STRUCTURE-FUNCTION STUDIES ON THE ESCHERICHIA-COLI CYTOCHROME BO COMPLEX .13. OBSERVATION OF THE FE-O-2 AND FE-IV=O STRETCHING RAMAN BANDS FOR DIOXYGEN REDUCTION INTERMEDIATES OF CYTOCHROME BO ISOLATED FROM ESCHERICHIA-COLI

Reaction intermediates in dioxygen reduction by the E. coli cytochrome bo-type ubiquinol oxidase were studied by time-resolved resonance Ram an spectroscopy using the artificial cardiovascular system. At 0-20 mu s following photolysis of the enzyme-CO adduct in the presence of O-2 , we observed the Fe-O-2 stretching Raman band at 568 cm(-1) which shi fted to 535 cm(-1) with the O-18(2) derivative. These frequencies are remarkably close to those of other oxyhemoproteins including dioxygen- bound hemoglobin and aa(3)-type cytochrome c oxidase. In the later tim e range (20-40 mu s), other oxygen-isotope-sensitive Raman bands were observed at 788 and 361 cm(-1). Since the 781 cm(-1) band exhibited a downshift by 37 cm(-1) upon O-18(2) substitution, we assigned it to th e Fe-IV=O stretching mode. This band is considered to arise from the f erryl intermediate, but its appearance was much earlier than the corre sponding intermediate of bovine cytochrome c oxidase (> 100 mu s). The 361 cm(-1) band showed the O-16/O-18 isotopic frequency shift of 14 c m(-1) similar to the case of bovine cytochrome c oxidase reaction.