Biochemical and Biophysical Research Communications Spectral Intermediates in the Reaction of Oxygen with Purified Liver Microsomal Cytochrome P-450 F

Stopped flow spectrophotometry has shown the occurrence of two distinct spectral intermediates in the reaction of oxygen with the reduced form of highly purified cytochrome P-450 from liver microsomes. As indicated by difference spectra, Complex I (with maxima at 430 and 450 nm) is rapidly formed and then decays to form Complex II (with a broad maximum at 440 nm), which resembles the intermediate seen in steady state experiments. In the reaction sequence, P-450%'*Complex I+Complex II+P-450@., the last step is rate-limiting. The rate of that step is inadequate to account for the known turnover number of the enzyme in benzphetamine hydroxylation unless NADPH-cytochrome P-450 reductase or cytochrome b is added. The latter protein does not appear to function as an electron carrier in this process.