Observation of the O-O stretching raman band for cytochrome P-450(cam) under catalytic conditions

Dioxygen stretching (ν(oo)) Raman band was observed for the oxy form of Pseudomonas putida cytochrome P-450 (P-450(cam)) generated at room temperature under catalytic conditions, that is, in the presence of D-camphor, β-NADH, putidaredoxin, and putidaredoxin reductase, by using the mixed flow transient Raman apparatus. At the same time the visible absorption spectra were monitored for the transient species. It was found that the ν(oo) frequency is little altered by binding of putidaredoxin to P-450(cam), although the reduction rate of the oxy form becomes faster. Another intermediate with an oxygen isotope-sensitive band was not found in a time region until 2 s after mixing of the reduced enzyme with oxygen.