The Na+-translocating NADH:quinone oxidoreductase (Na+-NQR) from Vibrio cholerae enhances insertion of FeS in overproduced NqrF subunit

Tao, Minli; Fritz, Günter; Steuber, Julia

doi:10.1016/j.jinorgbio.2008.01.009

Cited by 5 publications

(2 citation statements)

References 29 publications

(4 reference statements)

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“…Experiments with the water-soluble form of the V. harveyi NqrF subunit indicated, at variance with the data of Tao et al (35), that this subunit can be separately expressed in E. coli and that its [2Fe-2S] cluster can form in E. coli in the absence of NqrM. Indeed, E. coli and other bacterial cells contain the so-called ironsulfur cluster (ISC) and sulfur mobilization (SUF) systems, which permit the maturation of various Fe/S proteins (36).…”

Section: Discussionsupporting

confidence: 49%

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

et al. 2016

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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 ␣-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 Cys 4 [Fe] center during Na ؉ -NQR assembly. Besides highlighting Na ؉ -NQR biogenesis, these findings suggest a novel drug target to combat Na ؉ -NQR-containing bacteria.N a ϩ -translocating NADH:quinone oxidoreductase (Na ϩ -NQR) is a redox-driven sodium pump operating in the respiratory chains of various bacteria, including several pathogens (1). This enzyme is a functional analog of H ϩ -translocating NADH: quinone oxidoreductase, known as mitochondrial complex I or bacterial NDH-1...

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Section: Discussionsupporting

confidence: 49%

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

et al. 2016

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“…In Fig. 3D the spectrum of the fourth phase is shown together with reduced-minus-oxidized spectra of the 2Fe-2S center from Na ϩ -NQR of V. cholerae obtained by expression of a fragment of the NqrF subunit (blue line) (19) as well as the spectrum of one-electron reduc- tion of an anionic flavosemiquinone to the fully reduced state (F . 3 FH 2 ) obtained from glucose oxidase (18).…”

Section: Resultsmentioning

confidence: 99%

The Electron Transfer Pathway of the Na+-pumping NADH:Quinone Oxidoreductase from Vibrio cholerae

Juárez

Morgan

Barquera

2009

Journal of Biological Chemistry

117

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The Na ؉ -pumping NADH:quinone oxidoreductase (Na ؉ -NQR) is the only respiratory enzyme that operates as a Na ؉ pump. This redox-driven Na ؉ pump is amenable to experimental approaches not available for H ؉ pumps, providing an excellent system for mechanistic studies of ion translocation. An understanding of the internal electron transfer steps and their Na ؉ dependence is an essential prerequisite for such studies. To this end, we analyzed the reduction kinetics of the wild type Na ؉ -NQR, as well as site-directed mutants of the enzyme, which lack specific cofactors. NADH and ubiquinol were used as reductants in separate experiments, and a full spectrum UVvisible stopped flow kinetic method was employed. The results make it possible to define the complete sequence of redox carriers in the electrons transfer pathway through the enzyme. Electrons flow from NADH to quinone through the FAD in subunit F, the 2Fe-2S center, the FMN in subunit C, the FMN in subunit B, and finally riboflavin. The reduction of the FMN C to its anionic flavosemiquinone state is the first Na ؉ -dependent process, suggesting that reduction of this site is linked to Na The Na ϩ -pumping NADH: ubiquinone oxidoreductase (Na ϩ -NQR) 2 is a molecular energy transducer present in the membrane of many marine and pathogenic bacteria, including Vibrio cholerae (1). The enzyme accepts electrons from NADH and donates them to ubiquinone. This redox reaction releases a significant amount of energy, which is used to pump Na ϩ across the cell membrane, creating a Na ϩ gradient as well as an electrical potential (2, 3). Na ϩ -NQR catalyzes the same redox reaction as the H ϩ -pumping NADH:quinone oxidoreductase (complex I), but there is nothing to indicate a common mechanism. The two proteins have significantly different cofactor compositions and do not share any homology beyond substrate/cofactor motifs. In fact the pumping stoichiometry of the two enzymes is reportedly different; Na ϩ -NQR translocates one Na ϩ /electron, whereas as complex I moves at least 2 H ϩ /e Ϫ (4 -6).In Na ϩ -NQR electrons are carried through the enzyme by means of at least five redox cofactors: a noncovalently bound FAD and a 2Fe-2S center, in the NqrF subunit, where the binding site for NADH is also located, two covalently attached FMNs in subunits B and C (FMN B and FMN C , respectively), and a noncovalently bound riboflavin that has not been definitely localized but that is believed to reside in the B or C subunit (see Fig. 1) (7-12). Describing the way in which these redox cofactors work together to move electrons from NADH to quinone is a necessary step in understanding how Na ϩ -NQR functions as a molecular energy converter. It is important to know the sequence of redox carriers along the path taken by electrons as they flow through the enzyme, as well as the kinetics and energetics of each step and how they are influenced by Na ϩ and the electrical potential.The reaction of the oxidized enzyme with excess NADH, leading to fully reduced state, can be used to investigate t...

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Mechanism and impact of catecholamine conversion by Vibrio cholerae

Toulouse

Schmucker

Metesch

et al. 2019

Biochimica et Biophysica Acta (BBA) - Bioenergetics

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The Na+-translocating NADH:quinone oxidoreductase (Na+-NQR) from Vibrio cholerae enhances insertion of FeS in overproduced NqrF subunit

Cited by 5 publications

References 29 publications

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

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

The Electron Transfer Pathway of the Na+-pumping NADH:Quinone Oxidoreductase from Vibrio cholerae

Mechanism and impact of catecholamine conversion by Vibrio cholerae

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The Na+-translocating NADH:quinone oxidoreductase (Na+-NQR) from Vibrio cholerae enhances insertion of FeS in overproduced NqrF subunit

Cited by 5 publications

References 29 publications

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

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

The Electron Transfer Pathway of the Na+-pumping NADH:Quinone Oxidoreductase from Vibrio cholerae

Mechanism and impact of catecholamine conversion by Vibrio cholerae

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

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