Fluoride-binding to the Escherichia coli bd-type ubiquinol oxidase studied by visible absorption and EPR spectroscopies.
Cytochrome bd-type ubiquinol oxidase in the aerobic respiratory chain of Escherichia coli contains two hemes b (b558 and b595) and one heme d as redox metal centers. To clarify the structure of the reaction center, we analyzed the fully oxidized enzyme by visible and EPR spectroscopies using fluoride ion as a monitoring probe. The visible spectral changes upon fluoride-binding were typical of ferric iron-chlorine species, indicating heme d as a primary binding site. The negative peak at 645 nm in the difference spectrum indicates that heme b595 also provides the low-affinity fluoride-binding site. Fluoride-binding caused a complete disappearance from the EPR spectra of the low-spin signals ascribable to heme d and spectral changes in both rhombic and axial high-spin signals. After fluoride-binding, each component of the rhombic high-spin signal showed superhyperfine splitting arising from the interaction of the unpaired spin of the heme d iron with the nuclear magnetic moment of 19F. The axial high-spin species was converted to a new rhombic high-spin species assignable to heme b595-fluoride. The g = 2 component of this new species also gave 19F-superhyperfine splitting. These results indicate that both heme d and heme b595 can coordinate with a fluoride ion with different affinities in the fully oxidized state.
Department of Life Science, Faculty of Science, Himeji Institute of Technology, Kamigoori-cho, Akou-gun, Hyogo, 678-1297, Japan. tsubaki@sci.himeji-tech.ac.jp
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D001665:Binding Sites; D020134:Catalytic Domain; D003573:Cytochrome b Group; D003580:Cytochromes / Q000737:chemistry* / Q000378:metabolism*; D004578:Electron Spin Resonance Spectroscopy / Q000379:methods; D045222:Electron Transport Chain Complex Proteins; D004926:Escherichia coli / Q000201:enzymology*; D029968:Escherichia coli Proteins; D005459:Fluorides / Q000378:metabolism*; D010088:Oxidoreductases / Q000737:chemistry* / Q000378:metabolism*; D013057:Spectrum Analysis / Q000379:methods
D003573:Cytochrome b Group; D003580:Cytochromes; D045222:Electron Transport Chain Complex Proteins; D029968:Escherichia coli Proteins; D010088:Oxidoreductases; C040077:cytochrome bd terminal oxidase complex, E coli; D005459:Fluorides