title: The conformational changes induced by ubiquinone binding in the Na+-pumping NADH:ubiquinone oxidoreductase (Na+-NQR) are kinetically controlled by conserved glycines 140 and 141 of the NqrB subunit.
abstract: Na(+)-pumping NADH:ubiquinone oxidoreductase (Na(+)-NQR) is responsible for maintaining a sodium gradient across the inner bacterial membrane. This respiratory enzyme, which couples sodium pumping to the electron transfer between NADH and ubiquinone, is not present in eukaryotes and as such could be a target for antibiotics. In this paper it is shown that the site of ubiquinone reduction is conformationally coupled to the NqrB subunit, which also hosts the final cofactor in the electron transport chain, riboflavin. Previous work showed that mutations in conserved NqrB glycine residues 140 and 141 affect ubiquinone reduction and the proper functioning of the sodium pump. Surprisingly, these mutants did not affect the dissociation constant of ubiquinone or its analog HQNO (2-n-heptyl-4-hydroxyquinoline N-oxide) from Na(+)-NQR, which indicates that these residues do not participate directly in the ubiquinone binding site but probably control its accessibility. Indeed, redox-induced difference spectroscopy showed that these mutations prevented the conformational change involved in ubiquinone binding but did not modify the signals corresponding to bound ubiquinone. Moreover, data are presented that demonstrate the NqrA subunit is able to bind ubiquinone but with a low non-catalytically relevant affinity. It is also suggested that Na(+)-NQR contains a single catalytic ubiquinone binding site and a second site that can bind ubiquinone but is not active.
authors.affiliation: From the Laboratoire de Bioélectrochimie et Spectroscopie, UMR 7140, CNRS Université de Strasbourg, Strasbourg, France, 67000 and.
authors.forename: Madeleine
authors.initials: M
authors.lastname: Strickland
authors.affiliation: Department of Biological Sciences, Center for Biotechnology and Interdisciplinary Studies, Rensselaer Polytechnic Institute, Troy, New York 12180.
authors.forename: Oscar
authors.initials: O
authors.lastname: Juárez
authors.affiliation: From the Laboratoire de Bioélectrochimie et Spectroscopie, UMR 7140, CNRS Université de Strasbourg, Strasbourg, France, 67000 and.
authors.forename: Yashvin
authors.initials: Y
authors.lastname: Neehaul
authors.affiliation: Department of Biological Sciences, Center for Biotechnology and Interdisciplinary Studies, Rensselaer Polytechnic Institute, Troy, New York 12180.
authors.forename: Darcie A
authors.initials: DA
authors.lastname: Cook
authors.affiliation: Department of Biological Sciences, Center for Biotechnology and Interdisciplinary Studies, Rensselaer Polytechnic Institute, Troy, New York 12180 barqub@rpi.edu.
authors.forename: Blanca
authors.initials: B
authors.lastname: Barquera
authors.affiliation: From the Laboratoire de Bioélectrochimie et Spectroscopie, UMR 7140, CNRS Université de Strasbourg, Strasbourg, France, 67000 and hellwig@unistra.fr.
authors.forename: Petra
authors.initials: P
authors.lastname: Hellwig
mesh_terms: D001483:Base Sequence; D017124:Conserved Sequence; D017931:DNA Primers; D042967:Electron Transport Complex I / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D005998:Glycine / Q000737:chemistry*; D007700:Kinetics; D011485:Protein Binding; D011487:Protein Conformation; D000254:Sodium-Potassium-Exchanging ATPase / Q000378:metabolism*; D017550:Spectroscopy, Fourier Transform Infrared; D014451:Ubiquinone / Q000378:metabolism*
chemical_list: D017931:DNA Primers; D014451:Ubiquinone; D042967:Electron Transport Complex I; D000254:Sodium-Potassium-Exchanging ATPase; D005998:Glycine
keywords: Enzyme Inhibitor; HQNO; Infrared Spectroscopy (IR spectroscopy); Membrane Proteins; NQR; Oxidation-Reduction (redox); Respiratory Chain; Sodium Pumping; Ubiquinone