Role of the Proximal Region of CYP1A2 in the heteromeric P450 complex with POR and Cytochrome-b 5

• Published in: The FASEB journal Vol. 36 Suppl 1; no. S1
• Main Authors: Saha, Aratrika, Reed, J R, Connick, J P, Backes, Wayne L
• Format: Journal Article
• Language: English
• Published: United States 01-05-2022
• ISSN: 0892-6638; 1530-6860
• DOI: 10.1096/fasebj.2022.36.S1.R4071

Previous studies have shown that CYP1A2 is capable of forming a CYP1A2-CYP1A2 homomeric complex that involves the proximal region of the enzyme. Using molecular cloning, specific residues on the proximal face of CYP1A2 (residues L91-K106: P
region, residues P131-S155: P
region and residues T434-K447: P
region) were swapped with homologous residues from CYP2B4 (CYP2B4: T92-S107 for P1, E132-E163 for P2 and D438-R448 for P3). CYP2B4 was chosen as we have evidence that shows that CYP2B4 does not form a homomeric complex in vitro. These constructs were then expressed in HEK-293T cells and bioluminescence resonance energy transfer (BRET) was used to measure complex formation and disruption. The P
region of the proximal face was shown to be involved in the homomeric interaction. Interestingly, when we examined the activity of the P
chimera, lowered activity was observed which suggested that the CYP1A2-POR (NADPH-cytochrome P450 reductase) interaction had been affected. POR interacts with P450s on the proximal face. The activity data suggested that the P
mutation had affected the ability of the chimera to associate with POR. Consequently, BRET competition studies established that the P
region was involved in the CYP1A2-POR complex. Subsequent studies with other residues from the proximal face (regions P
and P
) suggested that these residues were not involved in either the CYP1A2-CYP1A2 homomeric complex or the CYP1A2-POR heteromeric complex. We have extended this analysis to examine the effects of these proximal CYP1A2 modifications on its ability to interact with cytochrome b
. BRET competition studies were performed with wild-type CYP1A2 and wild-type b
(tagged with GFP and Rluc respectively) co-transfected at a high GFP: Rluc ratio with increasing amounts of unlabeled CYP1A2, P
, P
and P
chimera as the competitor. Preliminary data suggests that the unlabeled P
, P
and P
chimera were unable to disrupt the wild-type CYP1A2-b
complex as well as the unlabeled CYP1A2, thereby suggesting that the proximal face of CYP1A2 may be involved in the CYP1A2- b
complex. Studies are underway to establish the role of the proximal face in the CYP1A2-b
complex and to elucidate the interactions between CYP1A2, POR and cytochrome b
.