title: Aspartic acid 397 in subunit B of the Na+-pumping NADH:quinone oxidoreductase from Vibrio cholerae forms part of a sodium-binding site, is involved in cation selectivity, and affects cation-binding site cooperativity.
abstract: The Na(+)-pumping NADH:quinone complex is found in Vibrio cholerae and other marine and pathogenic bacteria. NADH:ubiquinone oxidoreductase oxidizes NADH and reduces ubiquinone, using the free energy released by this reaction to pump sodium ions across the cell membrane. In a previous report, a conserved aspartic acid residue in the NqrB subunit at position 397, located in the cytosolic face of this protein, was proposed to be involved in the capture of sodium. Here, we studied the role of this residue through the characterization of mutant enzymes in which this aspartic acid was substituted by other residues that change charge and size, such as arginine, serine, lysine, glutamic acid, and cysteine. Our results indicate that NqrB-Asp-397 forms part of one of the at least two sodium-binding sites and that both size and charge at this position are critical for the function of the enzyme. Moreover, we demonstrate that this residue is involved in cation selectivity, has a critical role in the communication between sodium-binding sites, by promoting cooperativity, and controls the electron transfer step involved in sodium uptake (2Fe-2S → FMNC).
authors.affiliation: From the Department of Biology and Center of Biotechnology and Interdisciplinary Studies, Rensselaer Polytechnic Institute, Troy, New York 121801.
authors.forename: Michael E
authors.initials: ME
authors.lastname: Shea
authors.forename: Oscar
authors.initials: O
authors.lastname: Juárez
authors.forename: Jonathan
authors.initials: J
authors.lastname: Cho
authors.forename: Blanca
authors.initials: B
authors.lastname: Barquera
mesh_terms: D019943:Amino Acid Substitution; D001224:Aspartic Acid / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D001665:Binding Sites; D004579:Electron Transport / Q000502:physiology; D017136:Ion Transport / Q000502:physiology; D020125:Mutation, Missense; D011808:Quinone Reductases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D012964:Sodium / Q000737:chemistry / Q000378:metabolism*; D014734:Vibrio cholerae / Q000201:enzymology* / Q000235:genetics
chemical_list: D001426:Bacterial Proteins; D001224:Aspartic Acid; D012964:Sodium; C041831:NADH dehydrogenase (quinone); D011808:Quinone Reductases
keywords: Bacteria; Bacterial Metabolism; Bioenergetics-Electron Transfer Complex; Redox; Sodium Transport
references.citation: Biochemistry. 2012 May 15;51(19):4072-7
references.pmid: 22533880
references.citation: Biochemistry. 2013 May 7;52(18):3085-93
references.pmid: 23566241
references.citation: Proc Natl Acad Sci U S A. 2010 Jul 13;107(28):12505-10
references.pmid: 20616050
references.citation: J Bacteriol. 2006 Dec;188(24):8343-51
references.pmid: 17041063
references.citation: Science. 2005 Dec 2;310(5753):1461-5
references.pmid: 16322449
references.citation: J Biol Chem. 2009 Feb 27;284(9):5533-8
references.pmid: 19117949
references.citation: Biochim Biophys Acta. 1986 Jul 23;850(3):458-65
references.pmid: 2942186
references.citation: J Biol Chem. 2009 Mar 27;284(13):8963-72
references.pmid: 19155212
references.citation: J Biol Chem. 2011 Jul 29;286(30):26383-90
references.pmid: 21652714
references.citation: Biochim Biophys Acta. 2009 Jun;1787(6):691-6
references.pmid: 19167341
references.citation: Biochemistry (Mosc). 2005 Feb;70(2):143-9
references.pmid: 15807651
references.citation: Biochim Biophys Acta. 2001 May 1;1505(1):37-44
references.pmid: 11248187
references.citation: Eur J Biochem. 1997 Nov 1;249(3):770-6
references.pmid: 9395325
references.citation: Biochemistry. 2002 Mar 19;41(11):3781-9
references.pmid: 11888296
references.citation: Biochemistry. 2004 Sep 28;43(38):12322-30
references.pmid: 15379571
references.citation: Biochemistry. 2009 Oct 13;48(40):9516-24
references.pmid: 19694431
references.citation: J Bacteriol. 2001 Jul;183(14):4142-8
references.pmid: 11418553
references.citation: Biochemistry. 1999 Dec 7;38(49):16084-91
references.pmid: 10587431
references.citation: J Bacteriol. 1999 Mar;181(6):1927-30
references.pmid: 10074090