Redox Partner Adrenodoxin Allosterically Alters the Function of the Human Steroidogenic Cytochrome P450 11B Enzymes

Abstract ID 94134 Poster Board 430 Human cytochrome P450 (CYP) enzymes are a group of heme monooxygenases responsible for a range of biological functions, including the generation of steroid hormones. The CYP11B family synthesizes the major human corticosteroids cortisol, produced by CYP11B1, and aldosterone, produced by CYP11B2. Both enzymes are drug targets for different disease states: Cushing's disease (CYP11B1) and primary aldosteronism (CYP11B2). Selective drug development for either disease has been impeded by the 93% shared sequence identity between the CYP11B enzymes. Further structure/function information is needed to identify differences between CYP11B1 and CYP11B2 and develop drugs that selectively target either enzyme. Previous work has demonstrated that the common redox partner adrenodoxin is a transient but allosteric modulator of both CYP11B enzymes in addition to its role as an electron donor. In equilibrium binding studies, adrenodoxin increases both substrate saturation and affinity, and in steady-state kinetic studies, increasing adrenodoxin concentrations decrease the apparent Km (1,2). The mechanism by which adrenodoxin mediates these allosteric effects for CYP11B enzymes is unknown. Herein, stopped-flow revealed that adrenodoxin increases substrate affinity for both enzymes through a two-pronged effect: accelerating binding and decelerating release. This dual influence supports a complex and dynamic CYP11B conformational change upon adrenodoxin binding. With or without adrenodoxin, substrate binding is monophasic for CYP11B1 and biphasic for CYP11B2. Kinetic modeling experiments verified that CYP11B1 follows a simple two-step substrate binding mechanism, whereas CYP11B2 follows a more complex three-step model which could be either conformational selection or induced fit. Further stopped-flow experiments at high and ligand concentrations produced two pieces of evidence supporting conformational selection. First, experiments with an expanded substrate concentration range demonstrated biphasic binding at maximally high substrate concentrations. Secondly, experiments measuring CYP11B2 substrate dissociation demonstrated that the binding condensed to one phase at very low substrate concentrations. Both are indicative of a conformational selection mechanism. Future directions for this project includes repeating these studies in the more biologically relevant reduced state. References Brixius-Anderko S & Scott EE. JBC, 2021. Loomis CL, Brixius-Anderko S, and Scott EE. JIB, 2022.