The cytochrome P450 24A1 interaction with adrenodoxin relies on multiple recognition sites that vary among species.
Mitochondrial cytochromes P450 (P450s) are responsible for important metabolic reactions, including steps involved in steroid and vitamin D metabolism. The mitochondrial P450 24A1 (CYP24A1) is responsible for deactivation of the bioactive form of vitamin D, 1,25(OH)2D3. Its function relies on formation of a P450-redox partner complex with the ferredoxin and electron donor adrenodoxin (Adx). However, very little is known about how the Adx-CYP24A1 complex forms. In this study, we report the results of solution NMR in which we monitor isotopically labeled full-length Adx as it binds CYP24A1 in complex with the P450 inhibitor clotrimazole. The NMR titration data suggested a mode for P450-Adx interactions in which formation of the complex relies on contributions from multiple recognition sites on the Adx core domain, some of which have not previously been reported. To evaluate differences among CYP24A1-Adx complexes from different mammalian species and displaying distinct regioselectivity for 1,25(OH)2D3, all bound spectra were acquired in parallel for human (carbon-23 and -24 hydroxylase), rat (carbon-24 hydroxylase), and opossum (carbon-23 hydroxylase) CYP24A1 isoforms. Binding data from a series of single and double charge-neutralizing substitutions of Adx confirmed that species-specific CYP24A1 isoforms differ in binding to Adx, providing evidence that variations in redox partner interactions correlate with P450 regioselectivity. In summary, these findings reveal that CYP24A1-Adx interactions rely on several recognition sites and that variations in CYP24A1 isoforms modulate formation of the complex, thus providing insight into the variable and complex nature of mitochondrial P450-Adx interactions.
['Estrada|D Fernando|DF|']
D000325:Adrenodoxin D000818:Animals D001665:Binding Sites D002417:Cattle D000072498:Cytochrome P450 Family 24 D006801:Humans D008958:Models, Molecular D016297:Mutagenesis, Site-Directed D009154:Mutation D019906:Nuclear Magnetic Resonance, Biomolecular D009893:Opossums D010084:Oxidation-Reduction D011487:Protein Conformation D051381:Rats D013045:Species Specificity
cytochrome P450 adrenodoxin vitamin D CYP24A1 ferredoxin P450-ferredoxin complex membrane protein mitochondria nuclear magnetic resonance (NMR)