The heme-regulatory motif of nuclear receptor Rev-erbβ is a key mediator of heme and redox signaling in circadian rhythm maintenance and metabolism.

Rev-erbβ is a heme-responsive transcription factor that regulates genes involved in circadian rhythm maintenance and metabolism, effectively bridging these critical cellular processes. Heme binding to Rev-erbβ indirectly facilitates its interaction with the nuclear receptor co-repressor (NCoR1), resulting in repression of Rev-erbβ target genes. Fe3+-heme binds in a 6-coordinate complex with axial His and Cys ligands, the latter provided by a heme-regulatory motif (HRM). Rev-erbβ was thought to be a heme sensor based on a weak Kd value for the Rev-erbβ·heme complex of 2 μm determined with isothermal titration calorimetry. However, our group demonstrated with UV-visible difference titrations that the Kd value is in the low nanomolar range, and the Fe3+-heme off-rate is on the order of 10-6 s-1 making Rev-erbβ ineffective as a sensor of Fe3+-heme. In this study, we dissected the kinetics of heme binding to Rev-erbβ and provided a Kd for Fe3+-heme of ∼0.1 nm Loss of the HRM axial thiolate via redox processes, including oxidation to a disulfide with a neighboring cysteine or dissociation upon reduction of Fe3+- to Fe2+-heme, decreased binding affinity by >20-fold. Furthermore, as measured in a co-immunoprecipitation assay, substitution of the His or Cys heme ligands in Rev-erbβ was accompanied by a significant loss of NCoR1 binding. These results demonstrate the importance of the Rev-erbβ HRM in regulating interactions with heme and NCoR1 and advance our understanding of how signaling through HRMs affects the major cellular processes of circadian rhythm maintenance and metabolism.