A Redox-Regulated, Heterodimeric NADH:cinnamate Reductase in Vibrio ruber.

Genes of putative reductases of α,ß-unsaturated carboxylic acids are abundant among anaerobic and facultatively anaerobic microorganisms, yet substrate specificity has been experimentally verified for few encoded proteins. Here, we co-produced in Escherichia coli a heterodimeric protein of the facultatively anaerobic marine bacterium Vibrio ruber (GenBank SJN56019 and SJN56021; annotated as NADPH azoreductase and urocanate reductase, respectively) with Vibrio cholerae flavin transferase. The isolated protein (named Crd) consists of the sjn56021-encoded subunit CrdB (NADH:flavin, FAD binding 2, and FMN bind domains) and an additional subunit CrdA (SJN56019, a single NADH:flavin domain) that interact via their NADH:flavin domains (Alphafold2 prediction). Each domain contains a flavin group (three FMNs and one FAD in total), one of the FMN groups being linked covalently by the flavin transferase. Crd readily reduces cinnamate, p-coumarate, caffeate, and ferulate under anaerobic conditions with NADH or methyl viologen as the electron donor, is moderately active against acrylate and practically inactive against urocanate and fumarate. Cinnamates induced Crd synthesis in V. ruber cells grown aerobically or anaerobically. The Crd-catalyzed reduction started by NADH demonstrated a time lag of several minutes, suggesting a redox regulation of the enzyme activity. The oxidized enzyme is inactive, which apparently prevents production of reactive oxygen species under aerobic conditions. Our findings identify Crd as a regulated NADH-dependent cinnamate reductase, apparently protecting V. ruber from (hydroxy)cinnamate poisoning.

Acrylate Reductase of an Anaerobic Electron Transport Chain of the Marine Bacterium Shewanella woodyi.

Many microorganisms are capable of anaerobic respiration in the absence of oxygen, by using different organic compounds as terminal acceptors in electron transport chain. We identify here an anaerobic respiratory chain protein responsible for acrylate reduction in the marine bacterium Shewanella woodyi. When the periplasmic proteins of S. woodyi were separated by ion exchange chromatography, acrylate reductase activity copurified with an ArdA protein (Swoo_0275). Heterologous expression of S. woodyi ardA gene (swoo_0275) in Shewanella oneidensis MR-1 cells did not result in the appearance in them of periplasmic acrylate reductase activity, but such activity was detected when the ardA gene was co-expressed with an ardB gene (swoo_0276). Together, these genes encode flavocytochrome c ArdAB, which is thus responsible for acrylate reduction in S. woodyi cells. ArdAB was highly specific for acrylate as substrate and reduced only methacrylate (at a 22-fold lower rate) among a series of other tested 2-enoates. In line with these findings, acrylate and methacrylate induced ardA gene expression in S. woodyi under anaerobic conditions, which was accompanied by the appearance of periplasmic acrylate reductase activity. ArdAB-linked acrylate reduction supports dimethylsulfoniopropionate-dependent anaerobic respiration in S. woodyi and, possibly, other marine bacteria.

A Novel, NADH-Dependent Acrylate Reductase in Vibrio harveyi.

Bacteria coping with oxygen deficiency use alternative terminal electron acceptors for NADH regeneration, particularly fumarate. Fumarate is reduced by the FAD_binding_2 domain of cytoplasmic fumarate reductase in many bacteria. The variability of the primary structure of this domain in homologous proteins suggests the existence of reducing activities with different specificities. Here, we produced and characterized one such protein encoded in the Vibrio harveyi genome (GenBank ID: AIV07243) and found it to be a specific NADH:acrylate oxidoreductase (ARD). This previously unknown enzyme is formed by the OYE-like, FMN_bind, and FAD_binding_2 domains and contains covalently bound flavin mononucleotide (FMN) and noncovalently bound flavin adenine dinucleotide (FAD) and FMN in a ratio of 1:1:1. The covalently bound FMN is absolutely required for activity and is attached by the specific flavin transferase, ApbE, to the FMN_bind domain. Quantitative reverse transcription PCR (RT-qPCR) and activity measurements indicated dramatic stimulation of ARD biosynthesis by acrylate in the V. harveyi cells grown aerobically. In contrast, the ard gene expression in the cells grown anaerobically without acrylate was higher than that in aerobic cultures and increased only 2-fold in the presence of acrylate. These findings suggest that the principal role of ARD in Vibrio is energy-saving detoxification of acrylate coming from the environment. IMPORTANCE The benefits of the massive genomic information accumulated in recent years for biological sciences have been limited by the lack of data on the function of most gene products. Approximately half of the known prokaryotic genes are annotated as "proteins with unknown functions," and many other genes are annotated incorrectly. Thus, the functional and structural characterization of the products of such genes, including identification of all existing enzymatic activities, is a pressing issue in modern biochemistry. In this work, we have shown that the product of the V. harveyi ard gene exhibits a yet-undescribed NADH:acrylate oxidoreductase activity. This activity may allow acrylate detoxification and its use as a terminal electron acceptor in anaerobic or substrate in aerobic respiration of marine and other bacteria.

