Observation of the O-O stretching Raman band for cytochrome P-450cam under catalytic conditions

Dioxygen stretching (voo) Raman band was observed for the oxy form of Pseudomonas putida cytochrome P-450 (P-450,,,) generated at room temperature under catalytic conditions, that is, in the presence of Dcamphor, &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 voo frequency is little altered by binding of putidaredoxin to P-450,,,, 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.Cytochrome P-450 (P-450)' is a generic name of heme proteins which have the catalytic activity for monooxygenation of a variety of organic compounds (1).In their catalytic reaction one oxygen atom of the heme-bound dioxygen is incorporated into a substrate while the other oxygen atom is converted into water.The unique reactivity of the dioxygen bound to P-450 and the effect from its trans ligand, which is a sulfur anion of cysteinate common to all P-450s, have been a matter of physicochemical as well as organometallic concerns (2).The question to be answered is whether the dioxygen bound to P-450 differs from those of other heme proteins.The 0-0 stretching (vOo) frequency is expected to reflect most sensitively the nature of the 0-0 bond.Champion and co-workers (3) succeeded in observing the v00 resonance Raman (RR) band for noncatalytic 0, adduct of frozen P-450 with no electron donor present at -60 "C, and this was recently confirmed for a solution at -20 "C by Nishimura et al. (4).The observed vo0 frequency was, unexpectedly, not greatly different from that of oxy-myoglobin (oxyMb) in which the heme-bound dioxygen is nonreactive.The visible absorption (5) and Mossbauer spectra (6) of oxyP-450 were also close to those of oxyMb although magnetic circular dichroism spectra of oxyP-450 and oxyMb were different ( 7 ) .We thought it important to detect the voo RR band for oxyP-450 under catalytic conditions, that is, at room temperature in the