Functional Interactions Between Cytochromes P450 1A2 and 2B4 Require Both Enzymes to Reside in the Same Phospholipid Vesicle: Evidence for Physical Complex Formation

Overview

Journal J Biol Chem

Specialty Biochemistry

Date 2010 Jan 15

PMID 20071338

Citations 30

Authors

James R Reed

Marilyn Eyer

Wayne L Backes

Affiliations

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Abstract

Previous studies have shown that the combined presence of two cytochrome P450 enzymes (P450s) can affect the function of both enzymes, results that are consistent with the formation of heteromeric P450.P450 complexes. The goal of this study was to provide direct evidence for a physical interaction between P450 1A2 (CYP1A2) and P450 2B4 (CYP2B4), by determining if the interactions required both enzymes to reside in the same lipid vesicles. When NADPH-cytochrome P450 reductase (CPR) and a single P450 were incorporated into separate vesicles, extremely slow reduction rates were observed, demonstrating that the enzymes were anchored in the vesicles. Next, several reconstituted systems were prepared: 1) CPR.CYP1A2, 2) CPR.CYP2B4, 3) a mixture of CPR.CYP1A2 vesicles with CPR.CYP2B4 vesicles, and 4) CPR.CYP1A2.CYP2B4 in the same vesicles (ternary system). When in the ternary system, CYP2B4-mediated metabolism was significantly inhibited, and CYP1A2 activities were stimulated by the presence of the alternate P450. In contrast, P450s in separate vesicles were unable to interact. These data demonstrate that P450s must be in the same vesicles to alter metabolism. Additional evidence for a physical interaction among CPR, CYP1A2, and CYP2B4 was provided by cross-linking with bis(sulfosuccinimidyl) suberate. The results showed that after cross-linking, antibody to CYP1A2 was able to co-immunoprecipitate CYP2B4 but only when both proteins were in the same phospholipid vesicles. These results clearly demonstrate that the alterations in P450 function require both P450s to be present in the same vesicles and support a mechanism whereby P450s form a physical complex in the membrane.

Citing Articles

Functional characterization of CYP1 enzymes: Complex formation, membrane localization and function.

Connick J, Reed J, Cawley G, Saha A, Backes W J Inorg Biochem. 2023; 247:112325.

PMID: 37479567 PMC: 10529082. DOI: 10.1016/j.jinorgbio.2023.112325.

The Influence of Lipid Microdomain Heterogeneity on Protein-Protein Interactions: Proteomic Analysis of Co-Immunoprecipitated Binding Partners of P450 1A2 and P450 3A in Rat Liver Microsomes.

Reed J, Guidry J, Eyer M, Backes W Drug Metab Dispos. 2023; 51(9):1196-1206.

PMID: 37349115 PMC: 10449098. DOI: 10.1124/dmd.123.001287.

Identification of the contact region responsible for the formation of the homomeric CYP1A2•CYP1A2 complex.

Saha A, Connick J, Reed J, Lott C, Backes W Biochem J. 2021; 478(11):2163-2178.

PMID: 34032264 PMC: 8522333. DOI: 10.1042/BCJ20210269.

Heteromeric complex formation between human cytochrome P450 CYP1A1 and heme oxygenase-1.

Connick J, Reed J, Cawley G, Backes W Biochem J. 2021; 478(2):377-388.

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Effects of alcohol-induced increase in CYP2E1 content in human liver microsomes on the activity and cooperativity of CYP3A4.

Dangi B, Davydova N, Maldonado M, Abbasi A, Vavilov N, Zgoda V Arch Biochem Biophys. 2020; 698:108677.

PMID: 33197431 PMC: 7856178. DOI: 10.1016/j.abb.2020.108677.

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