Mechanism of Ion Translocation by Na+-Rhodopsin.

This review provides a brief description of the structure and transport function of the recently discovered family of retinal-containing Na+-translocating rhodopsins. The main emphasis is put on the kinetics of generation of electric potential difference in the membrane during a single transporter turnover. According to the proposed transport mechanism of Na+-rhodopsin, the driving force for the Na+ translocation from the cytoplasm is the local electric field created by the H+ movement from the Schiff base.

The Mechanism of Energy Coupling in H+/Na+-Pumping Membrane Pyrophosphatase-Possibilities and Probabilities.

Membrane pyrophosphatases (mPPases) found in plant vacuoles and some prokaryotes and protists are ancient cation pumps that couple pyrophosphate hydrolysis with the H+ and/or Na+ transport out of the cytoplasm. Because this function is reversible, mPPases play a role in maintaining the level of cytoplasmic pyrophosphate, a known regulator of numerous metabolic reactions. mPPases arouse interest because they are among the simplest membrane transporters and have no homologs among known ion pumps. Detailed phylogenetic studies have revealed various subtypes of mPPases and suggested their roles in the evolution of the "sodium" and "proton" bioenergetics. This treatise focuses on the mechanistic aspects of the transport reaction, namely, the coupling step, the role of the chemically produced proton, subunit cooperation, and the relationship between the proton and sodium ion transport. The available data identify H+-PPases as the first non-oxidoreductase pump with a "direct-coupling" mechanism, i.e., the transported proton is produced in the coupled chemical reaction. They also support a "billiard" hypothesis, which unifies the H+ and Na+ transport mechanisms in mPPase and, probably, other transporters.

Catalytic Asymmetry in Homodimeric H+-Pumping Membrane Pyrophosphatase Demonstrated by Non-Hydrolyzable Pyrophosphate Analogs.

Membrane-bound inorganic pyrophosphatase (mPPase) resembles the F-ATPase in catalyzing polyphosphate-energized H+ and Na+ transport across lipid membranes, but differs structurally and mechanistically. Homodimeric mPPase likely uses a "direct coupling" mechanism, in which the proton generated from the water nucleophile at the entrance to the ion conductance channel is transported across the membrane or triggers Na+ transport. The structural aspects of this mechanism, including subunit cooperation, are still poorly understood. Using a refined enzyme assay, we examined the inhibition of K+-dependent H+-transporting mPPase from Desulfitobacterium hafniensee by three non-hydrolyzable PPi analogs (imidodiphosphate and C-substituted bisphosphonates). The kinetic data demonstrated negative cooperativity in inhibitor binding to two active sites, and reduced active site performance when the inhibitor or substrate occupied the other active site. The nonequivalence of active sites in PPi hydrolysis in terms of the Michaelis constant vanished at a low (0.1 mM) concentration of Mg2+ (essential cofactor). The replacement of K+, the second metal cofactor, by Na+ increased the substrate and inhibitor binding cooperativity. The detergent-solubilized form of mPPase exhibited similar active site nonequivalence in PPi hydrolysis. Our findings support the notion that the mPPase mechanism combines Mitchell's direct coupling with conformational coupling to catalyze cation transport across the membrane.

A simple strategy to differentiate between H+- and Na+-transporting NADH:quinone oxidoreductases.

