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Physical Interactions between Heme Oxygenase‐1 and the Cytochromes P450 Lead to Complex Changes in Enzyme Activity
Author(s) -
Connick John,
Reed James,
Cawley George,
Backes Wayne
Publication year - 2021
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.2021.35.s1.03022
Subject(s) - heme , cyp1a2 , chemistry , biochemistry , cytochrome p450 , heme oxygenase , cyp2b6 , enzyme , biophysics , homomeric , microbiology and biotechnology , biology , protein subunit , gene
The cytochromes P450 (P450s) catalyze the oxygenation of endogenous and exogenous organic substrates, including most prescribed drugs. Heme oxygenase‐1 (HO‐1) is an inducible enzyme responsible for the rate‐limiting step of heme catabolism. HO‐1 and the P450s are membrane‐bound proteins expressed on the cytosolic face of the endoplasmic reticulum where they must interact with NADPH‐cytochrome P450 reductase (POR) for activity. Because POR levels are often limiting, HO‐1 and the P450s must compete for POR binding in order to function. However, simple competition for POR is often complicated by homomeric and heteromeric P450•P450 interactions. We hypothesized that HO‐1 might participate with the P450s in their competition for POR and that this competition might lead to changes in P450 and/or HO‐1 activity. We were also interested in determining whether HO‐1 could physically interact with P450s and measuring what functional effects any P450•HO‐1 complex might have. Using bioluminescence resonance energy transfer (BRET), we were able to establish that HO‐1 formed physical complexes with CYP1A1, CYP1A2, and CYP2D6 in transiently transfected HEK293T cells. The stability of these complexes was determined by measuring the BRET signal generated by GFP‐ and Renilla luciferase‐tagged P450 and HO‐1 in the presence and absence of untagged POR. The presence of POR did not have a significant effect on the interactions of HO‐1 with CYP1A2 and CYP2D6 but caused an increase in the BRET signal generated by the HO‐1/CYP1A1 pair. Further BRET studies of these three P450s showed that the presence of HO‐1 inhibited the formation of POR•P450 complexes, while, conversely, the presence of untagged P450 caused marginal disruption of POR•HO‐1 complex formation. The differences in the response of the HO‐1•CYP1A1 complex and the HO‐1•CYP1A2 complex to POR indicated the possibility of different functional consequences of these P450/HO‐1 interactions. Activity measurements of purified enzymes in lipid reconstituted systems confirmed that this was the case. CYP1A1 and HO‐1 had large inhibitory effects on each other over a large range of POR concentrations. HO‐1 activity in the presence of CYP1A1 approached that of HO‐1 alone only in the presence of a large excess of POR. The parallel experiments with HO‐1 and CYP1A2 yielded results that differed both qualitatively and quantitatively from those seen with CYP1A1. Like CYP1A1, CYP1A2 caused inhibition of HO‐1 activity, though the effect of CYP1A2 was much milder. The effect of HO‐1 on CYP1A2 was also smaller in magnitude than was observed with CYP1A1, but CYP1A2 activity increased in the presence of HO‐1 and subsaturating POR. In the presence of HO‐1 and excess POR, CYP1A2 activity was slightly inhibited. Kinetic analysis of these results showed that they were inconsistent with a simple model of competition between HO‐1 and P450 for POR.