TIME-RESOLVED RESONANCE RAMAN EVIDENCE FOR TIGHT COUPLING BETWEEN ELECTRON-TRANSFER AND PROTON-PUMPING OF CYTOCHROME-C-OXIDASE UPON THE CHANGE FROM THE FE-V OXIDATION LEVEL TO THE FE-IV OXIDATION LEVEL

The mechanism of dioxygen reduction catalyzed by cytochrome c oxidase, the terminal enzyme of the respiration chain of aerobic organisms, ha s been investigated with time-resolved resonance Raman spectroscopy. F ive oxygen isotope sensitive Raman bands have been identified for O-16 (2)/O-18(2) intermediates at 571/544, 804/764, 356/342, 785/750, and 4 50/425 cm(-1) in the order of appearance. The first and last ones, whi ch have been assigned to the Fe-III-O-2(-) and Fe-III-OH- stretching m odes, respectively, are in general consensus, but the remaining three bands are currently under debate. This study establishes that the 804/ 764 cm(-1) species is generated prior to the 785/750 cm(-1) species, a nd the latter is not observed when we start from the mixed valence enz yme. Therefore, the enzyme oxidation state of the 804/764 cm(-1) speci es is higher by one oxidative equivalent than that of the 785/750 cm(- 1) species, although both bands have been assigned to the Fe=O stretch ing mode. The excitation wavelength dependences of these two sets of R aman bands are distinctly different, suggesting that the electronic pr operties of the two hemes are quite different. We found that the conve rsion from the 804/764 cm(-1) species to the 785/750 cm(-1) species wa s significantly slower in D2O than in H2O, suggesting strong coupling between electron and vector cm proton transfers at this stage. Presuma bly, Cu-B at the binuclear site causes heterolytic cleavage of the O-O bond, giving rise to an oxoiron in the Fe-v oxidation level of the en zyme with the 804/764 cm(-1) bands, and the electron transfer to this oxoiron is accompanied by some protein conformational changes, which c ause distortion of the oxoheme and thus appearance of the His-Fe=O ben ding Raman bands at 356/342 cm(-1) and simultaneously active transport of protons.