Bacteroides fragilis Maintains Concurrent Capability for Anaerobic and Nanaerobic Respiration.

Bacteroides species can use fumarate and oxygen as terminal electron acceptors during cellular respiration. In the human gut, oxygen diffuses from intestinal epithelial cells supplying "nanaerobic" oxygen levels. Many components of the anaerobic respiratory pathway have been determined, but such analyses have not been performed for nanaerobic respiration. Here, we present genetic, biochemical, enzymatic, and mass spectrometry analyses to elucidate the nanaerobic respiratory pathway in Bacteroides fragilis. Under anaerobic conditions, the transfer of electrons from NADH to the quinone pool has been shown to be contributed by two enzymes, NQR and NDH2. We find that the activity contributed by each under nanaerobic conditions is 77 and 23%, respectively, similar to the activity levels under anaerobic conditions. Using mass spectrometry, we show that the quinone pool also does not differ under these two conditions and consists of a mixture of menaquinone-8 to menaquinone-11, with menaquinone-10 predominant under both conditions. Analysis of fumarate reductase showed that it is synthesized and active under anaerobic and nanaerobic conditions. Previous RNA sequencing data and new transcription reporter assays show that expression of the cytochrome bd oxidase gene does not change under these conditions. Under nanaerobic conditions, we find both increased CydA protein and increased cytochrome bd activity. Reduced-minus-oxidized spectra of membranes showed the presence of heme d when the bacteria were grown in the presence of protoporphyrin IX and iron under both anaerobic and nanaerobic conditions, suggesting that the active oxidase can be assembled with or without oxygen. IMPORTANCE By performing a comprehensive analysis of nanaerobic respiration in Bacteroides fragilis, we show that this organism maintains capabilities for anaerobic respiration on fumarate and nanaerobic respiration on oxygen simultaneously. The contribution of the two NADH:quinone oxidoreductases and the composition of the quinone pool are the same under both conditions. Fumarate reductase and cytochrome bd are both present, and which of these terminal enzymes is active in electron transfer depends on the availability of the final electron acceptor: fumarate or oxygen. The synthesis of cytochrome bd and fumarate reductase under both conditions serves as an adaptation to an environment with low oxygen concentrations so that the bacteria can maximize energy conservation during fluctuating environmental conditions or occupation of different spatial niches.

Nanaerobic growth enables direct visualization of dynamic cellular processes in human gut symbionts.

Mechanistic studies of anaerobic gut bacteria have been hindered by the lack of a fluorescent protein system to track and visualize proteins and dynamic cellular processes in actively growing bacteria. Although underappreciated, many gut "anaerobes" are able to respire using oxygen as the terminal electron acceptor. The oxygen continually released from gut epithelial cells creates an oxygen gradient from the mucus layer to the anaerobic lumen [L. Albenberg et al., Gastroenterology 147, 1055-1063.e8 (2014)], with oxygen available to bacteria growing at the mucus layer. Here, we show that Bacteroides species are metabolically and energetically robust and do not mount stress responses in the presence of 0.10 to 0.14% oxygen, defined as nanaerobic conditions [A. D. Baughn, M. H. Malamy, Nature 427, 441-444 (2004)]. Taking advantage of this metabolic capability, we show that nanaerobic growth provides sufficient oxygen for the maturation of oxygen-requiring fluorescent proteins in Bacteroides species. Type strains of four different Bacteroides species show bright GFP fluorescence when grown nanaerobically versus anaerobically. We compared four different red fluorescent proteins and found that mKate2 yields the highest red fluorescence intensity in our assay. We show that GFP-tagged proteins can be localized in nanaerobically growing bacteria. In addition, we used time-lapse fluorescence microscopy to image dynamic type VI secretion system processes in metabolically active Bacteroides fragilis The ability to visualize fluorescently labeled Bacteroides and fluorescently linked proteins in actively growing nanaerobic gut symbionts ushers in an age of imaging analyses not previously possible in these bacteria.

Genetic and Biochemical Characterization of the Na+/H+ Antiporters of Pseudomonas aeruginosa.

