Tight binding of cytochrome b to cytochrome P450 17A1 is a critical feature of stimulation of C21 steroid lyase activity and androgen synthesis.

It has been recognized for >50 years that cytochrome b (b) stimulates some cytochrome P450 (P450)-catalyzed oxidations, but the basis of this function is still not understood well. The strongest stimulation of catalytic activity by b is in the P450 17A1 lyase reaction, an essential step in androgen synthesis from 21-carbon (C21) steroids, making this an excellent model system to interrogate b function. One of the issues in studying b-P450 interactions has been the limited solution assay methods. We constructed a fluorescently labeled variant of human b that can be used in titrations. The labeled b bound to WT P450 17A1 with a K of 2.5 nM and rapid kinetics, on the order of 1 s. Only weak binding was observed with the clinical P450 17A1 variants E305G, R347H, and R358Q; these mutants are deficient in lyase activity, which has been hypothesized to be due to attenuated b binding. K values were not affected by the presence of P450 17A1 substrates. A peptide containing the P450 17A1 Arg-347/Arg-358 region attenuated Alexa 488-T70C-b fluorescence at higher concentrations. The addition of NADPH-P450 reductase (POR) to an Alexa 488-T70C-b:P450 17A1 complex resulted in a concentration-dependent partial restoration of b fluorescence, indicative of a ternary P450:b:POR complex, which was also supported by gel filtration experiments. Overall, these results are interpreted in the context of a dynamic and tight P450 17A1:b complex that also binds POR to form a catalytically competent ternary complex, and variants that disrupt this interaction have low catalytic activity.