SELECTIVE RESONANCE RAMAN OBSERVATION OF THE 607 NM FORM GENERATED INTHE REACTION OF OXIDIZED CYTOCHROME-C-OXIDASE WITH HYDROGEN-PEROXIDE

Resonance Raman spectra were measured selectively for the ''607 nm'' f orm, which had been assigned to a peroxy intermediate formed in the re action of oxidized cytochrome c oxidase with hydrogen peroxide at ambi ent temperature. A single oxygen isotope-sensitive band was found at 8 03 cm(-1) for the reaction with (H2O2)-O-16 (at 769 cm(-1) with (H2O2) -O-18) upon excitation at 607 nm, the wave-length of the difference ab sorption maximum characteristic of the ''peroxy'' intermediate. Upon e xcitation at shorter wavelengths (down to 580 nm), the Raman spectrum simply became weaker without yielding any new features. When (H2OO)-O- 16-O-18 was used, two bands were observed at 803 and 769 cm(-1) (withi n an accuracy of 0.5 cm(-1)), but with only half the intensity of thos e observed with (H2O2)-O-16 or (H2O2)-O-18, which ruled out the possib ility that the 803 cm(-1) band arose from the O-O or Fe-O-2 stretching of the Fe-II(O-O-) heme. Conversely the 34-cm(-1) downshift with O-18 is in good agreement with the calculated O-16/O-18 Shift (35 cm(-1)) expected for the diatomic Fe=O-16 oscillator at 803 cm(-1). This band exhibited an upshift by 1.3 cm(-1) in (H2O)-H-2, similar to the case o f compound II of horseradish peroxidase at neutral pH, and indicative of the presence of a hydrogen bond to the Fe-IV=O oxygen. The 803/769 cm(-1) pair of resonance Raman bands were also observed upon blue exci tation, as is the case for the bands found in the dioxygen cycle of th is enzyme (Ogura, T., Takahashi, S., Hirota, S., Shinzawa-Itoh, K., Yo shikawa, S., Appelman, E. H., and Kitagawa, T. (1993) J. Am. Chem. Sec . 115, 8527-8536). This observation provides the first direct characte rization of the 607 nm form of this enzyme in its reaction with H2O2.