The cytochrome P450 24A1 interaction with adrenodoxin relies on multiple recognition sites that vary among species

Abstract

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) 2 D3. 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) 2 D3, 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.Mitochondrial cytochromes P450 (P450) are responsible for a host of biological reactions, including steps necessary in steroid and vitamin D metabolism. Similar to microsomal P450s, their function requires the transfer of two electrons, delivered sequentially, in order to generate the active oxidizing species necessary for completion of one cycle of P450 catalysis (1). However, in contrast to microsomal P450 enzymes, for which reduction of P450 can be achieved by binding either a P450 oxidoreductase or, in some cases, the small heme protein cytochrome b 5 , mitochondrial enzymes rely entirely on formation of a transient complex with the soluble ferredoxin protein, Adrenodoxin (Adx). Adx folds into a compact structure consisting of three short α-helices and five anti-parallel β strands, with a [2Fe-2S] cluster coordinated near solvent accessible surfaces between helix-1 and helix-3 (2,3). Changes in the C-terminal region of bovine Adx have been shown to participate in mediating a monomer to dimer transition in response to reduction of the [2Fe-2S] cluster (2), thereby suggesting that dimers of Adx are functionally relevant.While the prevailing evidence suggests that complex formation between Adx with P450 relies on salt bridge interactions between the side chains of acidic residues on helix-3 (Asp-72 - Cytoch...

Increased Phenacetin Oxidation upon the L382V Substitution in Cytochrome P450 1A2 is Associated with Altered Substrate Binding Orientation

“…Except for the –OCH 2 – group, the substrate proton-heme iron distances for CYP1A2 WT, L382V, N312L, and L382V/N312L were very similar, ranging from 6.1 to 8.2 Å. These values are similar to the distances between hydroxylation sites and the heme iron reported for other P450 enzymes [ 15 , 17 , 22 , 23 , 24 , 25 , 26 ]. The distance between the protons of the –OCH 2 – group and heme iron in CYP1A2 N312L was about 7.84 Å, which is much longer than that in CYP1A2 WT (6.55 Å).…”

Surface hydrophobics mediate functional dimerization of CYP121A1 of Mycobacterium tuberculosis

“…Expression and purification of WT and mutant CYP121A1 is described further under Supplementary data . Expression and purification of bovine adrenodoxin reductase and bovine adrenodoxin used in the activity assays were carried out as described previously 33 , 34 .…”

“…Affinity constants were calculated by fitting the ligand concentrations plotted against the difference spectra showing either a red-shifted, type-I response (cYY) or a blue-shifted, type-II response (ketoconazole and imidazole). Data fitting was carried out using Prism GraphPad v7.05 using a single binding mode equation for hyperbolic binding as described previously 33 . Carbon monoxide (CO) binding spectra were performed by first reducing samples of CYP121A1 with excess sodium dithionite, followed by bubbling of CO gas and immediately recording spectra between 250 and 700 nm at regular intervals of one scan every 5–10 min for one hour.…”

Evidence of Allosteric Coupling between Substrate Binding and Adx Recognition in the Vitamin D Carbon-24 Hydroxylase CYP24A1

“…This hypothesis is supported by two of our previous studies. First, we utilized protein nuclear magnetic resonance (NMR) to demonstrate that CYP24A1 enzymes from species that are known to display distinctive substrate regioselectivity also rely on different secondary interactions to bind Adx . Subsequently, we also demonstrated that in a carbon-24 hydroxylase system (from rat), allosteric communication exists between the active site and the proximal Adx recognition surface of CYP24A1 …”