The novel purification and biochemical characterization of a reversible CYP24A1:adrenodoxin complex

▸ We developed a novel method for purifying mitochondrial P450s in complex with their redox partner protein Adx, by exploiting their unique biological interaction. ▸ A truncated form of rat CYP24A1 (Δ51) displayed enhanced ligand binding properties when in complex with Adx, hinting at a role for the N-terminus in substrate binding. ▸ Adx binding to CYP24A1, reduced substrate binding affinity over 9-fold, but stabilized the formation of the enzyme:substrate complex. ▸ Common crystallization detergents (e.g. CHAPS) were found to inhibit ligand binding to the CYP24A1:Adx complex at concentrations well below their reported CMC values.