Physical Interactions between Heme Oxygenase 1 and the Cytochromes P450 in the Endoplasmic Reticulum Membrane

Abstract

Heme oxygenase 1 (HO‐1) and the cytochromes P450 (P450s) are endoplasmic reticulum (ER)‐bound enzymes that rely on NADPH‐cytochrome P450 reductase (POR) to provide the electrons that fuel their catalytic cycle. Previous work has shown that under normal conditions POR levels are limiting and P450s must compete for POR binding. Our lab and others have also shown that, in addition to simple competition, certain P450s can bind POR as multimeric P450•P450 complexes that are associated with altered P450 activity. Because HO‐1 also relies on physical interaction with POR in the ER, we hypothesized that it too would compete with the P450s for POR binding. Additionally, the prevalence of P450•P450 interactions led us to consider the possibility that HO‐1 might form analogous homomeric (HO‐1•HO‐1) or heteromeric (HO‐1•P450) complexes that could affect POR binding and enzyme activity. To gain insight into how these enzymes are physically organized, we used bioluminescence resonance energy transfer (BRET). We implemented BRET by expressing a pair of tagged proteins, one with a green fluorescent protein (GFP) tag and one with a Renilla luciferase tag, in HEK293T cells. Energy transfer only occurs if the tags are in close proximity, allowing us to detect protein complexes by observing changes in the tag's emission spectrum. We are also able to assess the relative stability of protein complexes by observing how co‐expression of a third, untagged protein affects the BRET signal of the tagged pair. Using this technique, we have determined that HO‐1 forms homomeric complexes that are more stable when POR is limiting. We have also found that HO‐1 forms heteromeric complexes with several P450s including CYP1A1 and CYP2D6. The BRET signal for the HO‐1•CYP2D6 complex was robust in both the presence and absence of co‐transfected untagged POR, showing that the complex is stable when POR is present. Interestingly, the BRET signal for the HO‐1•CYP1A1 complex was low in the absence of POR but increased significantly when cells were co‐transfected with untagged POR. Doubling the amount of POR led to an attenuation of the BRET signal. Current and future work will use BRET to determine whether HO‐1 affects the physical interaction between POR and P450s and, conversely, whether the presence of a P450 can affect the HO‐1•POR complex.Support or Funding InformationNIH ES004344, NIH ES013648, NIH GM123253This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.