Pseudomonas aeruginosa has four Na+/H+ antiporters that interconvert and balance Na+ and H+ gradients across the membrane. These gradients are important for bioenergetics and ionic homeostasis. To understand these transporters, we constructed four strains, each of which has only one antiporter, i.e., NhaB, NhaP, NhaP2, and Mrp. We also constructed a quadruple deletion mutant that has no Na+/H+ antiporters. Although the antiporters of P. aeruginosa have been studied previously, the strains constructed here present the opportunity to characterize their kinetic properties in their native membranes and their roles in the physiology of P. aeruginosa. The strains expressing only NhaB or Mrp, the two electrogenic antiporters, were able to grow essentially like the wild-type strain across a range of Na+ concentrations and pH values. Strains with only NhaP or NhaP2, which are electroneutral, grew more poorly at increasing Na+ concentrations, especially at high pH values, with the strain expressing NhaP being more sensitive. The strain with no Na+/H+ antiporters was extremely sensitive to the Na+ concentration and showed essentially no Na+(Li+)/H+ antiporter activity, but it retained most K+/H+ antiporter activity of the wild-type strain at pH 7.5 and approximately one-half at pH 8.5. We also used the four strains that each express one of the four antiporters to characterize the kinetic properties of each transporter. Transcriptome sequencing analysis of the quadruple deletion strain showed widespread changes, including changes in pyocyanin synthesis, biofilm formation, and nitrate and glycerol metabolism. Thus, the strains constructed for this study will open a new door to understanding the physiological roles of these proteins and their activities in P. aeruginosa. IMPORTANCE Pseudomonas aeruginosa has four Na+/H+ antiporters that connect and interconvert its Na+ and H+ gradients. We have constructed four deletion mutants, each of which has only one of the four Na+/H+ antiporters. These strains made it possible to study the properties and physiological roles of each antiporter independently in its native membrane. Mrp and NhaB are each able to sustain growth over a wide range of pH values and Na+ concentrations, whereas the two electroneutral antiporters, NhaP and NhaP2, are most effective at low pH values. We also constructed a quadruple mutant lacking all four antiporters, in which the H+ and Na+ gradients are disconnected. This will make it possible to study the role of the two gradients independently.

Inhibitors of a Na+-pumping NADH-ubiquinone oxidoreductase play multiple roles to block enzyme function.

The Na+-pumping NADH-ubiquinone (UQ) oxidoreductase (Na+-NQR) is present in the respiratory chain of many pathogenic bacteria and is thought to be a promising antibiotic target. Whereas many details of Na+-NQR structure and function are known, the mechanisms of action of potent inhibitors is not well-understood; elucidating the mechanisms would not only advance drug design strategies but might also provide insights on a terminal electron transfer from riboflavin to UQ. To this end, we performed photoaffinity labeling experiments using photoreactive derivatives of two known inhibitors, aurachin and korormicin, on isolated Vibrio cholerae Na+-NQR. The inhibitors labeled the cytoplasmic surface domain of the NqrB subunit including a protruding N-terminal stretch, which may be critical to regulate the UQ reaction in the adjacent NqrA subunit. The labeling was blocked by short-chain UQs such as ubiquinone-2. The photolabile group (2-aryl-5-carboxytetrazole (ACT)) of these inhibitors reacts with nucleophilic amino acids, so we tested mutations of nucleophilic residues in the labeled region of NqrB, such as Asp49 and Asp52 (to Ala), and observed moderate decreases in labeling yields, suggesting that these residues are involved in the interaction with ACT. We conclude that the inhibitors interfere with the UQ reaction in two ways: the first is blocking structural rearrangements at the cytoplasmic interface between NqrA and NqrB, and the second is the direct obstruction of UQ binding at this interfacial area. Unusual competitive behavior between the photoreactive inhibitors and various competitors corroborates our previous proposition that there may be two inhibitor binding sites in Na+-NQR.

Metabolic engineering of E. coli for pyocyanin production.

