Observation of the O-O stretching raman band for cytochrome P-450_cam under catalytic conditions

Dioxygen stretching (v_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 Dcamphor, 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 v_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.