Publications by authors named "Yulia V Bertsova"

26 Publications

  • Page 1 of 1

A new water-soluble bacterial NADH: fumarate oxidoreductase.

FEMS Microbiol Lett 2020 11;367(20)

Belozersky Institute of Physico-Chemical Biology, Lomonosov Moscow State University, Vorobievy Gory 1/40, Moscow 119234, Russia.

The cytoplasmic fumarate reductase of Klebsiella pneumoniae (FRD) is a monomeric protein which contains three prosthetic groups: noncovalently bound FMN and FAD plus a covalently bound FMN. In the present work, NADH is revealed to be an inherent electron donor for this enzyme. We found that the fumarate reductase activity of FRD significantly exceeds its NADH dehydrogenase activity. During the catalysis of NADH:fumarate oxidoreductase reaction, FRD turnover is limited by a very low rate (∼10/s) of electron transfer between the noncovalently and covalently bound FMN moieties. Induction of FRD synthesis in K. pneumoniae cells was observed only under anaerobic conditions in the presence of fumarate or malate. Enzymes with the FRD-like domain architecture are widely distributed among various bacteria and apparently comprise a new type of water-soluble NADH:fumarate oxidoreductases.
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http://dx.doi.org/10.1093/femsle/fnaa175DOI Listing
November 2020

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

Arch Biochem Biophys 2020 03 15;681:108266. Epub 2020 Jan 15.

Belozersky Institute of Physico-Chemical Biology, Lomonosov Moscow State University, Moscow, 119234, Russia. Electronic address:

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.
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http://dx.doi.org/10.1016/j.abb.2020.108266DOI Listing
March 2020

Mutational analysis of the flavinylation and binding motifs in two protein targets of the flavin transferase ApbE.

FEMS Microbiol Lett 2019 11;366(22)

Belozersky Institute of Physico-Chemical Biology, Lomonosov Moscow State University, Vorobievy Gory 1/40, Moscow 119234, Russia.

Many flavoproteins belonging to three domain types contain an FMN residue linked through a phosphoester bond to a threonine or serine residue found in a conserved seven-residue motif. The flavinylation reaction is catalyzed by a specific enzyme, ApbE, which uses FAD as a substrate. To determine the structural requirements of the flavinylation reaction, we examined the effects of single substitutions in the flavinylation motif of Klebsiella pneumoniae cytoplasmic fumarate reductase on its modification by its own ApbE in recombinant Escherichia coli cells. The replacement of the flavin acceptor threonine with alanine completely abolished the modification reaction, whereas the replacements of conserved aspartate and serine had only minor effects. Effects of other substitutions, including replacing the acceptor threonine with serine, (a 10-55% decrease in the flavinylation degree) pinpointed important glycine and alanine residues and suggested an excessive capacity of the ApbE-based flavinylation system in vivo. Consistent with this deduction, drastic replacements of conserved leucine and threonine residues in the binding pocket that accommodates FMN residue still allowed appreciable flavinylation of the NqrC subunit of Vibrio harveyi Na+-translocating NADH:quinone oxidoreductase, despite a profound weakening of the isoalloxazine ring binding and an increase in its exposure to solvent.
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http://dx.doi.org/10.1093/femsle/fnz252DOI Listing
November 2019

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

Photosynth Res 2019 Nov 13;142(2):127-136. Epub 2019 Jul 13.

Belozersky Institute of Physico-Chemical Biology, Lomonosov Moscow State University, Moscow, Russia, 119234.

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.
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http://dx.doi.org/10.1007/s11120-019-00658-1DOI Listing
November 2019

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

Biochem Soc Trans 2018 10 28;46(5):1161-1169. Epub 2018 Aug 28.

Belozersky Institute of Physico-Chemical Biology, Lomonosov Moscow State University, Moscow 119234, Russia.

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 DgxtsA/ 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 (E) is found in many bacteria that have no known target protein, suggesting the presence of yet unknown flavinylation targets.
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http://dx.doi.org/10.1042/BST20180524DOI Listing
October 2018

EPR evidence for a fast-relaxing iron center in Na-translocating NADH:quinone-oxidoreductase.

