PT - JOURNAL ARTICLE AU - Amit Kumar AU - D. Fernando Estrada TI - Specificity of the redox complex between cytochrome P450 24A1 and adrenodoxin relies on carbon-25 hydroxylation of vitamin-D substrate AID - 10.1124/dmd.119.087759 DP - 2019 Jan 01 TA - Drug Metabolism and Disposition PG - dmd.119.087759 4099 - http://dmd.aspetjournals.org/content/early/2019/07/09/dmd.119.087759.short 4100 - http://dmd.aspetjournals.org/content/early/2019/07/09/dmd.119.087759.full AB - Metabolic deactivation of 1,25(OH)2D3 is initiated by modification of the vitamin-D side chain, as carried out by the mitochondrial cytochrome P450 24A1 (CYP24A1). In addition to its role in endogenous vitamin-D metabolism, CYP24A1 is involved in catabolism of vitamin-D analogs, thereby reducing their efficacy. Like all mitochondrial CYP enzymes, CYP24A1 function relies on electron transfer from the soluble ferredoxin protein adrenodoxin (Adx). Recent structural evidence suggests that regioselectivity of the CYP24A1 reaction may correlate with distinct modes of Adx recognition. In this study, we use nuclear magnetic resonance (NMR) spectroscopy to monitor the structure of 15N labeled full-length Adx from rat while forming the complex with rat CYP24A1 in the ligand-free state or bound to either 1,25(OH)2D3 or the vitamin-D supplement 1α(OH)D3. While both vitamin-D ligands were found to induce a reduction in overall NMR peak broadening, thereby suggesting ligand-induced disruption of the complex, a cross-linking analysis suggests that ligand does not have a significant effect on the relative association affinities of the redox complexes. However, a key observation is that, while the presence of primary CYP24A1 substrate was found to induce NMR peak broadening focused on the putative recognition site α-helix 3 of Adx, the interaction in the presence of 1α(OH)D3, which is lacking the carbon-25 hydroxyl, results in disruption of the NMR peak broadening pattern, thus indicating a ligand-induced non-specific protein interaction. These findings provide a structural basis for the poor substrate turnover of side chain modified vitamin-D analogs, while also confirming that specificity of the CYP24A1-ligand interaction influences specificity of CYP24A1-Adx recognition.SIGNIFICANCE STATEMENT Mitochondrial cytochrome P450 enzymes, such as CYP24A1 responsible for catabolizing vitamin-D and its analogs, rely on a protein-protein interaction with a ferredoxin in order to receive delivery of the electrons required for catalysis. In this study, we demonstrate that this protein interaction is influenced by the enzyme-ligand interaction that precedes it. Specifically, vitamin-D missing carbon-25 hydroxylation binds the enzyme active site with high affinity, but results in a loss of CYP-ferredoxin binding specificity.