Summary
Cytochrome bd is a terminal quinol:O2 oxidoreductase of respiratory chains of many bacteria. It contains three hemes, b558, b595, and d. The role of heme b595 remains obscure. A CO photolysis/recombination study of the membranes of Escherichia coli containing either wild type cytochrome bd or inactive E445A mutant was performed using nanosecond absorption spectroscopy. We compared photoinduced changes of heme d-CO complex in one-electron-reduced, two-electron-reduced, and fully-reduced states of cytochromes bd. The line shape of spectra of photodissociation of one-electron-reduced and two-electron-reduced enzymes is strikingly different from that of the fully-reduced enzyme. The difference demonstrates that in the fully-reduced enzyme photolysis of CO from heme d perturbs ferrous heme b595 causing loss of an absorption band centered at 435 nm, thus supporting interactions between heme b595 and heme d in the di-heme oxygen-reducing site, in agreement with previous works. Photolyzed CO recombines with the fully-reduced enzyme monoexponentially with τ ~12 µs, whereas recombination of CO with one-electron-reduced cytochrome bd shows three kinetic phases, with τ ~14 ns, 14 µs, and 280 µs. The spectra of the absorption changes associated with these components are different in line shape. The 14 ns phase, absent in the fully-reduced enzyme, reflects geminate recombination of CO with part of heme d. The 14 µs component reflects bimolecular recombination of CO with heme d and electron backflow from heme d to hemes b in ~4% of the enzyme population. The final, 280 µs component, reflects return of the electron from hemes b to heme d and bimolecular recombination of CO in that population. The fact that even in the two-electron-reduced enzyme, a nanosecond geminate recombination is observed, suggests that namely the redox state of heme b595, and not that of heme b558, controls the pathway(s) by which CO migrates between heme d and the medium.