J Inorg Biochem 2018 07 6;184:15-18. Epub 2018 Apr 6.

Belozersky Institute of Physico-Chemical Biology, Lomonosov Moscow State University, Moscow 119234, Russia. Electronic address:

A paramagnetic Cys[Fe] center was detected by pulse EPR in Na-translocating NADH:quinone-oxidoreductase (Na-NQR) by influence of this center on transverse and longitudinal spin relaxation of Na-NQR flavin radicals. The oxidation state of the Cys[Fe] center was Fe in the oxidized and Fe in the reduced Na-NQR, as deduced from the temperature dependence of spin relaxation rates of different flavin radicals. A high-spin state of iron in the Cys[Fe] center was assigned to both forms of Na-NQR.
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http://dx.doi.org/10.1016/j.jinorgbio.2018.04.004DOI Listing
July 2018

Catalytically important flavin linked through a phosphoester bond in a eukaryotic fumarate reductase.

Biochimie 2018 Jun 3;149:34-40. Epub 2018 Apr 3.

Belozersky Institute of Physico-Chemical Biology, Lomonosov Moscow State University, Moscow 119234, Russia. Electronic address:

One of the three domains of kinetoplastid NADH:fumarate oxidoreductase (FRD) is homologous to bacterial flavin transferase that catalyzes transfer of FMN residue from FAD to threonine in flavoproteins. Leptomonas pyrrhocoris FRD produced in yeast cells, which lack flavin transferase gene in their proteome, reduces fumarate in the presence of NADH and contains an FMN residue covalently linked to a Ser9 residue. The conserved flavinylation motif of FRD, D(g/s)x(s/t)(s/g)AS, is similar to the Dxx(s/t)gAT motif recognized by flavin transferase in prokaryotic proteins. Ser9 replacement abolished the flavinylation and fumarate reductase activity of FRD. These findings suggest that the flavinylation is important for the activity of FRD and that this post-translational modification is carried out by the own flavin transferase domain.
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http://dx.doi.org/10.1016/j.biochi.2018.03.013DOI Listing
June 2018

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

Biochem Biophys Res Commun 2018 05 30;499(3):600-604. Epub 2018 Mar 30.

Belozersky Institute of Physico-Chemical Biology, Lomonosov Moscow State University, Moscow 119234, Russia. Electronic address:

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.
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http://dx.doi.org/10.1016/j.bbrc.2018.03.196DOI Listing
May 2018

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

Photosynth Res 2018 May 5;136(2):161-169. Epub 2017 Oct 5.

Belozersky Institute of Physico-Chemical Biology, Lomonosov Moscow State University, Moscow, Russia, 119234.

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.
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http://dx.doi.org/10.1007/s11120-017-0453-0DOI Listing
May 2018

A single mutation converts bacterial Na(+) -transporting rhodopsin into an H(+) transporter.

FEBS Lett 2016 09 5;590(17):2827-35. Epub 2016 Aug 5.

Belozersky Institute of Physico-Chemical Biology, Lomonosov Moscow State University, Russia.

Na(+) -rhodopsins are light-driven pumps used by marine bacteria to extrude Na(+) ions from the cytoplasm. We show here that replacement of Gln123 on the cytoplasmic side of the ion-conductance channel with aspartate or glutamate confers H(+) transport activity to the Na(+) -rhodopsin from Dokdonia sp. PRO95. The Q123E variant could transport H(+) out of Escherichia coli cells in a medium containing 100 mm Na(+) and SCN(-) as the penetrating anion. The rates of the photocycle steps of this variant were only marginally dependent on Na(+) , and the major electrogenic steps were the decays of the K and O intermediates.
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http://dx.doi.org/10.1002/1873-3468.12324DOI Listing
September 2016

Real-time kinetics of electrogenic Na(+) transport by rhodopsin from the marine flavobacterium Dokdonia sp. PRO95.

Sci Rep 2016 Feb 11;6:21397. Epub 2016 Feb 11.

Belozersky Institute of Physico-Chemical Biology, Lomonosov Moscow State University, Moscow 119234, Russia.

