Heme-, Redox-, and CO-dependent Regulation of Heme Homeostasis

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Heme oxygenases (HO2 and HO2) and Rev-Erbβ play critical roles in cytoprotection and in heme-dependent CO metabolism and signaling. HO2 and Rev-Erbβ exhibit similarities in their modes of heme and thiol-disulfide redox regulation via their heme responsive motifs (HRMs). HO1 and HO2 are the sole heme degrading systems in mammals. The HOs are cytoprotective, catalyzing heme degradation to avert toxic heme buildup (>1 µM). In addition, HO produces both the cytoprotective signaling molecule CO and biliverdin (precursor of the antioxidant, bilirubin). Thus, HO1 and HO2 play a protective role against cardiovascular, renal, and central nervous system pathologies induced by heme/Fe-related oxidative stress. Rev-Erbβ, is a transcriptional repressor, exerting wide control of metabolism, inflammatory responses, and the circadian rhythm. Decreased Rev-Erb levels disrupt lipid homeostasis and cause abnormalities in heart functions like cardiac mitochondrial function, metabolism, signaling, and contractile function. HO2 binds three equivalents of heme, one at its catalytic site and another at each of its two regulatory heme responsive motifs (HRMs), whereas Rev-Erbβ binds a single heme at its HRM. HO1 binds a single heme at its catalytic core, which is highly conserved with HO2. We recently described novel roles for the HO2 catalytic core in regulating cellular heme bioavailability via heme sequestration and committing HO2 to lysosomal degradation under heme deficiency. We will now tackle how HO2 coordinates these processes for achieving cellular heme homeostasis and to determine if the inducible HO1 plays a similar sequestration role in heme bioavailability. We also uncovered novel roles for the HRMs in HO2 in shuttling heme to the catalytic site and in interacting with heme chaperones. We will now address the cellular relevance of these in vitro data and whether the chaperone transfers heme directly to the catalytic core or, as we propose, to the HRM-containing tail of HO2. Having successfully incorporated full-length-HO2 with its membrane-binding region into nanodiscs and liposomes, we will now compare the properties of this most physiologically relevant form of HO in its membrane environment with those of the soluble protein. We recently discovered that the cellular form of heme, as well as the most bioavailable form, is oxidized (in the Fe3+ state). We also identified a coupling mechanism within hemeproteins that drives conversion of their resting Fe3+ state to the Fe2+-CO state. We next intend to elucidate the cellular mechanism of Rev-Erbβ regulation starting from heme loading to carbonylation. Furthermore, we plan to reveal whether cytoprotective CO, which is generated by HO, is transferred locally and directly through a protein-protein complex formed between HO and Rev-Erb or if HO-generated CO travels to target proteins by passive cellular diffusion. Our research will help us to understand HO’s involvement in cellular protection against oxidative stress-induced cardiovascular, renal, and central nervous system pathologies and why dysfunction in Rev-erbβ is associated with disorders in metabolism, circadian rhythm, and inflammation.