Abstract
Heme oxygenase 1 (HO-1) and the cytochromes P450 (P450s) are endoplasmic reticulum–bound enzymes that rely on the same protein, NADPH-cytochrome P450 reductase (POR), to provide the electrons necessary for substrate metabolism. Although the HO-1 and P450 systems are interconnected owing to their common electron donor, they generally have been studied separately. As the expressions of both HO-1 and P450s are affected by xenobiotic exposure, changes in HO-1 expression can potentially affect P450 function and, conversely, changes in P450 expression can influence HO-1. The goal of this study was to examine interactions between the P450 and HO-1 systems. Using bioluminescence resonance energy transfer (BRET), HO-1 formed HO-1•P450 complexes with CYP1A2, CYP1A1, and CYP2D6, but not all P450s. Studies then focused on the HO-1–CYP1A2 interaction. CYP1A2 formed a physical complex with HO-1 that was stable in the presence of POR. As expected, both HO-1 and CYP1A2 formed BRET-detectable complexes with POR. The POR•CYP1A2 complex was readily disrupted by the addition of HO-1, whereas the POR•HO-1 complex was not significantly affected by the addition of CYP1A2. Interestingly, enzyme activities did not follow this pattern. BRET data suggested substantial inhibition of CYP1A2-mediated 7-ethoxyresorufin de-ethylation in the presence of HO-1, whereas its activity was actually stimulated at subsaturating POR. In contrast, HO-1–mediated heme metabolism was inhibited at subsaturating POR. These results indicate that HO-1 and CYP1A2 form a stable complex and have mutual effects on the catalytic behavior of both proteins that cannot be explained by a simple competition for POR.
Highlights
Cytochrome P450 (EC 1.14.14.1) is a heme-containing, membrane-bound protein that catalyzes the oxygenation of a wide variety of exogenous and endogenous compounds [1, 2]
In an effort to determine if interactions between different P450s and Heme oxygenase 1 (HO-1) were selective for the individual enzymes, an initial search was performed with different green fluorescent protein (GFP)–tagged P450s and Rluc-tagged HO-1
Higher bioluminescence resonance energy transfer (BRET) signals were observed where HO-1 was paired with CYP1A1, CYP2D6, and CYP1A2, with much weaker responses observed with CYP2A6 and CYP2C9 (Fig. 1)
Summary
Cytochrome P450 (EC 1.14.14.1) is a heme-containing, membrane-bound protein that catalyzes the oxygenation of a wide variety of exogenous and endogenous compounds [1, 2]. Heme oxygenase 1 (EC 1.14.14.18; HO-1) is responsible for the first step of heme degradation, converting the substrate to biliverdin [5, 6] This enzyme protects cells from oxidative damage and is induced by agents that are related to oxidative stress, including many xenobiotics, such as aspirin, statins, niacin, etc. The HO-1 and P450 systems are interconnected by their common electron donor, the systems generally have been studied separately. The existence of both physical and functional P450P450 interactions has been well established, whereas potential interactions between HO-1 and the P450s have attracted relatively little attention. The results show that HO-1 and CYP1A2 form a stable complex both in reconstituted systems and in natural membranes, suggesting that the change in both CYP1A2 and HO-1 function is the result of this complex
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