TIME-RESOLVED RESONANCE RAMAN ELUCIDATION OF THE PATHWAY FOR DIOXYGEN REDUCTION BY CYTOCHROME-C-OXIDASE

Time-resolved resonance Raman (RR) spectra were observed for catalytic intermediates of bovine cytochrome c oxidase at room temperature by using an Artificial Cardiovascular System for Enzymatic Reactions and Raman/Absorption Simultaneous Measurement Device. The isotope-frequency-shift data using an asymmetrically labeled dioxygen, (OO)-O-16-O-18, established that the primary intermediate (compound A) is an end-on-type dioxygen adduct of cytochrome a3. Higher resolution RR experiments revealed that the approximately 800-cm-1 band assigned previously to the Fe(IV)=O stretching mode of the subsequent intermediate (compound B) was actually composed of two bands that behaved differently upon deuteration. The 804/764-cm-1 pair for the O-16(2)/O-18(2) derivatives were insensitive to deuteration, and these bands became noticeably stronger at higher temperatures. In contrast, the 785/751-cm-1 pair for the O-16(2)/O-18(2) derivatives was shifted to 796/766 cm-1 in D2O, and its intensity became virtually zero at 30-degrees-C. The corresponding bands for the (OO)-O-16-O-18 derivative appeared at nearly the same frequencies as those of the O-16(2) and O-18(2) derivatives but not at an intermediate frequency. Therefore, neither of these bands could be attributed to the O-O stretching mode. Normal coordinate calculations using a set of suitable force constants of the Urey-Bradley-Shimanouchi force field for an isolated Fe-O-O-H group could explain the 785/751-cm-1 bands in terms of the Fe-O stretching vibration, although this model assumed a relatively strengthened Fe-O bond and weakened O-O bond for the Fe-O-O-H group. The 804/764-cm-1 bands were assigned to the Fe(IV)=O stretching mode of the ferryl intermediate. The 356/342-cm-1 pair for the O-16(2)/O-18(2) derivatives was insensitive to deuteration, and its frequencies were the same as those obtained with (OO)-O-16-O-18. The intensities of the components of this pair were appreciable even when those of the 785/751-cm-1 pair were zero. Therefore, contrary to the previous assignment, the 356/342-cm-1 pair cannot be ascribed to the hydroperoxy intermediate, but its intensity behavior with temperature was also not coincident with that of the 804/764-cm-1 pair. Although the assignment of the 356/342-cm-1 pair is yet to be determined, the present experiments have conclusively established that the main pathway for dioxygen reduction by cytochrome c oxidase is Fe(II)-O2 --> Fe(III)-O-O-H --> Fe(IV)=O --> Fe(III)-OH and that their key bands for the O-16(2)/O-18(2) pair are at 571/544, 785/751, 804/764, and 450/425 cm-1, respectively.