Proteomics, modeling, and fluorescence assays delineate cytochrome b residues involved in binding and stimulation of cytochrome P450 17A1 17,20-lyase.

Cytochrome b (b) is known to stimulate some catalytic activities of cytochrome P450 (P450, CYP) enzymes, although mechanisms still need to be defined. The reactions most strongly enhanced by b are the 17,20-lyase reactions of P450 17A1 involved in steroid biosynthesis. We had previously used a fluorescently labeled human b variant (Alexa 488-T70C-b) to characterize human P450 17A1-b interactions, but subsequent proteomic analyses indicated that lysines in b were also modified with Alexa 488 maleimide in addition to Cys-70, due to disulfide dimerization of the T70C mutant. A series of b variants were constructed with Cys replacements for the identified lysine residues and labeled with the dye. Fluorescence attenuation and the function of b in the steroid lyase reaction depended on the modified position. Apo-b (devoid of heme group) studies revealed the lack of involvement of the b heme in the fluorescence attenuation. A structural model of b with P450 17A1 was predicted using AlphaFold-Multimer algorithms/Rosetta docking, based upon the individual structures, which predicted several new contacts not previously reported, that is, interactions of b Glu-48:17A1 Arg-347, b Glu-49:17A1 Arg-449, b Asp-65:17A1 Arg-126, b Asp-65:17A1 Arg-125, and b Glu-61:17A1 Lys-91. Fluorescence polarization assays with two modified b variants yielded K values (for b-P450 17A1) of 120 to 380 nM, the best estimate of binding affinity. We conclude that both monomeric and dimeric b can bind to P450 17A1 and stimulate activity. Results with the mutants indicate that several Lys residues in b are sensitive to the interaction with P450 17A1, including Lys-88 and Lys-91.