Localization of Ubiquinone-8 in the Na+-pumping NADH:Quinone Oxidoreductase from Vibrio cholerae

Casutt, Marco S.; Nedielkov, Ruslan; Wendelspiess, Severin; Vossler, Sara; Gerken, Uwe; Murai, Masatoshi; Miyoshi, Hideto; Möller, Heiko M.; Steuber, Julia

doi:10.1074/jbc.m111.224980

Cited by 42 publications

(42 citation statements)

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“…Additionally, the crystallographic model lacks an anticipated tightly bound quinone, which has been reported in the enzyme preparations from different laboratories (4,6,8) and suggested to be located in NqrA on the basis of photoaffinity labeling and NMR studies (7,9). In the crystallographic model, the NqrA subunit includes a deep cavity that is large enough to accommodate a ubiquinone molecule, but the cavity is ϳ20 Å above the predicted membrane surface and the distance between the cavity and the riboflavin in NqrB subunit is too large (Ͼ40 Å) to be consistent with electron transfer during turnover (Fig.…”

Section: Edited By Ruma Banerjeementioning

confidence: 99%

“…Na ϩ -NQR that has been purified using the non-ionic detergent dodecyl maltoside (DDM) is reported to contain tightly bound ubiquinone-8 (Q 8 ) in substoichiometric quantities that can be removed by washing the enzyme preparation with a buffer containing the zwitterionic detergent lauryldimethylamine N-oxide (LDAO) (7,10). We used LDAO-washed Na ϩ -NQR throughout this study.…”

Section: ؉ -Nqr By Puq-3mentioning

confidence: 99%

“…For instance, the spatial distances between several pairs of redox cofactors in the proposed electron transfer pathway are too large to support physiological rates of electron transfer; for example, the edge-toedge distance between the [2Fe-2S] cluster in NqrF and the Fe(Cys) 4 in NqrD (33.4 Å) and between the FMN and the riboflavin cofactor in NqrB (29.3 Å). The fact that electron transfer between these cofactors takes place indicates that the subunits harboring the cofactors undergo large conformational changes during turnover that decrease these spatial gaps (13).Additionally, the crystallographic model lacks an anticipated tightly bound quinone, which has been reported in the enzyme preparations from different laboratories (4,6,8) and suggested to be located in NqrA on the basis of photoaffinity labeling and NMR studies (7,9). In the crystallographic model, the NqrA subunit includes a deep cavity that is large enough to accommodate a ubiquinone molecule, but the cavity is ϳ20 Å above the predicted membrane surface and the distance between the cavity and the riboflavin in NqrB subunit is too large (Ͼ40 Å) to be consistent with electron transfer during turnover (Fig.…”

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

Ito

Murai

Ninokura

et al. 2017

Journal of Biological Chemistry

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Edited by Ruma BanerjeeThe 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. I]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.The Na ϩ -pumping NADH-quinone oxidoreductase (Na ϩ -NQR) 2 is the first enzyme of the respiratory chain and the main ion transporter in many marine and pathogenic bacteria, such as Vibrio cholerae and Haemophilus influenzae (1, 2). Na ϩ -NQR obtains energy by oxidizing NADH and reducing ubiquinone, which allows it to generate an electrochemical Na ϩ gradient across the inner bacterial membrane. The enzyme is an integral membrane complex consisting of six subunits (NqrA-F) encoded by the nqr operon (2). There is a consensus that the electron transfer takes place through a series of five redox cofactors as follows: a FAD; a 2Fe-2S center; two covalently bound FMN, and a riboflavin (3-5). Different studies have intensively investigated the locations and redox properties of the cofactors of the enzyme (6 -12); however, the exact locations of the ubiquinone-binding site(s) and the Na ϩ transport pathway, as well as the mechanism that couples Na ϩ transport to the electron transfer, still remain elusive.A recently published X-ray crystallographic study provided important information about the structure of V. cholerae Na ϩ -NQR (13), but the model includes several features that are difficult to reconcile with previous biochemical and/or biophysical functional characterizations (11). For instance, the spatial distances between several pairs of redox cofactors in the proposed electron transfer pathway are too large to support physiological rates of electron transfer; for example, the edge-toedge distance between the [2Fe-2S] cluster in NqrF and the Fe(Cys) 4 in NqrD (33.4 Å) and between the FMN and the riboflavin cofactor in NqrB (29.3 Å). The fact that electron transfer between these cofactors takes place indicates that the subunits harboring the cofactors undergo large conformational changes during turnover that decrease these spatial gaps (13).Additionally, the crystallographic model lacks an anticipated tightly bound quinone, which has been reported in the enzyme preparations from different laboratories (4,6,8) and suggested to be located in NqrA on the basis of photoaffinity labeling and NMR studies (7,9). In the crystallographic model, the NqrA subunit includes a deep cavity that is large enough to accommodate a ubiquinone molecule, but the cavity is ϳ20 Å above the predicted membrane surface and the distance between the cavity and the riboflavin in NqrB subunit is too large (Ͼ40 Å) to be consistent with electron transfer during tu...

