The Na⁺-translocating NADH:ubiquinone oxidoreductase (Na⁺-NQR) of Vibrio cholerae is a respiratory complex that couples the exergonic oxidation of NADH to the transport of Na⁺ across the cytoplasmic membrane. It is composed of six different subunits, NqrA, NqrB, NqrC, NqrD, NqrE, and NqrF, which harbor FAD, FMN, riboflavin, quinone, and two FeS centers as redox co-factors. We recently determined the X-ray structure of the entire Na⁺-NQR complex at 3.5-Å resolution and complemented the analysis by high-resolution structures of NqrA, NqrC, and NqrF. The position of flavin and FeS co-factors both at the cytoplasmic and the periplasmic side revealed an electron transfer pathway from cytoplasmic subunit NqrF across the membrane to the periplasmic NqrC, and via NqrB back to the quinone reduction site on cytoplasmic NqrA. A so far unknown Fe site located in the midst of membrane-embedded subunits NqrD and NqrE shuttles the electrons over the membrane. Some distances observed between redox centers appear to be too large for effective electron transfer and require conformational changes that are most likely involved in Na⁺ transport. Based on the structure, we propose a mechanism where redox induced conformational changes critically couple electron transfer to Na⁺ translocation from the cytoplasm to the periplasm through a channel in subunit NqrB.

Escherichia coli is a convenient host for the expression of proteins, but the heterologous production of large membrane protein complexes often is hampered by the lack of specific accessory genes required for membrane insertion or cofactor assembly. In this study we introduce the non-pathogenic and fast-growing Vibrio natriegens as a suitable expression host for membrane-bound proteins from Vibrio cholerae. We achieved production of the primary Na+ pump, the NADH:quinone oxidoreductase (NQR), from V. cholerae in an active state, as indicated by increased overall NADH:quinone oxidoreduction activity of membranes from the transformed V. natriegens, and the sensitivity toward Ag+, a specific inhibitor of the NQR. Complete assembly of V. cholerae NQR expressed in V. natriegens was demonstrated by BN PAGE followed by activity staining. The secondary transport system Mrp from V. cholerae, another membrane-bound multisubunit complex, was also produced in V. natriegens in a functional state, as demonstrated by in vivo Li+ transport. V. natriegens is a promising expression host for the production of membrane protein complexes from Gram-negative pathogens.

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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The Na+-translocating NADH:quinone oxidoreductase (NQR) is the entry site for electrons into the respiratory chain of Vibrio cholerae, the causative agent of cholera disease. NQR couples the electron transfer from NADH to ubiquinone to the translocation of sodium ions across the membrane. We investigated the pH dependence of electron transfer and generation of a transmembrane voltage (ΔΨ) by NQR reconstituted in liposomes with Na+ or Li+ as coupling cation. ΔΨ formation was followed with the voltage-sensitive dye oxonol. With Na+, ΔΨ was barely influenced by pH (6.5-8.5), while Q reduction activity exhibited a maximum at pH 7.5-8.0. With Li+, ΔΨ was generally lower, and the pH profile of electron transfer activity did not reveal a pronounced maximum. We conclude that the coupling efficiency of NQR is influenced by the nature of the transported cation, and by the concentration of protons. The 3D structure of NQR reveals a transmembrane channel in subunit NqrB. It is proposed that partial uncoupling of the NQR observed with the smaller Li+, or with Na+ at pH 7.5-8.0, is caused by the backflow of the coupling cation through the channel in NqrB.