title: The single NqrB and NqrC subunits in the Na(+)-translocating NADH: quinone oxidoreductase (Na(+)-NQR) from Vibrio cholerae each carry one covalently attached FMN.
abstract: The Na(+)-translocating NADH:quinone oxidoreductase (Na(+)-NQR) is the prototype of a novel class of flavoproteins carrying a riboflavin phosphate bound to serine or threonine by a phosphodiester bond to the ribityl side chain. This membrane-bound, respiratory complex also contains one non-covalently bound FAD, one non-covalently bound riboflavin, ubiquinone-8 and a [2Fe-2S] cluster. Here, we report the quantitative analysis of the full set of flavin cofactors in the Na(+)-NQR and characterize the mode of linkage of the riboflavin phosphate to the membrane-bound NqrB and NqrC subunits. Release of the flavin by β-elimination and analysis of the cofactor demonstrates that the phosphate group is attached at the 5'-position of the ribityl as in authentic FMN and that the Na(+)-NQR contains approximately 1.7mol covalently bound FMN per mol non-covalently bound FAD. Therefore, each of the single NqrB and NqrC subunits in the Na(+)-NQR carries a single FMN. Elimination of the phosphodiester bond yields a dehydro-2-aminobutyrate residue, which is modified with β-mercaptoethanol by Michael addition. Proteolytic digestion followed by mass determination of peptide fragments reveals exclusive modification of threonine residues, which carry FMN in the native enzyme. The described reactions allow quantification and localization of the covalently attached FMNs in the Na(+)-NQR and in related proteins belonging to the Rhodobacter nitrogen fixation (RNF) family of enzymes. This article is part of a Special Issue entitled: 17th European Bioenergetics Conference (EBEC 2012).
authors.affiliation: Department of Neuropathology, University of Freiburg, Freiburg, Germany. marco.casutt@uniklinik-freiburg.de
authors.forename: Marco S
authors.initials: MS
authors.lastname: Casutt
authors.forename: Andreas
authors.initials: A
authors.lastname: Schlosser
authors.forename: Wolfgang
authors.initials: W
authors.lastname: Buckel
authors.forename: Julia
authors.initials: J
authors.lastname: Steuber
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry / Q000378:metabolism; D020134:Catalytic Domain; D005486:Flavin Mononucleotide / Q000737:chemistry / Q000378:metabolism; D017136:Ion Transport / Q000502:physiology; D009247:NADH, NADPH Oxidoreductases / Q000737:chemistry / Q000378:metabolism; D010455:Peptides; D059748:Proteolysis; D012964:Sodium / Q000737:chemistry / Q000378:metabolism; D014451:Ubiquinone / Q000737:chemistry / Q000378:metabolism; D014734:Vibrio cholerae / Q000235:genetics*
chemical_list: D001426:Bacterial Proteins; D010455:Peptides; D014451:Ubiquinone; D005486:Flavin Mononucleotide; D012964:Sodium; C030778:ubiquinone 8; D009247:NADH, NADPH Oxidoreductases