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Redox partner adrenodoxin alters cytochrome P450 11B1 ligand binding and inhibition.

ABSTRACT: Human cytochrome P450 11B1 (CYP11B1) generation of the major glucocorticoid cortisol requires two electrons delivered sequentially by the iron‑sulfur protein adrenodoxin. While the expected adrenodoxin binding site is on the opposite side of the heme and 15-20 Å away, evidence is provided that adrenodoxin allosterically impacts CYP11B1 ligand binding and catalysis. The presence of adrenodoxin both decreases the dissociation constant (K_d) for substrate binding and increases the proportion of substrate that is bound at saturation. Adrenodoxin additionally decreases the Michaelis-Menten constant for the native substrate. Similar studies with several inhibitors also demonstrate the ability of adrenodoxin to modulate inhibition (IC₅₀ values). Somewhat similar allosterism has recently been observed for the closely related CYP11B2/aldosterone synthase, but there are several marked differences in adrenodoxin effects on the two CYP11B enzymes. Comparison of the sequences and structures of these two CYP11B enzymes helps identify regions likely responsible for the functional differences. The allosteric effects of adrenodoxin on CYP11B enzymes underscore the importance of considering P450/redox partner interactions when evaluating new inhibitors.

SUBMITTER: Loomis CL

PROVIDER: S-EPMC9907956 | biostudies-literature | 2022 Oct

REPOSITORIES: biostudies-literature

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Redox partner adrenodoxin alters cytochrome P450 11B1 ligand binding and inhibition.

Loomis Cara L CL Brixius-Anderko Simone S Scott Emily E EE

Journal of inorganic biochemistry 20220714

Human cytochrome P450 11B1 (CYP11B1) generation of the major glucocorticoid cortisol requires two electrons delivered sequentially by the iron‑sulfur protein adrenodoxin. While the expected adrenodoxin binding site is on the opposite side of the heme and 15-20 Å away, evidence is provided that adrenodoxin allosterically impacts CYP11B1 ligand binding and catalysis. The presence of adrenodoxin both decreases the dissociation constant (K<sub>d</sub>) for substrate binding and increases the proport ...[more]

PMID: 35952394

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Project description: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 vitamin-D metabolism, CYP24A1 is involved in catabolism of vitamin-D analogs, thereby reducing their efficacy. 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. Here we used 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. Although both vitamin-D ligands were found to induce a reduction in overall NMR peak broadening, thereby suggesting ligand-induced disruption of the complex, a crosslinking analysis suggested that ligand does not have a significant effect on the relative association affinities of the redox complexes. However, a key finding is that, whereas the presence of primary CYP24A1 substrate was found to induce NMR peak broadening focused on the putative recognition site α-helix 3 of rat adrenodoxin, 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 nonspecific 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 P450-ferredoxin binding specificity.

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Redox partner adrenodoxin alters cytochrome P450 11B1 ligand binding and inhibition.

Publications

Redox partner adrenodoxin alters cytochrome P450 11B1 ligand binding and inhibition.

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