Premium
Preliminary Investigation by NMR of the Electron Transfer Complex Between the Vitamin D Metabolizing Cytochrome P450 24A1 and Adrenodoxin
Author(s) -
Estrada D. Fernando,
Tu Youbin
Publication year - 2017
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.31.1_supplement.lb112
Subject(s) - adrenodoxin , chemistry , cytochrome p450 , cyp24a1 , vitamin d and neurology , enzyme , hydroxylation , biochemistry , stereochemistry , calcitriol receptor , biology , endocrinology , receptor
Background Deactivation of the bio‐activated form of vitamin D (1,25‐dihydroxy vitamin D3) represents a key metabolic step that helps determine the bioavailability of the active hormone. Deactivation is accomplished by hydroxylation of carbon 23 or carbon 24 of the vitamin D side chain as mediated by the mitochondrial cytochrome P450 enzyme CYP24A1. CYP24A1 belongs to a sub‐set of P450 enzymes that function in the mitochondrial membrane, where interactions with the soluble [2FE‐2S] cluster protein adrenodoxin (Adx) triggers the electron transfer required for P450 catalysis. Adx also serves as the principle redox partner for all other mitochondrial P450 enzymes and their respective reactions, including the activation of vitamin D by CYP27B1. In light of the importance of this electron transfer step in activation and deactivation of vitamin D, there remain basic questions regarding the formation of the Adx‐P450 enzyme complex. Among these are 1) the interface of the protein complex has yet to be mapped out for a vitamin D metabolizing P450, and 2) it is unknown whether Adx binding influences other regions of CYP24A1 that relate to substrate recognition and access. Objective In this preliminary study, we have set out to characterize the Adx‐CYP24A1 complex via a combination of spectrophotometric and nuclear magnetic resonance techniques. Results Our spectral CYP24A1‐ligand binding data indicate that the affinity between rat CYP24A1 and the vitamin D analog alfacalcidol is strengthened by approximately two‐fold in the presence of adrenodoxin, thereby suggesting a long‐range effect between the putative Adx binding surface of CYP24A1 and the canonical P450 substrate recognition and binding sites. Additionally, we have also optimized the Adx‐CYP24A1 complex for analysis by solution NMR, in which titration with CYP24A1 induces differential line broadening of the spectrum of 15 N labeled Adx, with enhanced line broadening occurring in the short α‐helix adjacent to the [2FE‐2S] cluster. Conclusions The interaction of Adx with CYP24A1 induces a change in ligand binding, leading to greater affinity for alfacalcidol. Additionally, line broadening of residues in the short α‐helix adjacent to the [2FE‐2S] cluster is suggestive of participation of this region in formation of the complex with CYP24A1. These preliminary studies lay the groundwork for a comprehensive study of the Adx‐CYP24A1 complex under a variety of conditions, including a comparison between the oxidized and reduced states of Adx, and thereby providing insight toward this important step in vitamin D metabolism. Support or Funding Information This work was funded in combination by R00GM112862 and SUNY Buffalo startup funds.