Pyocyanin is a secondary metabolite from Pseudomonas aeruginosa that belongs to the class of phenazines, which are aromatic nitrogenous compounds with numerous biological functions. Besides its antifungal and antimicrobial activities, pyocyanin is a remarkable redox-active molecule with potential applications ranging from the pharma industry to the development of microbial fuel cells. Nevertheless, pyocyanin production has been restricted to P. aeruginosa strains, limiting its practical applicability. In this study, the pyocyanin biosynthetic pathway was engineered for the first time for high level production of this compound in a heterologous host. Escherichia coli cells harboring the nine-gene pathway divided into two plasmids were able to produce and secrete pyocyanin at higher levels than some Pseudomonas aeruginosa strains. The influence of culture and induction parameters were evaluated, and the optimized conditions led to an increase of 3.5-fold on pyocyanin accumulation. Pathway balancing was achieved by testing a set of plasmids with different copy numbers to optimize the expression levels of pyocyanin biosynthetic genes, resulting in a fourfold difference in product titer among the engineered strains. Further improvements were achieved by co-expression of Vitreoscilla hemoglobin Vhb, which relieved oxygen limitations and led to a final titer of 18.8 mg/L pyocyanin. These results show promise to use E. coli for phenazines production, and the engineered strain developed here has the potential to be used in electro-fermentation systems where pyocyanin plays a role as electron-shuttle.

Antibiotic Korormicin A Kills Bacteria by Producing Reactive Oxygen Species.

Korormicin is an antibiotic produced by some pseudoalteromonads which selectively kills Gram-negative bacteria that express the Na+-pumping NADH:quinone oxidoreductase (Na+-NQR.) We show that although korormicin is an inhibitor of Na+-NQR, the antibiotic action is not a direct result of inhibiting enzyme activity. Instead, perturbation of electron transfer inside the enzyme promotes a reaction between O2 and one or more redox cofactors in the enzyme (likely the flavin adenine dinucleotide [FAD] and 2Fe-2S center), leading to the production of reactive oxygen species (ROS). All Pseudoalteromonas contain the nqr operon in their genomes, including Pseudoalteromonas strain J010, which produces korormicin. We present activity data indicating that this strain expresses an active Na+-NQR and that this enzyme is not susceptible to korormicin inhibition. On the basis of our DNA sequence data, we show that the Na+-NQR of Pseudoalteromonas J010 carries an amino acid substitution (NqrB-G141A; Vibrio cholerae numbering) that in other Na+-NQRs confers resistance against korormicin. This is likely the reason that a functional Na+-NQR is able to exist in a bacterium that produces a compound that typically inhibits this enzyme and causes cell death. Korormicin is an effective antibiotic against such pathogens as Vibrio cholerae, Aliivibrio fischeri, and Pseudomonas aeruginosa but has no effect on Bacteroides fragilis and Bacteroides thetaiotaomicron, microorganisms that are important members of the human intestinal microflora.IMPORTANCE As multidrug antibiotic resistance in pathogenic bacteria continues to rise, there is a critical need for novel antimicrobial agents. An essential requirement for a useful antibiotic is that it selectively targets bacteria without significant effects on the eukaryotic hosts. Korormicin is an excellent candidate in this respect because it targets a unique respiratory enzyme found only in prokaryotes, the Na+-pumping NADH:quinone oxidoreductase (Na+-NQR). Korormicin is synthesized by some species of the marine bacterium Pseudoalteromonas and is a potent and specific inhibitor of Na+-NQR, an enzyme that is essential for the survival and proliferation of many Gram-negative human pathogens, including Vibrio cholerae and Pseudomonas aeruginosa, among others. Here, we identified how korormicin selectively kills these bacteria. The binding of korormicin to Na+-NQR promotes the formation of reactive oxygen species generated by the reaction of the FAD and the 2Fe-2S center cofactors with O2.

Glutamate 95 in NqrE Is an Essential Residue for the Translocation of Cations in Na+-NQR.

