Thiol-disulfide redox dependence of heme binding and heme ligand switching in nuclear hormone receptor rev-erb{beta}.
Rev-erbβ is a heme-binding nuclear hormone receptor that represses a broad spectrum of target genes involved in regulating metabolism, the circadian cycle, and proinflammatory responses. Here, we demonstrate that a thiol-disulfide redox switch controls the interaction between heme and the ligand-binding domain of Rev-erbβ. The reduced dithiol state of Rev-erbβ binds heme 5-fold more tightly than the oxidized disulfide state. By means of site-directed mutagenesis and by UV-visible and EPR spectroscopy, we also show that the ferric heme of reduced (dithiol) Rev-erbβ can undergo a redox-triggered switch from imidazole/thiol ligation (via His-568 and Cys-384, based on a prior crystal structure) to His/neutral residue ligation upon oxidation to the disulfide form. On the other hand, we find that change in the redox state of iron has no effect on heme binding to the ligand-binding domain of the protein. The low dissociation constant for the complex between Fe(3+)- or Fe(2+)-heme and the reduced dithiol state of the protein (K(d) = ∼ 20 nM) is in the range of the intracellular free heme concentration. We also determined that the Fe(2+)-heme bound to the ligand-binding domain of Rev-erbβ has high affinity for CO (K(d) = 60 nM), which replaces one of the internal ligands when bound. We suggest that this thiol-disulfide redox switch is one mechanism by which oxidative stress is linked to circadian and/or metabolic imbalance. Heme dissociation from Rev-erbβ has been shown to derepress the expression of target genes in response to changes in intracellular redox conditions. We propose that oxidative stress leads to oxidation of cysteine(s), thus releasing heme from Rev-erbβ and altering its transcriptional activity.
Department of Biological Chemistry, University of Michigan, Ann Arbor, Michigan 48109-0606, USA.
Nirupama
N
Gupta
Stephen W
SW
Ragsdale
D003545:Cysteine / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D004220:Disulfides / Q000737:chemistry / Q000378:metabolism; D006418:Heme / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D006801:Humans; D007501:Iron / Q000737:chemistry* / Q000378:metabolism; D008024:Ligands; D010084:Oxidation-Reduction; D018384:Oxidative Stress / Q000502:physiology*; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D018160:Receptors, Cytoplasmic and Nuclear / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D012097:Repressor Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D014158:Transcription, Genetic / Q000502:physiology*
D004220:Disulfides; D008024:Ligands; C090977:NR1D2 protein, human; D018160:Receptors, Cytoplasmic and Nuclear; D012097:Repressor Proteins; D006418:Heme; D007501:Iron; D003545:Cysteine