Acid Residues in the Transmembrane Helices of the Na⁺-Pumping NADH:Quinone Oxidoreductase from Vibrio cholerae Involved in Sodium Translocation

Abstract

Vibrio cholerae and many other marine and pathogenic bacteria posses a unique respiratory complex, the Na+-pumping NADH: quinone oxidoreductase (Na+-NQR)1, which pumps Na+ across the cell membrane using the energy released by the redox reaction between NADH and ubiquinone. In order to function as a selective sodium pump, Na+-NQR must contain structures that: 1) allow the sodium ion to pass through the hydrophobic core of the membrane, and 2) provide cation specificity to the translocation system. In other sodium transporting proteins, the structures that carry out these roles frequently include aspartate and glutamate residues. The negative charge of these residues facilitates binding and translocation of sodium. In this study we have analyzed mutants of acid residues located in the transmembrane helices of subunits B, D and E of Na+-NQR. The results are consistent with the participation of seven of these residues in the translocation process of sodium. Mutations at NqrB-D397, NqrD-D133 and NqrE-E95 produced a decrease of approximately ten times or more in the apparent affinity of the enzyme for sodium (Kmapp), which suggests that these residues may form part of a sodium-binding site. Mutation at other residues, including NqrB-E28, NqrB-E144, NqrB-E346 and NqrD-D88, had a large effect on the quinone reductase activity of the enzyme and its sodium sensitivity, but less effect on the apparent sodium affinity, consistent with a possible role in sodium conductance pathways.

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

“…This 50% fraction (12 mg) was further purified by reversed phase HPLC (20 ϫ 250 mm, COSMOSIL 5C 18 -MS-II, 80% methanol, 0.1% formic acid, detected at 254 nm) with a flow rate of 5.0 ml/min at 30°C to provide korormicin as a colorless oil (5.6 mg, 12 mol, retention time is 54.2 min). The following spectral data completely corresponded to those reported previously (16,17) Expression and purification of wild-type and mutants of Na ؉ -NQR Site-directed mutants were obtained using the QuikChange Lightning mutagenesis kit (Agilent) as reported before (29). The forward primers used for the mutants in NqrA are as follows: NqrA-Y36A, GCTTGGCGAAGAGGCCGTTGGCAT-GCGTC; NqrA-G38V, GGCGAAGAGTACGTTGTCATGC-GTCCTACTATG.…”

“…The primers for the mutants in NqrB were reported before (29). Recombinant wild-type Na ϩ -NQR and mutants were grown in LB medium as reported before (8) in 30-liter fermenters.…”

“…Membranes were obtained by ultracentrifugation (100,000 ϫ g) and washed with 5 mM imidazole, 300 mM NaCl, 0.05% glycerol. Wild-type and mutant proteins were purified by affinity chromatography as reported before (29).…”

Energy transducing redox steps of the Na + -pumping NADH:quinone oxidoreductase from Vibrio cholerae

“…Wild-type Na þ -NQR reconstituted in proteoliposomes is able to transport sodium (Fig. 2, trace i), and this process is insensitive to proton ionophores (CCCP) and abolished by sodium ionophores (ETH-157) (23). Surprisingly, the enzyme is also able to form an electrochemical gradient of sodium in the absence of CoQ (Fig.…”

Structure of the V. cholerae Na+-pumping NADH:quinone oxidoreductase

“…Despite the low sequence identity (5 12% ), the structural similarity is rather high.ln structural alignments ofN qrB with a urea transporter from Bos tauru~ (PDB code 4EZC ) Residue Asp 346 ofNqrB is located at the bottom of this vestibule (Fig. 2c), and mutational studies have suggested that this aspartic residue is involved in Na + transport 15 • At a position halfway through the membrane the channel narrows sharply and the access to the second half of the chan ne!, which opens to the periplasm, is blocked by side chains of hydro phobic residues: Phe 338 and Phe 342 located on helix VIII, Ile 164, Leu 165 and Leu 168 on helix m, and V al64 of helix I (Fig. 2c, d).…”