The Cytochrome P450 24A1 Interaction with Adrenodoxin Modulates Substrate Binding and Relies on Species‐variable Recognition Sites

Abstract

Redox partner and accessory protein interactions are known to modulate function in mammalian cytochrome P450 enzymes by exerting a level of allosteric control, most notably demonstrated by the interaction of cytochrome b5 with P450 enzymes in the endoplasmic reticulum. However, in the inner mitochondrial membrane, in which P450 electron transfer occurs solely via interaction with the ferredoxin protein adrenodoxin (Adx), the events that modulate P450 function are not clear. In this study, we report the use of combined spectroscopic techniques to demonstrate that Adx serves to modulate substrate binding in the mitochondrial vitamin D metabolizing enzyme CYP24A1. The presence of equal molar amounts of Adx potentiates substrate binding for 25(OH)D3 and 1,24,25(OH)3D3, but not for the primary substrate 1,25(OH)2D3. We follow up on the CYP24A1‐Adx interaction by utilizing solution nuclear magnetic resonance (NMR) spectroscopy to monitor the spectra of uniformly labeled 15N Adx while titrating with unlabeled CYP24A1 bound to the P450 inhibitor clotrimazole. The pattern of NMR line broadening, combined with single and double mutants of Adx, suggest the presence of multiple recognition sites between the proteins. In order to evaluate redox partner complexes for CYP24A1 isoforms displaying distinct regioselectivity for vitamin D, all of the binding data were acquired in parallel for a carbon 23 hydroxylase, a carbon 24 hydroxylase, and a combined carbon 23/24 hydroxylase. Interestingly, disruption of the CYP24A1‐Adx complex in response to Adx mutagenesis was variable between CYP24A1 isoforms, suggesting a correlation between redox partner binding and regioselectivity and consistent with the ability of Adx to modulate substrate binding. Therefore, this work provides structural evidence in support of Adx modulation of function in a mitochondrial P450 enzyme.Support or Funding InformationThis work was supported by R00GM112862 and the State University of New YorkThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.