Nuclear Receptors Homo sapiens Rev-erbβ and Drosophila melanogaster E75 Are Thiolate-Ligated Heme Proteins Which Undergo Redox-Mediated Ligand Switching and Bind CO and NO

Abstract

Nuclear receptors E75, which regulates development in Drosophila melanogaster, and Rev-erbbeta, which regulates circadian rhythm in humans, bind heme within their ligand binding domains (LBD). The heme-bound ligand binding domains of E75 and Rev-erbbeta were studied using electronic absorption, MCD, resonance Raman, and EPR spectroscopies. Both proteins undergo redox-dependent ligand switching and CO- and NO-induced ligand displacement. In the Fe(III) oxidation state, the nuclear receptor hemes are low spin and 6-coordinate with cysteine(thiolate) as one of the two axial heme ligands. The sixth ligand is a neutral donor, presumably histidine. When the heme is reduced to the Fe(II) oxidation state, the cysteine(thiolate) is replaced by a different neutral donor ligand, whose identity is not known. CO binds to the Fe(II) heme in both E75(LBD) and Rev-erbbeta(LBD) opposite a sixth neutral ligand, plausibly the same histidine that served as the sixth ligand in the Fe(III) state. NO binds to the heme of both proteins; however, the NO-heme is 5-coordinate in E75 and 6-coordinate in Rev-erbbeta. These nuclear receptors exhibit coordination characteristics that are similar to other known redox and gas sensors, suggesting that E75 and Rev-erbbeta may function in heme-, redox-, or gas-regulated control of cellular function.