Cytochrome b5 Binds Tightly to Several Human Cytochrome P450 Enzymes

Numerous studies have been reported in the past 50-plus years regarding the stimulatory role of cytochrome b(5) (b(5)) in some, but not all, microsomal cytochrome P450 (P450) reactions with drugs and steroids. A missing element in most of these studies has been a sensitive and accurate measure of binding affinities of b(5) with P450s. In the course of work with P450 17A1, we developed a fluorescent derivative of a human b(5) site-directed mutant, Alexa 488T70C-b(5), that could be used in binding assays at sub-mu M concentrations. Alexa 488-T70C-b(5 )bound to human P450s 1A2, 2B6, 2C8, 2C9, 2E1, 2S1, 4A11, 3A4, and 17A1, with estimated K-d values ranging from 2.5 to 61 nM. Only weak binding was detected with P450 2D6, and no fluorescence attenuation was observed with P450 2A6. All of the P450s that bound b(5) have some reported activity stimulation except for P450 2S1. The affinity of P450 3A4 for b(5) was decreased somewhat by the presence of a substrate or inhibitor. The fluorescence of a P450 3A4.Alexa 488-T70C-b(5) complex was partially restored by titration with NADPH-P450 reductase (POR) (K-d,K-apparent 89 nM), suggesting the existence of a ternary P450 3A4-b(5)-POR complex, as observed previously with P450 17A1. Gel filtration evidence was also obtained for this ternary complex with P450 3A4. Overall, the results indicated that the affinity of b(5) for many P450s is very high, and that ternary P450-b(5)-POR complexes are relevant in P450 3A4 reactions as opposed to a shuttle mechanism. SIGNIFICANCE STATEMENT High-affinity binding of cytochrome b(5) (b(5)) (K-d < 100 nM) was observed with many drug-metabolizing cytochrome P450 (P450) enzymes. There is some correlation of binding with reported stimulation, with several exceptions. Evidence is provided for a ternary P450 3A4-b(5)-NADPH-P450 reductase complex.