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, ¹⁶O¹⁸O, established that the primary intermediate (compound A) is an end-on-type dioxygen adduct of cytochrome a₃. Higher resolution RR experiments revealed that the ~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 ¹⁶O₂/ ¹⁸O₂ derivatives were insensitive to deuteration, and these bands became noticeably stronger at higher temperatures. In contrast, the 785/751-cm⁻¹ pair for the ¹⁶O₂/ ¹⁸O₂ derivatives was shifted to 796/766 cm⁻¹ in D₂O, and its intensity became virtually zero at 30 °C. The corresponding bands for the ¹⁶O¹⁸O derivative appeared at nearly the same frequencies as those of the ¹⁶O₂ and ¹⁸O₂ 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 ¹⁶O₂/¹⁸O₂ derivatives was insensitive to deuteration, and its frequencies were the same as those obtained with ¹⁶O¹⁸O. 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—?—?—H → Fe^IV=0 → Fe^III—OH and that their key bands for the ¹⁶O₂/¹⁸O₂ pair are at 571/544, 785/751, 804/764, and 450/425 cm⁻¹, respectively.