Spectral intermediates in the reaction of oxygen with purified liver microsomal cytochrome P-450

Abstract

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 LM red → O 2 Complex I→Complex II→P-450 LM ox 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 5 is added. The latter protein does not appear to function as an electron carrier in this process.