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Publication Detail

Title: Amphipol-facilitated elucidation of the functional tetrameric complex of full-length cytochrome P450 CYP2B4 and NADPH-cytochrome P450 oxidoreductase.

Authors: Cheng, Shen; Bo, Zhiyuan; Hollenberg, Paul; Osawa, Yoichi; Zhang, Haoming

Published In J Biol Chem, (2021 Jan-Jun)

Abstract: Interactions of membrane-bound mammalian cytochromes P450 (CYPs) with NADPH-cytochrome P450 oxidoreductase (POR), which are required for metabolism of xenobiotics, are facilitated by membrane lipids. A variety of membrane mimetics, such as phospholipid liposomes and nanodiscs, have been used to simulate the membrane to form catalytically active CYP:POR complexes. However, the exact mechanism(s) of these interactions are unclear because of the absence of structural information of full-length mammalian CYP:POR complexes in membranes. Herein, we report the use of amphipols (APols) to form a fully functional, soluble, homogeneous preparation of full-length CYP:POR complexes amenable to biochemical and structural study. Incorporation of CYP2B4 and POR into APols resulted in a CYP2B4:POR complex with a stoichiometry of 1:1, which was fully functional in demethylating benzphetamine at a turnover rate of 37.7 ± 2.2 min-1, with a coupling efficiency of 40%. Interestingly, the stable complex had a molecular weight (Mw) of 338 ± 22 kDa determined by multiangle light scattering, suggestive of a tetrameric complex of 2CYP2B4:2POR embedded in one APol nanoparticle. Moreover, negative stain electron microscopy (EM) validated the homogeneity of the complex and allowed us to generate a three-dimensional EM map and model consistent with the tetramer observed in solution. This first report of the full-length mammalian CYP:POR complex by transmission EM not only reveals the architecture that facilitates electron transfer but also highlights a potential use of APols in biochemical and structural studies of functional CYP complexes with redox partners.

PubMed ID: 33839156 Exiting the NIEHS site

MeSH Terms: Animals; Aryl Hydrocarbon Hydroxylases/chemistry; Aryl Hydrocarbon Hydroxylases/metabolism*; Catalysis; Cytochrome P450 Family 2/chemistry; Cytochrome P450 Family 2/metabolism; NADPH-Ferrihemoprotein Reductase/chemistry; NADPH-Ferrihemoprotein Reductase/metabolism*; Polymers/metabolism*; Propylamines/metabolism*; Protein Binding; Protein Conformation; Protein Multimerization; Rabbits

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