title: NMR reveals double occupancy of quinone-type ligands in the catalytic quinone binding site of the Na+-translocating NADH:Quinone oxidoreductase from Vibrio cholerae.
abstract: The sodium ion-translocating NADH:quinone oxidoreductase (Na(+)-NQR) from the pathogen Vibrio cholerae exploits the free energy liberated during oxidation of NADH with ubiquinone to pump sodium ions across the cytoplasmic membrane. The Na(+)-NQR consists of four membrane-bound subunits NqrBCDE and the peripheral NqrF and NqrA subunits. NqrA binds ubiquinone-8 as well as quinones with shorter prenyl chains (ubiquinone-1 and ubiquinone-2). Here we show that the quinone derivative 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone (DBMIB), a known inhibitor of the bc1 and b6f complexes found in mitochondria and chloroplasts, also inhibits quinone reduction by the Na(+)-NQR in a mixed inhibition mode. Tryptophan fluorescence quenching and saturation transfer difference NMR experiments in the presence of Na(+)-NQR inhibitor (DBMIB or 2-n-heptyl-4-hydroxyquinoline N-oxide) indicate that two quinone analog ligands are bound simultaneously by the NqrA subunit with very similar interaction constants as observed with the holoenzyme complex. We conclude that the catalytic site of quinone reduction is located on NqrA. The two ligands bind to an extended binding pocket in direct vicinity to each other as demonstrated by interligand Overhauser effects between ubiquinone-1 and DBMIB or 2-n-heptyl-4-hydroxyquinoline N-oxide, respectively. We propose that a similar spatially close arrangement of the native quinone substrates is also operational in vivo, enhancing the catalytic efficiency during the final electron transfer steps in the Na(+)-NQR.
authors.affiliation: From the Department of Chemistry, University of Konstanz, 78457 Konstanz, Germany and.
authors.forename: Ruslan
authors.initials: R
authors.lastname: Nedielkov
authors.affiliation: the Department of Microbiology, University of Hohenheim (Stuttgart), 70599 Stuttgart, Germany.
authors.forename: Wojtek
authors.initials: W
authors.lastname: Steffen
authors.affiliation: the Department of Microbiology, University of Hohenheim (Stuttgart), 70599 Stuttgart, Germany. Electronic address: julia.steuber@uni-hohenheim.de.
authors.forename: Julia
authors.initials: J
authors.lastname: Steuber
authors.affiliation: From the Department of Chemistry, University of Konstanz, 78457 Konstanz, Germany and. Electronic address: heiko.moeller@uni-konstanz.de.
authors.forename: Heiko M
authors.initials: HM
authors.lastname: Möller
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry*; D020134:Catalytic Domain; D003991:Dibromothymoquinone / Q000737:chemistry* / Q000378:metabolism; D006912:Hydroxyquinolines / Q000737:chemistry* / Q000378:metabolism; D009682:Magnetic Resonance Spectroscopy; D009243:NAD / Q000737:chemistry / Q000378:metabolism; D021122:Protein Subunits; D011808:Quinone Reductases / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000378:metabolism; D014451:Ubiquinone / Q000737:chemistry / Q000378:metabolism; D014734:Vibrio cholerae / Q000201:enzymology*
chemical_list: D001426:Bacterial Proteins; D006912:Hydroxyquinolines; D021122:Protein Subunits; D009243:NAD; D014451:Ubiquinone; C001333:2-(n-heptyl)-4-hydroxyquinoline N-oxide; D003991:Dibromothymoquinone; C041831:NADH dehydrogenase (quinone); D011808:Quinone Reductases
keywords: DBMIB; Interligand NOE; Membrane Enzymes; NMR; Redox; Sodium Transport; Ubiquinone