Physical Organization of Heme Oxygenase 1, NADPH‐Cytochrome P450 Reductase, and the Cytochromes P450 in the Endoplasmic Reticulum

Abstract

NADPH‐Cytochrome P450 reductase (POR) is an endoplasmic reticulum(ER)‐bound enzyme responsible for providing electrons to reactions catalyzed by the cytochromes P450 (P450s) as well as heme oxygenase‐1 (HO‐1). Previous work has revealed that some P450s form physical complexes with each other, leading to changes in their interactions with POR and subsequent modification of P450 activities. Because HO‐1 is expressed in the same location and must compete with the P450s for POR binding, we hypothesized that increased HO‐1 expression could inhibit the POR‐binding of individual P450s and that increased P450 expression could inhibit the POR‐binding of HO‐1. Additionally, HO‐1 has the potential to form homomeric complexes or heteromeric complexes with individual P450s as we have seen previously with the P450s. The goal of this study was to better understand the organization of these proteins, how they might interact in the membrane environment, and to determine if these interactions affected protein function. Bioluminescence resonance energy transfer (BRET) was used to examine these potential protein‐protein interactions. BRET involves expressing a pair of proteins, one with a green fluorescent protein tag and one with a Renilla luciferase tag, in HEK293T cells. Because energy transfer between the two protein tags requires them to be in close proximity, we can determine whether the tagged proteins exist as stable complexes in the cells. Co‐transfecting a third, untagged protein allows us to determine whether this protein destabilizes the complex formed by the tagged proteins. To date, we have determined that HO‐1 forms homomeric complexes that can be disrupted by excess POR, and that HO‐1 appears to form complexes with CYP1A1, CYP1A2, and CYP2D6, but not with CYP2A6 or CYP2C9. At least the HO‐1•CYP1A1 complex appears to be stable in the presence of POR. While further work is required to paint a complete picture of this system, the early results suggest the presence of a previously undescribed network of protein‐protein interactions involving HO‐1, POR, and the P450s.Support or Funding InformationNIH NIEHS ES004344NIH NIEHS ES013648This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.