The sodium-pumping NADH:quinone oxidoreductase (Na+-NQR) is a bacterial enzyme that oxidizes NADH, reduces ubiquinone, and translocates Na+ across the membrane. We previously identified three acidic residues in the membrane-spanning helices, near the cytosol, NqrB-D397, NqrD-D133, and NqrE-E95, as candidates likely to be involved in Na+ uptake, and replacement of any one of them by a non-acidic residue affects the Na+-dependent kinetics of the enzyme. Here, we have inquired further into the role of the NqrE-E95 residue by constructing a series of mutants in which this residue is replaced by amino acids with charges and/or sizes different from those of the glutamate of the wild-type enzyme. All of the mutants showed altered steady-state kinetics with the acceleration of turnover by Na+ greatly diminished. Selected mutants were studied by other physical methods. Membrane potential measurements showed that NqrE-E95D and A are significantly less efficient in ion transport. NqrE-E95A, Q, and D were studied by transient kinetic measurements of the reduction of the enzyme by NADH. In all three cases, the results indicated inhibition of the electron-transfer step in which the FMNC becomes reduced. This is the first Na+-dependent step and is associated with Na+ uptake by the enzyme. Electrochemical measurements on NqrE-E95Q showed that the Na+ dependence of the redox potential of the FMN cofactors has been lost. The fact that the mutations at the NqrE-E95 site have specific effects related to translocation of Na+ and Li+ strongly indicates a definite role for NqrE-E95 in the cation transport process of Na+-NQR.

Identification of the binding sites for ubiquinone and inhibitors in the Na+-pumping NADH-ubiquinone oxidoreductase from Vibrio cholerae by photoaffinity labeling.

The Na+-pumping NADH-quinone oxidoreductase (Na+-NQR) is the first enzyme of the respiratory chain and the main ion transporter in many marine and pathogenic bacteria, including Vibrio cholerae The V. cholerae Na+-NQR has been extensively studied, but its binding sites for ubiquinone and inhibitors remain controversial. Here, using a photoreactive ubiquinone PUQ-3 as well as two aurachin-type inhibitors [125I]PAD-1 and [125I]PAD-2 and photoaffinity labeling experiments on the isolated enzyme, we demonstrate that the ubiquinone ring binds to the NqrA subunit in the regions Leu-32-Met-39 and Phe-131-Lys-138, encompassing the rear wall of a predicted ubiquinone-binding cavity. The quinolone ring and alkyl side chain of aurachin bound to the NqrB subunit in the regions Arg-43-Lys-54 and Trp-23-Gly-89, respectively. These results indicate that the binding sites for ubiquinone and aurachin-type inhibitors are in close proximity but do not overlap one another. Unexpectedly, although the inhibitory effects of PAD-1 and PAD-2 were almost completely abolished by certain mutations in NqrB (i.e. G140A and E144C), the binding reactivities of [125I]PAD-1 and [125I]PAD-2 to the mutated enzymes were unchanged compared with those of the wild-type enzyme. We also found that photoaffinity labeling by [125I]PAD-1 and [125I]PAD-2, rather than being competitively suppressed in the presence of other inhibitors, is enhanced under some experimental conditions. To explain these apparently paradoxical results, we propose models for the catalytic reaction of Na+-NQR and its interactions with inhibitors on the basis of the biochemical and biophysical results reported here and in previous work.

Single Molecule Force Spectroscopy and Molecular Dynamics Simulations as a Combined Platform for Probing Protein Face-Specific Binding.