Discovery of the light-driven sodium-motive pump Na(+)-rhodopsin (NaR) has initiated studies of the molecular mechanism of this novel membrane-linked energy transducer. In this paper, we investigated the photocycle of NaR from the marine flavobacterium Dokdonia sp. PRO95 and identified electrogenic and Na(+)-dependent steps of this cycle. We found that the NaR photocycle is composed of at least four steps: NaR519 + hv → K585 → (L450↔M495) → O585 → NaR519. The third step is the only step that depends on the Na(+) concentration inside right-side-out NaR-containing proteoliposomes, indicating that this step is coupled with Na(+) binding to NaR. For steps 2, 3, and 4, the values of the rate constants are 4×10(4) s(-1), 4.7 × 10(3) M(-1) s(-1), and 150 s(-1), respectively. These steps contributed 15, 15, and 70% of the total membrane electric potential (Δψ ~ 200 mV) generated by a single turnover of NaR incorporated into liposomes and attached to phospholipid-impregnated collodion film. On the basis of these observations, a mechanism of light-driven Na(+) pumping by NaR is suggested.
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http://dx.doi.org/10.1038/srep21397DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4749991PMC
February 2016

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

Biochim Biophys Acta 2016 Feb 4;1857(2):141-149. Epub 2015 Dec 4.

Belozersky Institute of Physico-Chemical Biology, Lomonosov Moscow State University, Moscow 119992, Russia. Electronic address:

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.
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http://dx.doi.org/10.1016/j.bbabio.2015.12.001DOI Listing
February 2016

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

J Bacteriol 2015 Dec 7;198(4):655-63. Epub 2015 Dec 7.

Belozersky Institute of Physico-Chemical Biology, Lomonosov Moscow State University, Moscow, Russia

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.
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http://dx.doi.org/10.1128/JB.00757-15DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4751810PMC
December 2015

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

Biochim Biophys Acta 2014 Jul 20;1837(7):1122-9. Epub 2013 Dec 20.

Belozersky Institute of Physico-Chemical Biology, Lomonosov Moscow State University, Moscow 119992, Russia. Electronic address:

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.
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http://dx.doi.org/10.1016/j.bbabio.2013.12.006DOI Listing
July 2014

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

J Biol Chem 2013 May 4;288(20):14276-14286. Epub 2013 Apr 4.

Belozersky Institute of Physico-Chemical Biology, Lomonosov Moscow State University, Moscow 119992, Russia. Electronic address:

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.
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http://dx.doi.org/10.1074/jbc.M113.455402DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3656284PMC
May 2013

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

Mol Microbiol 2012 Dec 1;86(6):1452-63. Epub 2012 Nov 1.

Department of Molecular Energetics of Microorganisms, A.N. Belozersky Institute of Physico-Chemical Biology, Moscow State University, Moscow 119992, Russia.

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.
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http://dx.doi.org/10.1111/mmi.12067DOI Listing
December 2012

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

Biochemistry 2012 Jul 25;51(27):5414-21. Epub 2012 Jun 25.

Department of Molecular Energetics of Microorganisms, A.N. Belozersky Institute of Physico-Chemical Biology, Moscow State University, Russia.

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.
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http://dx.doi.org/10.1021/bi300322nDOI Listing
July 2012

Redox properties of the prosthetic groups of Na(+)-translocating NADH:quinone oxidoreductase. 2. Study of the enzyme by optical spectroscopy.

Biochemistry 2009 Jul;48(27):6299-304

Department of Molecular Energetics of Microorganisms, A. N. Belozersky Institute of Physico-Chemical Biology, Moscow State University, Moscow 119992, Russia.

