title: X- and W-band EPR and Q-band ENDOR studies of the flavin radical in the Na+ -translocating NADH:quinone oxidoreductase from Vibrio cholerae.
abstract: Na(+)-NQR is the entry point for electrons into the respiratory chain of Vibrio cholerae. It oxidizes NADH, reduces ubiquinone, and uses the free energy of this redox reaction to translocate sodium across the cell membrane. The enzyme is a membrane complex of six subunits that accommodates a 2Fe-2S center and several flavins. Both the oxidized and reduced forms of Na(+)-NQR exhibit a radical EPR signal. Here, we present EPR and ENDOR data that demonstrate that, in both forms of the enzyme, the radical is a flavin semiquinone. In the oxidized enzyme, the radical is a neutral flavin, but in the reduced enzyme the radical is an anionic flavin, where N(5) is deprotonated. By combining results of ENDOR and multifrequency continuous wave EPR, we have made an essentially complete determination of the g-matrix and all major nitrogen and proton hyperfine matrices. From careful analysis of the W-band data, the full g-matrix of a flavin radical has been determined. For the neutral radical, the g-matrix has significant rhombic character, but this is significantly decreased in the anionic radical. The out-of-plane component of the g-matrix and the nitrogen hyperfine matrices are found to be noncoincident as a result of puckering of the pyrazine ring. Two possible assignments of the radical signals are considered. The neutral and anionic forms of the radical may each arise from a different flavin cofactor, one of which is converted from semiquinone to flavohydroquinone, while the other goes from flavoquinone to semiquinone, at almost exactly the same redox potential, during reduction of the enzyme. Alternatively, both forms of the radical signal may arise from a single, extremely stable, flavin semiquinone, which becomes deprotonated upon reduction of the enzyme.
authors.affiliation: Department of Biochemistry. University of Illinois at Urbana-Champaign, 600 South Mathews Street, Urbana, IL 61801, USA.
authors.forename: Blanca
authors.initials: B
authors.lastname: Barquera
authors.forename: Joel E
authors.initials: JE
authors.lastname: Morgan
authors.forename: Dmitriy
authors.initials: D
authors.lastname: Lukoyanov
authors.forename: Charles P
authors.initials: CP
authors.lastname: Scholes
authors.forename: Robert B
authors.initials: RB
authors.lastname: Gennis
authors.forename: Mark J
authors.initials: MJ
authors.lastname: Nilges
mesh_terms: D001426:Bacterial Proteins; D004578:Electron Spin Resonance Spectroscopy / Q000379:methods; D005415:Flavins / Q000737:chemistry* / Q000378:metabolism; D005609:Free Radicals / Q000737:chemistry / Q000378:metabolism; D010084:Oxidation-Reduction; D011808:Quinone Reductases / Q000737:chemistry* / Q000378:metabolism; D014734:Vibrio cholerae / Q000201:enzymology*
chemical_list: D001426:Bacterial Proteins; D005415:Flavins; D005609:Free Radicals; C084142:sodium-translocating NADH-quinone reductase; D011808:Quinone Reductases