Conformational coupling of redox-driven Na-translocation in Vibrio cholerae NADH:quinone oxidoreductase.

In the respiratory chain, NADH oxidation is coupled to ion translocation across the membrane to build up an electrochemical gradient. In the human pathogen Vibrio cholerae, the sodium-pumping NADH:quinone oxidoreductase (Na-NQR) generates a sodium gradient by a so far unknown mechanism. Here we show that ion pumping in Na-NQR is driven by large conformational changes coupling electron transfer to ion translocation.

Respiratory Membrane Protein Complexes Convert Chemical Energy.

The invention of a biological membrane which is used as energy storage system to drive the metabolism of a primordial, unicellular organism represents a key event in the evolution of life. The innovative, underlying principle of this key event is respiration. In respiration, a lipid bilayer with insulating properties is chosen as the site for catalysis of an exergonic redox reaction converting substrates offered from the environment, using the liberated Gibbs free energy (ΔG) for the build-up of an electrochemical H (proton motive force, PMF) or Na gradient (sodium motive force, SMF) across the lipid bilayer.

as Host for Expression of Multisubunit Membrane Protein Complexes.

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 as a suitable expression host for membrane-bound proteins from . We achieved production of the primary Na pump, the NADH:quinone oxidoreductase (NQR), from in an active state, as indicated by increased overall NADH:quinone oxidoreduction activity of membranes from the transformed , and the sensitivity toward Ag, a specific inhibitor of the NQR.

A miniaturized assay for kinetic characterization of the Na-translocating NADH:ubiquinone oxidoreductase from Vibrio cholerae.

We demonstrate the miniaturization of an enzymatic assay for the determination of NADH oxidation and quinone reduction by the Na -translocating NADH quinone oxidoreductase (NQR) in the 96-well plate format. The assay is based on the spectrophotometric detection of NADH consumption and quinol formation. We validated the new method with known inhibitors of the NQR and optimized conditions for high-throughput screening as demonstrated by excellent Z-factors well above the accepted threshold (≥0.

Strong pH dependence of coupling efficiency of the Na+ - translocating NADH:quinone oxidoreductase (Na+-NQR) of Vibrio cholerae.

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.

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

Unlabelled: 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.

The structure of Na⁺-translocating of NADH:ubiquinone oxidoreductase of Vibrio cholerae: implications on coupling between electron transfer and Na⁺ transport.

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.

Continuous fluorescence-based measurement of redox-driven sodium ion translocation.

Investigation of the mechanism of sodium ion pumping enzymes requires methods to follow the translocation of sodium ions by the purified and reconstituted proteins in vitro. Here, we describe a protocol that allows following the accumulation of Na(+) in proteoliposomes by the Na(+)-translocating NADH:quinone oxidoreductase (Na(+)-NQR) from Vibrio cholerae using the sodium-sensitive fluorophor sodium green. In the presence of a regenerative system for its substrate NADH, the Na(+)-NQR accumulates Na(+) in the proteoliposomes which is visible as a change in fluorescence.