Redox titration of the electronic spectra of the prosthetic groups of the Na(+)-translocating NADH:quinone oxidoreductase (Na(+)-NQR) from Vibrio harveyi at different pH values showed five redox transitions corresponding to the four flavin cofactors of the enzyme and one additional transition reflecting oxidoreduction of the [2Fe-2S] cluster. The pH dependence of the measured midpoint redox potentials showed that the two-electron reduction of the FAD located in the NqrF subunit was coupled with the uptake of only one H(+). The one-electron reduction of neutral semiquinone of riboflavin and the formation of anion flavosemiquinone from the oxidized FMN bound to the NqrB subunit were not coupled to any proton uptake. The two sequential one-electron reductions of the FMN residue bound to the NqrC subunit showed pH-independent formation of anion radical in the first step and the formation of fully reduced flavin coupled to the uptake of one H(+) in the second step. All four flavins stayed in the anionic form in the fully reduced enzyme. None of the six redox transitions in Na(+)-NQR showed dependence of its midpoint redox potential on the concentration of sodium ions. A model of the sequence of electron transfer steps in the enzyme is suggested.
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http://dx.doi.org/10.1021/bi900525vDOI Listing
July 2009

Redox properties of the prosthetic groups of Na(+)-translocating nadh:quinone oxidoreductase. 1. Electron paramagnetic resonance study of the enzyme.

Biochemistry 2009 Jul;48(27):6291-8

Department of Molecular Energetics of Microorganisms, A. N. Belozersky Institute of Physico-Chemical Biology, Moscow State University, Moscow 119992, Russia.

Redox properties of all EPR-detectable prosthetic groups of Na(+)-translocating NADH:quinone oxidoreductase (Na(+)-NQR) from Vibrio harveyi were studied at pH 7.5 using cryo-EPR spectroelectrochemistry. Titration shows five redox transitions. One with E(m) = -275 mV belongs to the reduction of the [2Fe-2S] cluster, and the four others reflect redox transitions of flavin cofactors. Two transitions (E(m)(1) = -190 mV and E(m)(2) = -275 mV) originate from the formation of FMN anion radical, covalently bound to the NqrC subunit, and its subsequent reduction. The remaining two transitions arise from the two other flavin cofactors. A high potential (E(m) = -10 mV) transition corresponds to the reduction of riboflavin neutral radical, which is stable at rather high redox potentials. An E(m) = -130 mV transition reflects the formation of FMN anion radical from a flavin covalently bound to the NqrB subunit, which stays as a radical down to very low potentials. Taking into account the EPR-silent, two-electron transition of noncovalently bound FAD located in the NqrF subunit, there are four flavins in Na(+)-NQR all together. Defined by dipole-dipole magnetic interaction measurements, the interspin distance between the [2Fe-2S](+) cluster and the NqrB subunit-bound FMN anion radical is found to be 22.5 +/- 1.5 A, which means that for the functional electron transfer between these two centers another cofactor, most likely FMN bound to the NqrC subunit, should be located.
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http://dx.doi.org/10.1021/bi900524mDOI Listing
July 2009

Primary steps of the Na+-translocating NADH:ubiquinone oxidoreductase catalytic cycle resolved by the ultrafast freeze-quench approach.

J Biol Chem 2009 Feb 30;284(9):5533-8. Epub 2008 Dec 30.

Department of Molecular Energetics of Microorganisms, Belozersky Institute of Physico-Chemical Biology, Lomonosov Moscow State University, 119992 Moscow, Russia.

The Na(+)-translocating NADH:ubiquinone oxidoreductase (Na(+)-NQR) is a component of respiratory chain of various bacteria, and it generates a redox-driven transmembrane electrochemical Na(+) potential. Primary steps of the catalytic cycle of Na(+)-NQR from Vibrio harveyi were followed by the ultrafast freeze-quench approach in combination with conventional stopped-flow technique. The obtained sequence of events includes NADH binding ( approximately 1.5 x 10(7) m(-1) s(-1)), hydride ion transfer from NADH to FAD ( approximately 3.5 x 10(3) s(-1)), and partial electron separation and formation of equivalent fractions of reduced 2Fe-2S cluster and neutral semiquinone of FAD ( approximately 0.97 x 10(3) s(-1)). In the last step, a quasi-equilibrium is approached between the two states of FAD: two-electron reduced (50%) and one-electron reduced (the other 50%) species. The latter, neutral semiquinone of FAD, shares the second electron with the 2Fe-2S center. The transient midpoint redox potentials for the cofactors obtained during the fast kinetics measurements are very different from ones achieved during equilibrium redox titration and show that the functional states of the enzyme realized during its turning over cannot be modeled by the equilibrium approach.
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http://dx.doi.org/10.1074/jbc.M808984200DOI Listing
February 2009

Catalytic properties of Na+-translocating NADH:quinone oxidoreductases from Vibrio harveyi, Klebsiella pneumoniae, and Azotobacter vinelandii.