Biomolecular interactions frequently occur in orientation-specific manner. For example, prior nuclear magnetic resonance spectroscopy experiments in our lab have suggested the presence of a group of strongly binding residues on a particular face of the protein ubiquitin for interactions with Capto MMC multimodal ligands ("Capto" ligands) (Srinivasan, K.; et al. Langmuir 2014, 30 (44), 13205-13216). We present a clear confirmation of those studies by performing single-molecule force spectroscopy (SMFS) measurements of unbinding complemented with molecular dynamics (MD) calculations of the adsorption free energy of ubiquitin in two distinct orientations with self-assembled monolayers (SAMs) functionalized with "Capto" ligands. These orientations were maintained in the SMFS experiments by tethering ubiquitin mutants to SAM surfaces through strategically located cysteines, thus exposing the desired faces of the protein. Analogous orientations were maintained in MD simulations using suitable constraining methods. Remarkably, despite differences between the finer details of experimental and simulation methodologies, they confirm a clear preference for the previously hypothesized binding face of ubiquitin. Furthermore, MD simulations provided significant insights into the mechanism of protein binding onto this multimodal surface. Because SMFS and MD simulations both directly probe protein-surface interactions, this work establishes a key link between experiments and simulations at molecular scale through the determination of protein face-specific binding energetics. Our approach may have direct applications in biophysical systems where face- or orientation-specific interactions are important, such as biomaterials, sensors, and biomanufacturing.

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.

A mutation in Na(+)-NQR uncouples electron flow from Na(+) translocation in the presence of K(+).

The sodium-pumping NADH:ubiquinone oxidoreductase (Na(+)-NQR) is a bacterial respiratory enzyme that obtains energy from the redox reaction between NADH and ubiquinone and uses this energy to create an electrochemical Na(+) gradient across the cell membrane. A number of acidic residues in transmembrane helices have been shown to be important for Na(+) translocation. One of these, Asp-397 in the NqrB subunit, is a key residue for Na(+) uptake and binding. In this study, we show that when this residue is replaced with asparagine, the enzyme acquires a new sensitivity to K(+); in the mutant, K(+) both activates the redox reaction and uncouples it from the ion translocation reaction. In the wild-type enzyme, Na(+) (or Li(+)) accelerates turnover while K(+) alone does not activate. In the NqrB-D397N mutant, K(+) accelerates the same internal electron transfer step (2Fe-2S → FMNC) that is accelerated by Na(+). This is the same step that is inhibited in mutants in which Na(+) uptake is blocked. NqrB-D397N is able to translocate Na(+) and Li(+), but when K(+) is introduced, no ion translocation is observed, regardless of whether Na(+) or Li(+) is present. Thus, this mutant, when it turns over in the presence of K(+), is the first, and currently the only, example of an uncoupled Na(+)-NQR. The fact the redox reaction and ion pumping become decoupled from each other only in the presence of K(+) provides a switch that promises to be a useful experimental tool.

Complete topology of the RNF complex from Vibrio cholerae.

RNF is a redox-driven ion (Na(+) and in one case possibly H(+)) transporter present in many prokaryotes. It has been proposed that RNF performs a variety of reactions in different organisms, delivering low-potential reducing equivalents for specific cellular processes. RNF shares strong homology with the Na(+)-pumping respiratory enzyme Na(+)-NQR, although there are significant differences in subunit and redox cofactor composition. Here we report a topological analysis of the six subunits of RNF from Vibrio cholerae. Although individual subunits from other organisms have previously been studied, this is the first complete, experimentally derived, analysis of RNF from any one source. This has allowed us to identify and confirm key properties of RNF. The putative NADH binding site in RnfC is located on the cytoplasmic side of the membrane. FeS centers in RnfB and RnfC are also located on the cytoplasmic side. However, covalently attached FMNs in RnfD and RnfG are both located in the periplasm. RNF also contains a number of acidic residues that correspond to functionally important groups in Na(+)-NQR. The acidic residues involved in Na(+) uptake and many of those implicated in Na(+) translocation are topologically conserved. The topology of RNF closely matches the topology represented in the newly published structure of Na(+)-NQR, consistent with the close relation between the two enzymes. The topology of RNF is discussed in the context of the current structural model of Na(+)-NQR, and the proposed functionality of the RNF complex itself.

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.

Aspartic acid 397 in subunit B of the Na+-pumping NADH:quinone oxidoreductase from Vibrio cholerae forms part of a sodium-binding site, is involved in cation selectivity, and affects cation-binding site cooperativity.

