Observation of the O-O Stretching Resonance Raman Band for Cytochrome P-450cam Under Catalytic Conditions
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. 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 question to be answered is whether the dioxygen bound to P-450 differs from those of other heme proteins. The O-O stretching (v OO) frequency is expected to reflect most sensitively the nature of the O-O bond. Bangcharoenpaurpong et al.  succeeded in observing the v OO RR band for noncatalytic O2 adduct of frozen P-450cam with no electron donor present at -6° C and it was recently confirmed for a solution at -20° C by Nishimura et al. . The observed v OO frequency was, unexpectedly, very close to that of oxy-myoglobin (oxyMb) in which the heme bound dioxygen is nonreactive. We thought it important to detect the v OO RR band for oxy P-450cam under catalytic conditions, that is, at room temperature in the presence of electron donor (reduced putidaredoxin; Pdr) and substrate (D-camphor). Since the reaction becomes faster under such conditions (It is reported that Pdr and P-450cam is associate to form a bimolecular complex and the life time of oxy P-450 m is made significantly shorter by formation of the complex ), it is hard to detect oxy P-450cam with an ordinary technique. Accordingly, we applied a home made mixed-flow transient Raman apparatus to observe the v OO RR band successfully.
KeywordsElectron Donor Isotopic Shift Vibrational Spectroscopy Heme Protein Visible Absorption Spectrum
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