We describe here a simple strategy to characterize transport specificity of NADH:quinone oxidoreductases, using Na+-translocating (NQR) and H+-translocating (NDH-1) enzymes of the soil bacterium Azotobactervinelandii as the models. Submillimolar concentrations of Na+ and Li+ increased the rate of deaminoNADH oxidation by the inverted membrane vesicles prepared from the NDH-1-deficient strain. The vesicles generated carbonyl cyanide m-chlorophenyl hydrazone (CCCP)-resistant electric potential difference and CCCP-stimulated pH difference (alkalinization inside) in the presence of Na+. These findings testified a primary Na+-pump function of A. vinelandii NQR. Furthermore, ΔpH measurements with fluorescent probes (acridine orange and pyranine) demonstrated that A. vinelandii NQR cannot transport H+ under various conditions. The opposite results obtained in similar measurements with the vesicles prepared from the NQR-deficient strain indicated a primary H+-pump function of NDH-1. Based on our findings, we propose a package of simple experiments that are necessary and sufficient to unequivocally identify the pumping specificity of a bacterial Na+ or H+ transporter. The NQR-deficient strain, but not the NDH-1-deficient one, exhibited impaired growth characteristics under diazotrophic condition, suggesting a role for the Na+ transport in nitrogen fixation by A. vinelandii.

Flavodoxin with an air-stable flavin semiquinone in a green sulfur bacterium.

Flavodoxins are small proteins with a non-covalently bound FMN that can accept two electrons and accordingly adopt three redox states: oxidized (quinone), one-electron reduced (semiquinone), and two-electron reduced (quinol). In iron-deficient cyanobacteria and algae, flavodoxin can substitute for ferredoxin as the electron carrier in the photosynthetic electron transport chain. Here, we demonstrate a similar function for flavodoxin from the green sulfur bacterium Chlorobium phaeovibrioides (cp-Fld). The expression of the cp-Fld gene, found in a close proximity with the genes for other proteins associated with iron transport and storage, increased in a low-iron medium. cp-Fld produced in Escherichia coli exhibited the optical, ERP, and electron-nuclear double resonance spectra that were similar to those of known flavodoxins. However, unlike all other flavodoxins, cp-Fld exhibited unprecedented stability of FMN semiquinone to oxidation by air and difference in midpoint redox potentials for the quinone-semiquinone and semiquinone-quinol couples (- 110 and - 530 mV, respectively). cp-Fld could be reduced by pyruvate:ferredoxin oxidoreductase found in the membrane-free extract of Chl. phaeovibrioides cells and photo-reduced by the photosynthetic reaction center found in membrane vesicles from these cells. The green sulfur bacterium Chl. phaeovibrioides appears thus to be a new type of the photosynthetic organisms that can use flavodoxin as an alternative electron carrier to cope with iron deficiency.

Identification of the key determinant of the transport promiscuity in Na+-translocating rhodopsins.

Bacterial Na+-transporting rhodopsins convert solar energy into transmembrane ion potential difference. Typically, they are strictly specific for Na+, but some can additionally transport H+. To determine the structural basis of cation promiscuity in Na+-rhodopsins, we compared their primary structures and found a single position that harbors a cysteine in strictly specific Na+-rhodopsins and a serine in the promiscuous Krokinobacter eikastus Na+-rhodopsin (Kr2). A Cys253Ser variant of the strictly specific Dokdonia sp. PRO95 Na+-rhodopsin (NaR) was indeed found to transport both Na+ and H+ in a light-dependent manner when expressed in retinal-producing Escherichia coli cells. The dual specificity of the NaR variant was confirmed by analysis of its photocycle, which revealed an acceleration of the cation-capture step by comparison with the wild-type NaR in a Na+-deficient medium. The structural basis for the dependence of the Na+/H+ specificity in Na+-rhodopsin on residue 253 remains to be determined.

Flavin transferase: the maturation factor of flavin-containing oxidoreductases.

Flavins, cofactors of many enzymes, are often covalently linked to these enzymes; for instance, flavin adenine mononucleotide (FMN) can form a covalent bond through either its phosphate or isoalloxazine group. The prevailing view had long been that all types of covalent attachment of flavins occur as autocatalytic reactions; however, in 2013, the first flavin transferase was identified, which catalyzes phosphoester bond formation between FMN and Na+-translocating NADH:quinone oxidoreductase in certain bacteria. Later studies have indicated that this post-translational modification is widespread in prokaryotes and is even found in some eukaryotes. Flavin transferase can occur as a separate ∼40 kDa protein or as a domain within the target protein and recognizes a degenerate DgxtsAT/S motif in various target proteins. The purpose of this review was to summarize the progress already achieved by studies of the structure, mechanism, and specificity of flavin transferase and to encourage future research on this topic. Interestingly, the flavin transferase gene (apbE) is found in many bacteria that have no known target protein, suggesting the presence of yet unknown flavinylation targets.

