Cytochrome b(5) has been shown to Stimulate, inhibit or have no effect on catalysis by P450 cytochromes. Its action is known to depend on the isozyme of cytochrome P450, the Substrate, and experimental conditions. Cytochrome P450 2134 (CYP 2134) has been used in our laboratory as a model isozyme to study the role of cytochrome b(5) in cytochrome P450 catalysis using two substrates, methoxyflurane and bertzplictarnine. One substrate is the volatile anesthetic, methoxyflurane, whose metabolism is consistently markedly stimulated by cytochrome b(5). The other is benzphetamine, whose metabolism is minimally modified by cytochrome b(5). Determination of the stoichiometry of the metabolism of both substrates showed that the amount of product formed is the net result of the simultaneous stimulatory and inhibitory actions of cytochrome b(5) on catalysis. Site-directed mutagenesis Studies revealed that both cytochrome b(5) and cytochrome P450 reductase interact with cytochrome P450 on its proximal surface on overlapping but non-identical binding sites. Comparison of the rate of reduction of oxyferrous CYP 2134 and the rate of substrate oxidation by cyt b(5) and reductase with stopped-flow spectrophotometric and rapid chemical quench experiments has demonstrated that although cytochrome b(5) and reductase reduce oxyferrous CYP 2134 at the same rate, substrate oxidation proceeds more slowly in the presence of the reductase. (c) 2005 Elsevier Inc. All rights reserved.