sodium cycle in this human pathogen by in vivo 23Na-NMR spectroscopy. The Na+ extrusion activity of cells was monitored after adding glucose which stimulated respiration via the Na+-translocating NADH:quinone oxidoreductase (Na+-NQR). In a V. cholerae deletion mutant devoid of the Na+-NQR encoding genes (nqrA-F), rates of respiratory Na+ extrusion were decreased by a factor of four, but the cytoplasmic Na+ concentration was essentially unchanged. Furthermore, the mutant was impaired in formation of transmembrane voltage (ΔΨ, inside negative) and did not grow under hypoosmotic conditions at pH 8.2 or above. This growth defect could be complemented by transformation with the plasmid encoded nqr operon. In an alkaline environment, Na+/H+ antiporters acidify the cytoplasm at the expense of the transmembrane voltage. It is proposed that, at alkaline pH and limiting Na+ concentrations, the Na+-NQR is crucial for generation of a transmembrane voltage to drive the import of H+ by electrogenic Na+/H+ antiporters. Our study provides the basis to understand the role of the Na+-NQR in pathogenicity of V. cholerae and other pathogens relying on this primary Na+ pump for respiration.
Keywords: Nuclear magnetic resonance (NMR); Sodium transport; Vibrio cholerae; Respiration; Na+ homeostasis; Hypoosmotic stress;