Engineering a carotenoid-binding site in Dokdonia sp. PRO95 Na+-translocating rhodopsin by a single amino acid substitution.

Light-driven H+, Cl- and Na+ rhodopsin pumps all use a covalently bound retinal molecule to capture light energy. Some H+-pumping rhodopsins (xanthorhodopsins; XRs) additionally contain a carotenoid antenna for light absorption. Comparison of the available primary and tertiary structures of rhodopsins pinpointed a single Thr residue (Thr216) that presumably prevents carotenoid binding to Na+-pumping rhodopsins (NaRs). We replaced this residue in Dokdonia sp. PRO95 NaR with Gly, which is found in the corresponding position in XRs, and produced a variant rhodopsin in a ketocarotenoid-synthesising Escherichia coli strain. Unlike wild-type NaR, the isolated variant protein contained the tightly bound carotenoids canthaxanthin and echinenone. These carotenoids were visible in the absorption, circular dichroism and fluorescence excitation spectra of the Thr216Gly-substituted NaR, which indicates their function as a light-harvesting antenna. The amino acid substitution and the bound carotenoids did not affect the NaR photocycle. Our findings suggest that the antenna function was recently lost during NaR evolution but can be easily restored by site-directed mutagenesis.

Identification of the coupling step in Na(+)-translocating NADH:quinone oxidoreductase from real-time kinetics of electron transfer.

Bacterial Na(+)-translocating NADH:quinone oxidoreductase (Na(+)-NQR) uses a unique set of prosthetic redox groups-two covalently bound FMN residues, a [2Fe-2S] cluster, FAD, riboflavin and a Cys4[Fe] center-to catalyze electron transfer from NADH to ubiquinone in a reaction coupled with Na(+) translocation across the membrane. Here we used an ultra-fast microfluidic stopped-flow instrument to determine rate constants and the difference spectra for the six consecutive reaction steps of Vibrio harveyi Na(+)-NQR reduction by NADH. The instrument, with a dead time of 0.25 ms and optical path length of 1 cm allowed collection of visible spectra in 50-µs intervals. By comparing the spectra of reaction steps with the spectra of known redox transitions of individual enzyme cofactors, we were able to identify the chemical nature of most intermediates and the sequence of electron transfer events. A previously unknown spectral transition was detected and assigned to the Cys4[Fe] center reduction. Electron transfer from the [2Fe-2S] cluster to the Cys4[Fe] center and all subsequent steps were markedly accelerated when Na(+) concentration was increased from 20 µM to 25 mM, suggesting coupling of the former step with tight Na(+) binding to or occlusion by the enzyme. An alternating access mechanism was proposed to explain electron transfer between subunits NqrF and NqrC. According to the proposed mechanism, the Cys4[Fe] center is alternatively exposed to either side of the membrane, allowing the [2Fe-2S] cluster of NqrF and the FMN residue of NqrC to alternatively approach the Cys4[Fe] center from different sides of the membrane.

NqrM (DUF539) Protein Is Required for Maturation of Bacterial Na+-Translocating NADH:Quinone Oxidoreductase.

