Title: Characterization of Interactions Among CYP1A2, CYP2B4, and NADPH-cytochrome P450 Reductase: Identification of Specific Protein Complexes.
Authors: Connick, J Patrick; Reed, James R; Backes, Wayne L
Published In Drug Metab Dispos, (2018 03)
Abstract: Cytochromes P450s (P450s) catalyze oxygenation reactions via interactions with their redox partners. However, other proteins, particularly other P450s, also have been shown to form complexes that modulate P450 function. Previous studies showed that CYP1A2 and CYP2B4 form a complex when reconstituted into phospholipid vesicles; however, details of the interactions among the P450s and NADPH-cytochrome P450 reductase (POR) have not been fully characterized. The goal of this study was to examine P450 complex formation in living cells, using bioluminescence resonance energy transfer (BRET). Various pairs of P450 and POR constructs were tagged with either green fluorescent protein or Renilla luciferase, and transfected into human embryonic kidney 293T cells. Complexes were demonstrated by measuring energy transfer between the tags, and disruption of the complex was verified by cotransfection with unlabeled P450-system proteins. CYP1A2 and CYP2B4 formed a stable complex that could not be disrupted by cotransfection of untagged POR. Interactions of both P450s with POR were detected, with untagged CYP1A2 disrupting the POR-CYP2B4 interaction. In contrast, untagged CYP2B4 did not affect the POR-CYP1A2 interaction. These data are consistent with POR preferentially binding to the CYP1A2 moiety of CYP1A2-CYP2B4. BRET-detectable homomeric CYP1A2-CYP1A2 also was detected, and was disrupted by cotransfection of either POR or CYP2B4. Both CYP1A2 and CYP2B4 activities were affected by their coexpression in a manner consistent with formation of the high-affinity POR-CYP1A2-CYP2B4 complex. These findings demonstrate that CYP1A2 and CYP2B4 form a heteromeric POR-CYP1A2-CYP2B4 complex in living cells that has altered catalytic activities relative to the homomeric enzymes.
PubMed ID: 29233819
MeSH Terms: Aryl Hydrocarbon Hydroxylases/metabolism*; Catalysis; Cell Line; Cytochrome P-450 CYP1A2/metabolism*; Cytochrome P-450 Enzyme System/metabolism*; Cytochrome P450 Family 2/metabolism; HEK293 Cells; Humans; NADPH-Ferrihemoprotein Reductase/metabolism*; Oxidation-Reduction; Phospholipids/metabolism; Protein Binding/physiology