Abstract

Cytochrome P450 11B1 (CYP11B1), or 11β-hydroxylase, is a critical mitochondrial membrane-bound enzyme in the adrenal cortex responsible for the final step in cortisol biosynthesis. Its catalytic activity is dependent on the transfer of electrons from the soluble iron-sulfur protein Adrenodoxin (Adx). Understanding the precise molecular mechanism and kinetics of the Adx-CYP11B1 interaction is fundamental to comprehending the regulation of steroidogenesis. In this study, we employed Surface Plasmon Resonance (SPR) analysis to quantitatively investigate the binding kinetics of Adx to CYP11B1 embedded within a model lipid bilayer, closely mimicking the native mitochondrial membrane environment. This approach allowed for the direct measurement of the protein-protein interaction in a physiologically relevant context, overcoming limitations of studies conducted in detergent micelles or aqueous solution. Our results demonstrate a high-affinity interaction between Adx and membrane-anchored CYP11B1, characterized by a dissociation constant ($K_D$) in the nanomolar range. Furthermore, the kinetic analysis revealed a rapid association rate ($k_{on}$) and a slower dissociation rate ($k_{off}$), suggesting a stable, transient complex formation essential for efficient electron transfer. These findings provide crucial quantitative data on the membrane-dependent partnership between Adx and CYP11B1, offering structural and functional insights vital for the development of targeted therapeutics for steroid-related disorders.