UNLABELLED: Na(+)-translocating NADH:quinone oxidoreductase (Na(+)-NQR) catalyzes electron transfer from NADH to ubiquinone in the bacterial respiratory chain, coupled with Na(+) translocation across the membrane. Na(+)-NQR maturation involves covalent attachment of flavin mononucleotide (FMN) residues, catalyzed by flavin transferase encoded by the nqr-associated apbE gene. Analysis of complete bacterial genomes has revealed another putative gene (duf539, here renamed nqrM) that usually follows the apbE gene and is present only in Na(+)-NQR-containing bacteria. Expression of the Vibrio harveyi nqr operon alone or with the associated apbE gene in Escherichia coli, which lacks its own Na(+)-NQR, resulted in an enzyme incapable of Na(+)-dependent NADH or reduced nicotinamide hypoxanthine dinucleotide (dNADH) oxidation. However, fully functional Na(+)-NQR was restored when these genes were coexpressed with the V. harveyi nqrM gene. Furthermore, nqrM lesions in Klebsiella pneumoniae and V. harveyi prevented production of functional Na(+)-NQR, which could be recovered by an nqrM-containing plasmid. The Na(+)-NQR complex isolated from the nqrM-deficient strain of V. harveyi lacks several subunits, indicating that nqrM is necessary for Na(+)-NQR assembly. The protein product of the nqrM gene, NqrM, contains a single putative transmembrane α-helix and four conserved Cys residues. Mutating one of these residues (Cys33 in V. harveyi NqrM) to Ser completely prevented Na(+)-NQR maturation, whereas mutating any other Cys residue only decreased the yield of the mature protein. These findings identify NqrM as the second specific maturation factor of Na(+)-NQR in proteobacteria, which is presumably involved in the delivery of Fe to form the (Cys)4[Fe] center between subunits NqrD and NqrE. IMPORTANCE: Na(+)-translocating NADH:quinone oxidoreductase complex (Na(+)-NQR) is a unique primary Na(+) pump believed to enhance the vitality of many bacteria, including important pathogens such as Vibrio cholerae, Vibrio parahaemolyticus, Haemophilus influenzae, Neisseria gonorrhoeae, Pasteurella multocida, Porphyromonas gingivalis, Enterobacter aerogenes, and Yersinia pestis. Production of Na(+)-NQR in bacteria requires Na(+)-NQR-specific maturation factors. We earlier identified one such factor (ApbE) that covalently attaches flavin residues to Na(+)-NQR. Here we identify the other protein factor, designated NqrM, and show that NqrM and ApbE suffice to produce functional Na(+)-NQR from the Vibrio harveyi nqr operon. NqrM may be involved in Fe delivery to a unique Cys4[Fe] center during Na(+)-NQR assembly. Besides highlighting Na(+)-NQR biogenesis, these findings suggest a novel drug target to combat Na(+)-NQR-containing bacteria.

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

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

Alternative pyrimidine biosynthesis protein ApbE is a flavin transferase catalyzing covalent attachment of FMN to a threonine residue in bacterial flavoproteins.

Na(+)-translocating NADH:quinone oxidoreductase (Na(+)-NQR) contains two flavin residues as redox-active prosthetic groups attached by a phosphoester bond to threonine residues in subunits NqrB and NqrC. We demonstrate here that flavinylation of truncated Vibrio harveyi NqrC at Thr-229 in Escherichia coli cells requires the presence of a co-expressed Vibrio apbE gene. The apbE genes cluster with genes for Na(+)-NQR and other FMN-binding flavoproteins in bacterial genomes and encode proteins with previously unknown function. Experiments with isolated NqrC and ApbE proteins confirmed that ApbE is the only protein factor required for NqrC flavinylation and also indicated that the reaction is Mg(2+)-dependent and proceeds with FAD but not FMN. Inactivation of the apbE gene in Klebsiella pneumoniae, wherein the nqr operon and apbE are well separated in the chromosome, resulted in a complete loss of the quinone reductase activity of Na(+)-NQR, consistent with its dependence on covalently bound flavin. Our data thus identify ApbE as a novel modifying enzyme, flavin transferase.

Urocanate reductase: identification of a novel anaerobic respiratory pathway in Shewanella oneidensis MR-1.

Interpretation of the constantly expanding body of genomic information requires that the function of each gene be established. Here we report the genomic analysis and structural modelling of a previously uncharacterized redox-metabolism protein UrdA (SO_4620) of Shewanella oneidensis MR-1, which led to a discovery of the novel enzymatic activity, urocanate reductase. Further cloning and expression of urdA, as well as purification and biochemical study of the gene's product UrdA and redox titration of its prosthetic groups confirmed that the latter is indeed a flavin-containing enzyme catalysing the unidirectional reaction of two-electron reduction of urocanic acid to deamino-histidine, an activity not reported earlier. UrdA exhibits both high substrate affinity and high turnover rate (K(m) << 10 µM, k(cat) = 360 s(-1) ) and strong specificity in favour of urocanic acid. UrdA homologues are present in various bacterial genera, such as Shewanella, Fusobacterium and Clostridium, the latter including the human pathogen Clostridium tetani. The UrdA activity in S. oneidensis is induced by its substrate under anaerobic conditions and it enables anaerobic growth with urocanic acid as a sole terminal electron acceptor. The latter capability can provide the cells of UrdA-containing bacteria with a niche where no other bacteria can compete and survive.