FEMS Microbiol Lett 2008 Feb;279(1):116-23

Department of Molecular Energetics of Microorganisms, A.N. Belozersky Institute of Physico-Chemical Biology, Moscow State University, Moscow, Russia.

The catalytic properties of sodium-translocating NADH:quinone oxidoreductases (Na+-NQRs) from the marine bacterium Vibrio harveyi, the enterobacterium Klebsiella pneumoniae, and the soil microorganism Azotobacter vinelandii have been comparatively analyzed. It is shown that these enzymes drastically differ in their affinity to sodium ions. The enzymes also possess different sensitivity to inhibitors. Na+-NQR from A. vinelandii is not sensitive to low 2-n-heptyl-4-hydroxyquinoline N-oxide (HQNO) concentrations, while Na+-NQR from K. pneumoniae is fully resistant to either Ag+ or N-ethylmaleimide. All the Na+-NQR-type enzymes are sensitive to diphenyliodonium, which is shown to modify the noncovalently bound FAD of the enzyme.
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http://dx.doi.org/10.1111/j.1574-6968.2007.01015.xDOI Listing
February 2008

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

Biochemistry 2007 Sep 15;46(35):10186-91. Epub 2007 Aug 15.

Department of Molecular Energetics of Microorganisms, A.N. Belozersky Institute of Physico-Chemical Biology, Moscow State University, Moscow 119899, Russia.

Relaxation characteristics of the 23Na nuclei magnetization were used to determine the sodium-binding properties of the Na+-translocating NADH:quinone oxidoreductase from Vibrio harveyi (NQR). The dissociation constant of Na+ for the oxidized enzyme was found to be 24 mM and for the reduced enzyme about 30 microM. Such large (3 orders in magnitude) redox dependence of the NQR affinity to sodium ions shows that the molecular machinery was designed to use the drop in redox energy for creating an electrochemical sodium gradient. Redox titration of NQR monitored by changes in line width of the 23Na NMR signal at 2 mM Na+ showed that the enzyme affinity to sodium ions follows the Nernst law for a one-electron carrier with Em about -300 mV (vs SHE). The data indicate that energy conservation by NQR involves a mechanism modulating ion affinity by the redox state of an enzyme redox cofactor.
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http://dx.doi.org/10.1021/bi700440wDOI Listing
September 2007

Regulation of expression of Na+ -translocating NADH:quinone oxidoreductase genes in Vibrio harveyi and Klebsiella pneumoniae.

Arch Microbiol 2007 Oct 6;188(4):341-8. Epub 2007 Jun 6.

Department of Molecular Energetics of Microorganisms, A.N. Belozersky Institute of Physico-Chemical Biology, Moscow State University, Leninskie Gory, Moscow 119992, Russia.

The expression of genes encoding sodium-translocating NADH:quinone oxidoreductase (Na(+)-NQR) was studied in the marine bacterium Vibrio harveyi and in the enterobacterium Klebsiella pneumoniae. It has been shown that such parameters as NaCl concentration, pH value, and presence of an uncoupler in the growth media do not influence significantly the level of nqr expression. However, nqr expression depends on the growth substrates used by these bacteria. Na(+)-NQR is highly repressed in V. harveyi during anaerobic growth, and nqr expression is modulated by electron acceptors and values of their redox potentials. The latter effect was shown to be independent of the ArcAB regulatory system.
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http://dx.doi.org/10.1007/s00203-007-0254-5DOI Listing
October 2007

Thermodynamic properties of the redox centers of Na(+)-translocating NADH:quinone oxidoreductase.

Biochemistry 2006 Mar;45(10):3421-8

Department of Molecular Energetics of Microorganisms, A. N. Belozersky Institute of Physico-Chemical Biology, Moscow State University, Moscow 119899, Russia.

