Redox partner adrenodoxin induces substrate binding to steroidogenic cytochrome P450 11B2 and 11A1 by promoting a conformational change.

In humans, cytochrome P450 (CYP) 11A1 and 11B2 play crucial roles in the steroidogenic pathway. CYP11A1 is involved in the first step in the steroidogenic pathway, converting cholesterol to pregnenolone, whereas CYP11B2 completes the final step in aldosterone synthesis. Each is a drug target for a distinct disease, and detailed information about enzyme kinetics and function could inform drug development. Both enzymes require the common redox partner adrenodoxin. Adrenodoxin allosterically modulates CYP11B2 function separate from its role in electron transfer. Herein, stopped-flow investigations establish that adrenodoxin is also an allosteric modulator of CYP11A1 substrate binding. For both enzymes, a kinetic modeling approach was used to elucidate the substrate binding mechanisms in the absence and presence of adrenodoxin. This analysis determined that substrate binding to both enzymes is best described by complex, 4-state mechanisms. Substrate 11-deoxycorticosterone binds CYP11B2 primarily through a branched induced fit mechanism, and the presence of adrenodoxin shifts the mechanism to a 4-state closed model containing both induced fit and conformational selection steps. CYP11A1 primarily binds its substrate 20R,22R-dihydroxycholesterol through a 4-state closed mechanism, while increasing adrenodoxin results in enhancement of the conformational selection step. Overall, this demonstrates the complexity of both P450 substrate binding and its fine-tuning by interactions with redox partners.