Acid residues in the transmembrane helices of the Na+-pumping NADH:quinone oxidoreductase from Vibrio cholerae involved in sodium translocation.

Vibrio cholerae and many other marine and pathogenic bacteria possess a unique respiratory complex, the Na(+)-pumping NADH:quinone oxidoreductase (Na(+)-NQR), which pumps Na(+) across the cell membrane using the energy released by the redox reaction between NADH and ubiquinone. 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 >or=10-fold in the apparent affinity of the enzyme for sodium (Km(app)(Na+)), 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 strong effect on the quinone reductase activity of the enzyme and its sodium sensitivity, but a weaker effect on the apparent sodium affinity, consistent with a possible role in sodium conductance pathways.