title: A new flavin radical signal in the Na(+)-pumping NADH:quinone oxidoreductase from Vibrio cholerae. An EPR/electron nuclear double resonance investigation of the role of the covalently bound flavins in subunits B and C.
abstract: The Na(+)-pumping NADH-ubiquinone oxidoreductase has six polypeptide subunits (NqrA-F) and a number of redox cofactors, including a noncovalently bound FAD and a 2Fe-2S center in subunit F, covalently bound FMNs in subunits B and C, and a noncovalently bound riboflavin in an undisclosed location. The FMN cofactors in subunits B and C are bound to threonine residues by phosphoester linkages. A neutral flavin-semiquinone radical is observed in the oxidized enzyme, whereas an anionic flavin-semiquinone has been reported in the reduced enzyme. For this work, we have altered the binding ligands of the FMNs in subunits B and C by replacing the threonine ligands with other amino acids, and we studied the resulting mutants by EPR and electron nuclear double resonance spectroscopy. We conclude that the sodium-translocating NADH:quinone oxidoreductase forms three spectroscopically distinct flavin radicals as follows: 1) a neutral radical in the oxidized enzyme, which is observed in all of the mutants and most likely arises from the riboflavin; 2) an anionic radical observed in the fully reduced enzyme, which is present in wild type, and the NqrC-T225Y mutant but not the NqrB-T236Y mutant; 3) a second anionic radical, seen primarily under weakly reducing conditions, which is present in wild type, and the NqrB-T236Y mutant but not the NqrC-T225Y mutant. Thus, we can tentatively assign the first anionic radical to the FMN in subunit B and the second to the FMN in subunit C. The second anionic radical has not been reported previously. In electron nuclear double resonance spectra, it exhibits a larger line width and larger 8alpha-methyl proton splittings, compared with the first anionic radical.
authors.affiliation: Department of Biology and Center for Biotechnology and Interdisciplinary Studies, Rensselaer Polytechnic Institute, Troy, New York 12180, USA. barqub@rpi.edu
authors.forename: Blanca
authors.initials: B
authors.lastname: Barquera
authors.forename: Leticia
authors.initials: L
authors.lastname: Ramirez-Silva
authors.forename: Joel E
authors.initials: JE
authors.lastname: Morgan
authors.forename: Mark J
authors.initials: MJ
authors.lastname: Nilges
mesh_terms: D001483:Base Sequence; D004578:Electron Spin Resonance Spectroscopy; D005415:Flavins / Q000737:chemistry*; D005420:Flavoproteins / Q000737:chemistry; D005609:Free Radicals; D008024:Ligands; D015233:Models, Statistical; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D011485:Protein Binding; D011808:Quinone Reductases / Q000737:chemistry*; D012964:Sodium / Q000737:chemistry; D000254:Sodium-Potassium-Exchanging ATPase / Q000737:chemistry*; D013912:Threonine / Q000737:chemistry; D014734:Vibrio cholerae / Q000201:enzymology*
chemical_list: D005415:Flavins; D005420:Flavoproteins; D005609:Free Radicals; D008024:Ligands; D013912:Threonine; D012964:Sodium; C041831:NADH dehydrogenase (quinone); D011808:Quinone Reductases; D000254:Sodium-Potassium-Exchanging ATPase