Stopped-flow spectrophotometric measurements were carried out by the rate of cytochrome P-450 reduction by NADPH in a reconstituted system consisting of NADPH-cytochrome c (P-450) reductase and cytochrome P-450 purified from phenobarbital-induced rat and rabbit liver microsomes, respectively. The reduction rate was increased by the addition of hydroxylatable substrates and the apparent first-order rate constant of the reduction in the presence of 1 mM benzphetamine was determined to be 4-12 s−1, a value which corresponds to a potential rate of 240–720 nmol per min per nmol of cytochrome P-450. The rate constant of the binding of benzphetamine to cytochrome P-450 was also measured by stopped-flow spectrophotometry and found to be of the order of 50 s−1. The rate constants determined for the two partial reactions are much higher than the overall rate of benzphetamine-dependent NADPH oxidation of 50–70 nmol per min per nmol of cytochrome P-450. A difference spectrum attributable to an oxygenated form of ferrous cytochrome P-450 was observed during the steady state of the benzphetamine-dependent NADPH oxidation. It is concluded from these and other lines of evidence that the rate-limiting step of benzphetamine N-demethylation by the reconstituted system is at or after the introduction of the second electron. Although the reduction of cytochrome P-450 is not rate-limiting, it was affected in parallel with the overall NADPH oxidation in response to various substrates, pH, and benzphetamine concentration, suggesting that substrate binding to cytochrome P-450 causes parallel enhancement of the cytochrome reduction and the rate-limiting step of the overall process.
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Imai et al. (1977) studied this question.