title: A mutation in Na(+)-NQR uncouples electron flow from Na(+) translocation in the presence of K(+).
abstract: The sodium-pumping NADH:ubiquinone oxidoreductase (Na(+)-NQR) is a bacterial respiratory enzyme that obtains energy from the redox reaction between NADH and ubiquinone and uses this energy to create an electrochemical Na(+) gradient across the cell membrane. A number of acidic residues in transmembrane helices have been shown to be important for Na(+) translocation. One of these, Asp-397 in the NqrB subunit, is a key residue for Na(+) uptake and binding. In this study, we show that when this residue is replaced with asparagine, the enzyme acquires a new sensitivity to K(+); in the mutant, K(+) both activates the redox reaction and uncouples it from the ion translocation reaction. In the wild-type enzyme, Na(+) (or Li(+)) accelerates turnover while K(+) alone does not activate. In the NqrB-D397N mutant, K(+) accelerates the same internal electron transfer step (2Fe-2S → FMNC) that is accelerated by Na(+). This is the same step that is inhibited in mutants in which Na(+) uptake is blocked. NqrB-D397N is able to translocate Na(+) and Li(+), but when K(+) is introduced, no ion translocation is observed, regardless of whether Na(+) or Li(+) is present. Thus, this mutant, when it turns over in the presence of K(+), is the first, and currently the only, example of an uncoupled Na(+)-NQR. The fact the redox reaction and ion pumping become decoupled from each other only in the presence of K(+) provides a switch that promises to be a useful experimental tool.
authors.affiliation: Department of Biological Sciences and Center for Biotechnology and Interdisciplinary Studies, Rensselaer Polytechnic Institute , Troy, New York 12180, United States.
authors.forename: Michael E
authors.initials: ME
authors.lastname: Shea
authors.forename: Katherine G
authors.initials: KG
authors.lastname: Mezic
authors.forename: Oscar
authors.initials: O
authors.lastname: Juárez
authors.forename: Blanca
authors.initials: B
authors.lastname: Barquera
mesh_terms: D002414:Cations, Monovalent / Q000378:metabolism; D004579:Electron Transport; D042967:Electron Transport Complex I / Q000235:genetics* / Q000378:metabolism*; D004583:Electrons; D017136:Ion Transport; D009154:Mutation; D011188:Potassium / Q000378:metabolism*; D011808:Quinone Reductases / Q000378:metabolism; D012964:Sodium / Q000378:metabolism*; D014734:Vibrio cholerae / Q000201:enzymology* / Q000235:genetics* / Q000378:metabolism
chemical_list: D002414:Cations, Monovalent; D012964:Sodium; D011808:Quinone Reductases; D042967:Electron Transport Complex I; D011188:Potassium