The Na(+)-pumping NADH:quinone complex is found in Vibrio cholerae and other marine and pathogenic bacteria. NADH:ubiquinone oxidoreductase oxidizes NADH and reduces ubiquinone, using the free energy released by this reaction to pump sodium ions across the cell membrane. In a previous report, a conserved aspartic acid residue in the NqrB subunit at position 397, located in the cytosolic face of this protein, was proposed to be involved in the capture of sodium. Here, we studied the role of this residue through the characterization of mutant enzymes in which this aspartic acid was substituted by other residues that change charge and size, such as arginine, serine, lysine, glutamic acid, and cysteine. Our results indicate that NqrB-Asp-397 forms part of one of the at least two sodium-binding sites and that both size and charge at this position are critical for the function of the enzyme. Moreover, we demonstrate that this residue is involved in cation selectivity, has a critical role in the communication between sodium-binding sites, by promoting cooperativity, and controls the electron transfer step involved in sodium uptake (2Fe-2S → FMNC).

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.

Versatile roles of protein flavinylation in bacterial extracyotosolic electron transfer.

Bacteria perform diverse redox chemistries in the periplasm, cell wall, and extracellular space. Electron transfer for these extracytosolic activities is frequently mediated by proteins with covalently bound flavins, which are attached through post-translational flavinylation by the enzyme ApbE. Despite the significance of protein flavinylation to bacterial physiology, the basis and function of this modification remains unresolved. Here we apply genomic context analyses, computational structural biology, and biochemical studies to address the role of ApbE flavinylation throughout bacterial life. We find that ApbE flavinylation sites exhibit substantial structural heterogeneity. We identify two novel classes of flavinylation substrates that are related to characterized proteins with non-covalently bound flavins, providing evidence that protein flavinylation can evolve from a non-covalent flavoprotein precursor. We further find a group of structurally related flavinylation-associated cytochromes, including those with the domain of unknown function DUF4405, that presumably mediate electron transfer in the cytoplasmic membrane. DUF4405 homologs are widespread in bacteria and related to ferrosome iron storage organelle proteins that may facilitate iron redox cycling within ferrosomes. These studies reveal a complex basis for flavinylated electron transfer and highlight the discovery power of coupling comparative genomic analyses with high-quality structural models.

Infrared spectroscopic evidence of a redox-dependent conformational change involving ion binding residue NqrB-D397 in the Na(+)-pumping NADH:quinone oxidoreductase from Vibrio cholerae.

The Na(+)-pumping NADH:quinone oxidoreductase (Na(+)-NQR) is a unique respiratory enzyme that conserves energy by translocating Na(+) through the plasma membrane. Found only in prokaryotes, the enzyme serves as the point of entry of electrons into the respiratory chain in many pathogens, including Vibrio cholerae and Yersinia pestis. In this study, a combined electrochemical and Fourier transform infrared (FTIR) spectroscopic approach revealed that Na(+)-NQR undergoes significant conformational changes upon oxidoreduction, depending on the monovalent cation present (Na(+), Li(+), K(+), or Rb(+)). In the presence of the inhibitor Rb(+), additional conformational changes are evident, indicating a changed accessibility of the sodium binding sites. In electrochemically induced FTIR difference spectra, the involvement of deprotonated acid residues in the binding of cations, together with the spectral features, that point toward a monodentate binding mode for these acid residues in the oxidized form of the enzyme and bidentate binding in the reduced form could be identified. The measurements confirmed that NqrB-D397 is one of the acid residues involved in Na(+) and Li(+) binding. In the NqrB-D397E mutant, the spectral features characteristic of COO(-) groups are shifted, and a weakening of the hydrogen binding of the ion binding cluster is revealed. Finally, H-D exchange kinetics of amide protons confirmed that Na(+)-NQR adopts different conformations, with different accessibilities to the aqueous environment, depending on the cation present, which contributes to the selectivity mechanism of ion translocation.

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.

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).

Energy transducing redox steps of the Na+-pumping NADH:quinone oxidoreductase from Vibrio cholerae.

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 sodium pumping process do not have any redox cofactor in common. The significance of these results for the mechanism of coupling is discussed, and we proposed that Na(+)-NQR operates through a novel mechanism based on kinetic coupling, mediated by conformational changes.