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Section: Edited By Ruma Banerjeementioning

confidence: 99%

Section: ؉ -Nqr By Puq-3mentioning

confidence: 99%

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

Ito

Murai

Ninokura

et al. 2017

Journal of Biological Chemistry

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“…It is composed of six subunits (NQRA-F) pumping out Na+/H+ while electrons travel from NADH to quinone via a minimum of five redox carriers [68,69]: a FAD and a 2Fe-2S cluster in the peripheral NQRF subunit, an FMN in the hydrophilic NQRC subunit, and a FMN and riboflavin in the membrane integral NQRB subunit. NQRF is the site for NADH oxidation and NQRA has been identified as the quinol formation site.…”

Section: Respiratory Complexesmentioning

confidence: 99%

Genome-Guided Analysis of the Syntrophic Acetate Oxidizer <em>C. ultunense</em> and Comparative Genomics Reveal Different Strategies for Acetate Oxidation and Energy Conservation in Syntrophic Acetate-Oxidising Bacteria

Manzoor¹,

Schnürer²,

Bongcam‐Rudloff³

et al. 2018

Preprint

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ABSTRACTschinkii, were identified in C. ultunense and P. lettingae. Moreover, two respiratory complexes sharing similarities to the proton-translocating ferredoxin:NAD + oxidoreductase (Rnf) and the Na+ pumping NADH:quinone hydrogenase (NQR) were predicted. These might form a respiratory chain involved in reduction of electron acceptors other than protons.However, involvement of these complexes in acetate oxidation in C. ultunense and P.lettingae needs further study. This genome-based comparison provides a solid platform for future meta-proteomics and meta-transcriptomics studies and for metabolic engineering, control and monitoring of SAOB.

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“…The localization of the ubiquinone binding site in Na ϩ -NQR is controversial. Strickland and colleagues described a highaffinity quinone (Q) binding site on the NqrB subunit (12), whereas results from our group indicated binding of Q to the NqrA subunit (13). The recent three-dimensional (3D) structure of Na ϩ -NQR revealed that FMN C is the only cofactor in Na ϩ -NQR that is located in the periplasm and that all other cofactors are located in the cytoplasm or the transmembrane regions of the protein (3).…”

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The Na ⁺ -Translocating NADH:Quinone Oxidoreductase Enhances Oxidative Stress in the Cytoplasm of Vibrio cholerae

