NADH- and NADPH-dependent reconstituted p-nitroanisole O-demethylation system containing cytochrome P-450 with high...

A reconstituted system containing a form of cytochrome P-450, cytochrome b5, NADPH-cytochrome P-450 reductase, and NADH-cytochrome b5 reductase, all purified from rabbit liver microsomes, could catalyze O-demethylation of p-nitroanisole in the presence of both NADPH and NADH. Omission of either cytochrome P-450 or cytochrome b5 from the system led to complete loss of the activity. The reconstituted activity was sensitive to carbon monoxide, metyrapone, phenyl isocyanide, and cyanide, indicating that the cytochrome P-450 used is cyanide-sensitive and is involved in the catalytic process. The maximal demethylase activity was attained when the system contained cytochrome P-450 and cytochrome b5 at a 1 : 1 molar ratio. Trypsin digestion of cytochrome b5 abolished the capacity of this cytochrome to reconstitute the demethylase activity. These results suggest that O-demethylation of p-nitroanisole by this particular form of cytochrome P-450 absolutely requires the intact form of cytochrome b5 and that the second electron needed for the demethylation may be donated to the cytochrome P-450 only by way of cytochrome b5.