Thiol-disulfide redox dependence of heme binding and heme ligand switching in nuclear hormone receptor rev-erb{beta}

Abstract

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.

FIGURE 1.

Redox-dependent binding of Fe³⁺-heme to Rev-erbβ LBD. Difference absorption spectra and titration curves of oxidized (0. 4 μm) (A and B, respectively) and reduced (0.3 μm) (C and D, respectively) Rev-erbβ LBD are shown. The titrations were performed in Buffer B (see “Experimental Procedures”). Titration of reduced Rev-erbβ LBD with Fe³⁺-heme was performed anaerobically to avoid thiol-oxidation during the experiment. The lines in B and D were generated from fits to Equation 1.

FIGURE 2.

Oxidation of cysteines in oxidized Rev-erbβ LBD. OxICAT analysis of the redox state of purified and oxidized Rev-erbβ LBD reveals incorporation of heavy ICAT (¹³C) in Cys-384 (top panel), Cys-374 (middle panel), and Cys355 (bottom panel) in their respective peptides, which demonstrates that these cysteines are oxidized upon oxidation of the protein.

FIGURE 3.

Loss of redox dependence of binding of Fe³⁺-heme to the C374S variant. Fe³⁺-heme titration curves of oxidized (A, 0. 3 μm) and reduced (B, 0.2 μm) C374S variants. The titrations were performed in Buffer B (see “Experimental Procedures”). Titration of reduced Rev-erbβ LBD variant with Fe³⁺-heme was performed anaerobically to avoid thiol-oxidation during the experiment. The lines were generated from fits to Equation 1.

FIGURE 4.

Ligand switching associated with a change in redox state of Rev-erbβ LBD. A, EPR spectroscopic analysis of heme complexes with the reduced (Red) and oxidized (Ox) proteins (complexes were prepared at a ratio of 1:1.5 of Fe³⁺-heme:LBD). B, EPR analysis of E. coli cells overexpressing Rev-erbβ LBD (without or with oxidation by 30 mm diamide (DA) for 4 and 8 h). Inset, Western blot analysis of the EPR samples from the diamide treatment at 0, 4, and 8 h using anti-pentahistidine antibody. C, EPR analysis of C384A-heme and C374S-heme complexes as in A. Note that the in vitro experiments were performed in Buffer B. The minor peaks at g values of 2.0 and 1.99 are from the buffer.

FIGURE 5.

Similar affinity of reduced Rev-erbβ LBD toward Fe³⁺- and Fe²⁺-heme. The Rev-erbβ LBD (0. 3 μm) was anaerobically titrated with Fe²⁺-heme in Buffer B in the presence of 2.5 mm dithionite. The Soret peak for Fe²⁺-heme is at 428 nm and exhibits sharp alpha and beta peaks at 560 and 530 nm, respectively (A). The K_d values of the complexes of Fe³⁺- and Fe²⁺-heme with reduced Rev-erbβ LBD are 23 ± 2.7 nm (Fig. 1D) and 15.8 ± 4.1 nm (Fig. 5B), respectively. The line in B was generated from fits to Equation 1.

FIGURE 6.

High affinity of CO toward Fe²⁺ heme- Rev-erbβ LBD complex. Difference spectra following the binding of CO to the Fe²⁺-heme complex with the Rev-erbβ LBD. Inset, fitting the binding isotherm for the CO titration yielded a K_d value of 60 ± 15 nm. The CO titration was performed anaerobically in Buffer B in the presence of 2.5 mm dithionite with 2 μm Fe²⁺-heme-LBD complex, as described under “Experimental Procedures.” The line shown in the inset was generated from a fit to Equation 1.

FIGURE 7.

Model for redox modulation of heme binding and heme ligand switching in Rev-erbβ. Ferric heme is ligated via Cys-384 and His-568 in the ligand-binding domain of the thiol-reduced form of Rev-erbβ. Disulfides, formed after the oxidation of the protein, change the conformation of the protein, which results in His/neutral residue ligation (His/His or His/Met or His/Lys) of the heme. Formation of the disulfide bond between Cys-374 and Cys-384 interferes with heme binding. The LBD can bind ferrous heme with similar affinity to the ferric heme. In this state, Cys-384 appears to be replaced by a neutral ligand, suggested to be Met (45, 46). CO binds tightly to the ferrous state of Rev-erbβ, replacing the sulfur ligation from Cys-384.