Redox titration of all optically detectable prosthetic groups of Na(+)-translocating NADH:quinone oxidoreductase (Na(+)-NQR) at pH 7.5 showed that the functionally active enzyme possesses only three titratable flavin cofactors, one noncovalently bound FAD and two covalently bound FMN residues. All three flavins undergo different redox transitions during the function of the enzyme. The noncovalently bound FAD works as a "classical" two-electron carrier with a midpoint potential (E(m)) of -200 mV. Each of the FMN residues is capable of only one-electron reduction: one from neutral flavosemiquinone to fully reduced flavin (E(m) = 20 mV) and the other from oxidized flavin to flavosemiquinone anion (E(m) = -150 mV). The lacking second half of the redox transitions for the FMNs cannot be reached under our experimental conditions and is most likely not employed in the catalytic cycle. Besides the flavins, a [2Fe-2S] cluster was shown to function in the enzyme as a one-electron carrier with an E(m) of -270 mV. The midpoint potentials of all the redox transitions determined in the enzyme were found to be independent of Na(+) concentration. Even the components that exhibit very strong retardation in the rate of their reduction by NADH at low sodium concentrations experienced no change in the E(m) values when the concentration of the coupling ion was changed 1000 times. On the basis of these data, plausible mechanisms for the translocation of transmembrane sodium ions by Na(+)-NQR are discussed.
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http://dx.doi.org/10.1021/bi052422xDOI Listing
March 2006

The origin of the sodium-dependent NADH oxidation by the respiratory chain of Klebsiella pneumoniae.

FEBS Lett 2004 Apr;563(1-3):207-12

Department of Molecular Energetics of Microorganisms, A.N. Belozersky Institute of Physico-Chemical Biology, Moscow State University, Moscow 119899, Russia.

Properties of Klebsiella pneumoniae respiratory chain enzymes catalyzing NADH oxidation have been studied. Using constructed K. pneumoniae mutant strains, it was shown that three enzymes belonging to different families of NADH:quinone oxidoreductases operate in this bacterium. The NDH-2-type enzyme is not coupled with energy conservation, the NDH-1-type enzyme is a primary proton pump, and the NQR-type enzyme is homologous to the sodium-motive NADH dehydrogenase of Vibrio and is shown to be a primary Na(+) pump. It is concluded that the NQR-type enzyme, not the NDH-1-type enzyme, catalyzes sodium-dependent NADH oxidation in K. pneumoniae.
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http://dx.doi.org/10.1016/S0014-5793(04)00312-6DOI Listing
April 2004

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

Biochim Biophys Acta 2002 Dec;1556(2-3):113-20

Department of Bioenergetics, A.N. Belozersky Institute of Physico-Chemical Biology, Moscow State University, 119899, Moscow, Russia.

Two radical signals with different line widths are seen in the Na+-translocating NADH:ubiquinone oxidoreductase (Na+-NQR) from Vibrio harveyi by EPR spectroscopy. The first radical is observed in the oxidized enzyme, and is assigned as a neutral flavosemiquinone. The second radical is observed in the reduced enzyme and is assigned to be the anionic form of flavosemiquinone. The time course of Na+-NQR reduction by NADH, as monitored by stopped-flow optical spectroscopy, shows three distinct phases, the spectra of which suggest that they correspond to the reduction of three different flavin species. The first phase is fast both in the presence and absence of sodium, and is assigned to reduction of FAD to FADH2 at the NADH dehydrogenating site. The rates of the other two phases are strongly dependent on sodium concentration, and these phases are attributed to reduction of two covalently bound FMN's. Combination of the optical and EPR data suggests that a neutral FMN flavosemiquinone preexists in the oxidized enzyme, and that it is reduced to the fully reduced flavin by NADH. The other FMN moiety is initially oxidized, and is reduced to the anionic flavosemiquinone. One-electron transitions of two discrete flavin species are thus assigned as sodium-dependent steps in the catalytic cycle of Na+-NQR.
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http://dx.doi.org/10.1016/s0005-2728(02)00342-0DOI Listing
December 2002
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