Alginate synthesis in Azotobacter vinelandii is increased by reducing the intracellular production of ubiquinone.

Azotobacter vinelandii, a soil nitrogen fixing bacterium, produces alginate a polysaccharide with industrial and medical relevant applications. In this work, we characterized a miniTn5 mutant, named GG101, that showed a 14-fold increase in the specific production of alginate when grown diazotrophically on solid minimal medium comparing to the parental E strain (also named AEIV). Quantitative real-time reverse transcription PCR analysis indicated that this increased alginate production was due to higher expression levels of several biosynthetic alg genes such as algD. Sequencing of the locus interrupted in GG101 indicated that the miniTn5 was inserted in the positive strand, and 10 bp upstream the start codon of the gene ubiA, encoding the enzyme for the second step in the biosynthesis of ubiquinone (Q8). Both the transcription of ubiA and the content of Q8 are decreased in the mutant GG101 when compared to the wild-type strain E. Genetic complementation of mutant GG101 with a wild-type copy of the ubiCA genes restored the content of Q8 and reduced the production of alginate to levels similar to those of the parental E strain. Furthermore, respirometric analysis showed a reproducible decrease of about 8 % in the respiratory capacity of mutant GG101, at exponential phase of growth in liquid minimal medium. Collectively, our data show that a decreased content in Q8 results in higher levels of alginate in A. vinelandii.

Sodium-dependent movement of covalently bound FMN residue(s) in Na(+)-translocating NADH:quinone oxidoreductase.

Na(+)-translocating NADH:quinone oxidoreductase (Na(+)-NQR) is a component of respiratory electron-transport chain of various bacteria generating redox-driven transmembrane electrochemical Na(+) potential. We found that the change in Na(+) concentration in the reaction medium has no effect on the thermodynamic properties of prosthetic groups of Na(+)-NQR from Vibrio harveyi, as was revealed by the anaerobic equilibrium redox titration of the enzyme's EPR spectra. On the other hand, the change in Na(+) concentration strongly alters the EPR spectral properties of the radical pair formed by the two anionic semiquinones of FMN residues bound to the NqrB and NqrC subunits (FMN(NqrB) and FMN(NqrC)). Using data obtained by pulse X- and Q-band EPR as well as by pulse ENDOR and ELDOR spectroscopy, the interspin distance between FMN(NqrB) and FMN(NqrC) was found to be 15.3 Å in the absence and 20.4 Å in the presence of Na(+), respectively. Thus, the distance between the covalently bound FMN residues can vary by about 5 Å upon changes in Na(+) concentration. Using these results, we propose a scheme of the sodium potential generation by Na(+)-NQR based on the redox- and sodium-dependent conformational changes in the enzyme.

Pre-steady-state kinetics and solvent isotope effects support the "billiard-type" transport mechanism in Na+ -translocating pyrophosphatase.

Membrane-bound pyrophosphatase (mPPase) found in microbes and plants is a membrane H+ pump that transports the H+ ion generated in coupled pyrophosphate hydrolysis out of the cytoplasm. Certain bacterial and archaeal mPPases can in parallel transport Na+ via a hypothetical "billiard-type" mechanism, also involving the hydrolysis-generated proton. Here, we present the functional evidence supporting this coupling mechanism. Rapid-quench and pulse-chase measurements with [32 P]pyrophosphate indicated that the chemical step (pyrophosphate hydrolysis) is rate-limiting in mPPase catalysis and is preceded by a fast isomerization of the enzyme-substrate complex. Na+ , whose binding is a prerequisite for the hydrolysis step, is not required for substrate binding. Replacement of H2 O with D2 O decreased the rates of pyrophosphate hydrolysis by both Na+ - and H+ -transporting bacterial mPPases, the effect being more significant than with a non-transporting soluble pyrophosphatase. We also show that the Na+ -pumping mPPase of Thermotoga maritima resembles other dimeric mPPases in demonstrating negative kinetic cooperativity and the requirement for general acid catalysis. The findings point to a crucial role for the hydrolysis-generated proton both in H+ -pumping and Na+ -pumping by mPPases.

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

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