et al. 2016

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We searched for a source of reactive oxygen species (ROS) in the cytoplasm of the human pathogen Vibrio cholerae and addressed the mechanism of ROS formation using the dye 2=,7=-dichlorofluorescein diacetate (DCFH-DA) in respiring cells. By comparing V. cholerae strains with or without active Na ؉ -translocating NADH:quinone oxidoreductase (Na ؉ -NQR), this respiratory sodium ion redox pump was identified as a producer of ROS in vivo. The amount of cytoplasmic ROS detected in V. cholerae cells producing variants of Na ؉ -NQR correlated well with rates of superoxide formation by the corresponding membrane fractions. Membranes from wild-type V. cholerae showed increased superoxide production activity (9.8 ؎ 0.6 mol superoxide min ؊1 mg ؊1 membrane protein) compared to membranes from the mutant lacking Na ؉ -NQR (0.18 ؎ 0.01 mol min ؊1 mg ؊1 ). Overexpression of plasmid-encoded Na ؉ -NQR in the nqr deletion strain resulted in a drastic increase in the formation of superoxide (42.6 ؎ 2.8 mol min ؊1 mg ؊1 ). By analyzing a variant of Na ؉ -NQR devoid of quinone reduction activity, we identified the reduced flavin adenine dinucleotide (FAD) cofactor of cytoplasmic NqrF subunit as the site for intracellular superoxide formation in V. cholerae. The impact of superoxide formation by the Na ؉ -NQR on the virulence of V. cholerae is discussed. IMPORTANCEIn several studies, it was demonstrated that the Na ؉ -NQR in V. cholerae affects virulence in a yet unknown manner. We identified the reduced FAD cofactor in the NADH-oxidizing NqrF subunit of the Na ؉ -NQR as the site of superoxide formation in the cytoplasm of V. cholerae. Our study provides the framework to understand how reactive oxygen species formed during respiration could participate in the regulated expression of virulence factors during the transition from aerobic to microaerophilic (intestinal) habitats. This hypothesis may turn out to be right for many other pathogens which, like V. cholerae, depend on the Na ؉ -NQR as the sole electrogenic NADH dehydrogenase. Vibrio cholerae is a Gram-negative bacterium and the causative agent of cholera, a devastating diarrheal disease. It is found in estuaries, inhabiting seawater or brackish water where it is commonly associated with biofilms on phyto-or zooplankton (1). The high salinity in these habitats is counteracted by an electrochemical sodium motive force (SMF) generated by the respiratory chain of V. cholerae (2, 3). This SMF drives cellular processes like substrate transport (4) and motility (5). It is generated by the Na ϩ -translocating NADH:quinone oxidoreductase (Na ϩ -NQR), which couples the energy that is released by oxidation of NADH with ubiquinone to the transport of sodium ions out of the cytoplasm with a 1e Ϫ /1Na ϩ stoichiometry (6). The Na ϩ -NQR is a membrane-bound protein complex consisting of six subunits, NqrABCDEF, and contains a variety of different cofactors (3). Electron transfer from NADH to ubiquinone is mediated by a noncovalently bound flavin adenine dinucleotide (FAD) and a [2Fe-2...

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Localization of Ubiquinone-8 in the Na+-pumping NADH:Quinone Oxidoreductase from Vibrio cholerae

Cited by 42 publications

References 27 publications

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

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

Genome-Guided Analysis of the Syntrophic Acetate Oxidizer <em>C. ultunense</em> and Comparative Genomics Reveal Different Strategies for Acetate Oxidation and Energy Conservation in Syntrophic Acetate-Oxidising Bacteria

The Na ⁺ -Translocating NADH:Quinone Oxidoreductase Enhances Oxidative Stress in the Cytoplasm of Vibrio cholerae

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Localization of Ubiquinone-8 in the Na+-pumping NADH:Quinone Oxidoreductase from Vibrio cholerae

Cited by 42 publications

References 27 publications

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

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

Genome-Guided Analysis of the Syntrophic Acetate Oxidizer <em>C. ultunense</em> and Comparative Genomics Reveal Different Strategies for Acetate Oxidation and Energy Conservation in Syntrophic Acetate-Oxidising Bacteria

The Na + -Translocating NADH:Quinone Oxidoreductase Enhances Oxidative Stress in the Cytoplasm of Vibrio cholerae

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The Na ⁺ -Translocating NADH:Quinone Oxidoreductase Enhances Oxidative Stress in the Cytoplasm of Vibrio cholerae