Fluoride-binding to the Escherichia coli bd-type ubiquinol oxidase studiedby visible absorption and EPR spectroscopies

Cytochrome bd-type ubiquinol oxidase in the aerobic respiratory chain of Es cherichia coli contains two hemes b (b(558) and b(595)) and one heme d as r edox metal centers. To clarify the structure of the reaction center, we ana lyzed the fully oxidized enzyme by visible and EPR spectroscopies using flu oride 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 spec trum indicates that heme b(595) also provides the low-affinity fluoride-bin ding site. Fluoride-binding caused a complete disappearance from the EPR sp ectra of the low-spin signals ascribable to heme d and spectral changes in both rhombic and axial high-spin signals. After fluoride-binding, each comp onent of the rhombic high-spin signal showed superhyperfine splitting arisi ng from the interaction of the unpaired spin of the heme d iron with the nu clear magnetic moment of F-19. The axial high-spin species was converted to a new rhombic high-spin species assignable to heme b(595)-fluoride. The g = 2 component of this new species also gave F-19-superhyperfine splitting. These results indicate that both heme d and heme b(595) can coordinate with a fluoride ion with different affinities in the fully oxidized state.