Ferric heme as a CO/NO sensor in the nuclear receptor Rev-Erbß by coupling gas binding to electron transfer

Abstract

Rev-Erbβ is a nuclear receptor that couples circadian rhythm, metabolism, and inflammation. Heme binding to the protein modulates its function as a repressor, its stability, its ability to bind other proteins, and its activity in gas sensing. Rev-Erbβ binds Fe³⁺-heme more tightly than Fe²⁺-heme, suggesting its activities may be regulated by the heme redox state. Yet, this critical role of heme redox chemistry in defining the protein's resting state and function is unknown. We demonstrate by electrochemical and whole-cell electron paramagnetic resonance experiments that Rev-Erbβ exists in the Fe³⁺ form within the cell allowing the protein to be heme replete even at low concentrations of labile heme in the nucleus. However, being in the Fe³⁺ redox state contradicts Rev-Erb's known function as a gas sensor, which dogma asserts must be Fe²⁺ This paper explains why the resting Fe³⁺ state is congruent both with heme binding and cellular gas sensing. We show that the binding of CO/NO elicits a striking increase in the redox potential of the Fe³⁺/Fe²⁺ couple, characteristic of an EC mechanism in which the unfavorable Electrochemical reduction of heme is coupled to the highly favorable Chemical reaction of gas binding, making the reduction spontaneous. Thus, Fe³⁺-Rev-Erbβ remains heme-loaded, crucial for its repressor activity, and undergoes reduction when diatomic gases are present. This work has broad implications for proteins in which ligand-triggered redox changes cause conformational changes influencing its function or interprotein interactions (e.g., between NCoR1 and Rev-Erbβ). This study opens up the possibility of CO/NO-mediated regulation of the circadian rhythm through redox changes in Rev-Erbβ.

Keywords: Rev-Erb; electro-chemical coupling; heme; redox.

Fig. 1.

Rev-Erbβ LBD exists in Fe³⁺, not Fe²⁺, form in cellulo. (A) Crystal structure of Rev-Erbβ LBD complexed with Fe³⁺-heme (3CQV), ligand-binding pocket depicting the Fe³⁺-heme coordinated by Cys (red) and His (blue). (B) Spectral changes observed during potentiometric titration of Rev-Erbβ LBD (82 μM). (C) Fractional Fe²⁺-protein (calculated from relative absorption at 559 nm) versus applied potential and theoretical Nernst curves for a one-electron redox reaction. Individual data points are presented as mean ± SD. The redox titrations of the LBD are based on intensity changes in the α bands and include three and five datasets with Soret maxima at 422 nm and 427 nm, respectively. (D) EPR spectra of E. coli cells overexpressing Rev-Erbβ LBD without any treatment and after incubation with dithionite (20 mM) and H₂O₂ (20 mM) for 3 min. (E) EPR spectra of E. coli cells overexpressing Rev-Erbβ LBD without any treatment and with a pinch of solid dithionite for 5 min. The g-values for the rhombic feature of the LBD iron (2.47, 2.28, and 1.88), the adventitious iron (4.3), and the HS iron (5.96) are depicted in the EPR spectra. EPR conditions were as follows: temperature, 11 °K; microwave power, 208 µW; microwave frequencies for D, 9.3851 (black), 9.3881 (blue), and 9.3902 (red) GHz; for E, 9.3840 (red) and 9.3873 (blue) GHz; modulation frequency, 100 kHz; modulation amplitude, 7 G; two scans; and 327.68 ms time constant.

Fig. 2.

Redox properties of Rev-Erbβ LBD in presence of CO and NO. Spectral changes observed during potentiometric titration of Rev-Erbβ LBD , with low-potential redox mediators under CO atmosphere. [LBD] = 118.5 μM (A) and with high- and low-potential redox mediators under CO atmosphere. [LBD] = 95.5 μM (B). Gray traces represent absorption at potentials from 159 to 959 mV in A and −11 to 959 mV in B. (C) proposed scheme of CO-mediated Fe³⁺-LBD reduction. (D) Potentiometric titration of LBD with high- and low-potential redox mediators in presence of NO. Gray traces represent absorption at potentials from 189 to 789 mV. LBD = 99.1 μM.

Fig. 3.

Gas-driven reduction of Fe³⁺ Rev-Erbβ by the EC mechanism allowing Fe³⁺-heme to function as a gas sensor. (A) Thermodynamic box depicting redox potentials and gas-binding constants of different redox and gas-bound states of Rev-Erbβ LBD. Red and blue indicate thermodynamic parameters for CO and NO, respectively. E⁰ and E⁰ (Fe^3+/2+-CO/NO) represent the redox potentials of the Fe³⁺/Fe²⁺ couple in absence and presence of CO/NO, respectively. K_d1 and K_d2 represent the binding constants of the Fe²⁺ and Fe³⁺ protein to CO and NO, respectively. K_d1 for CO = 6 nM (14). Coupling of electron transfer with chemical reaction is depicted by the blue and green boxes; (B) Our experiments suggest that Rev-Erbβ predominantly exists in a Fe³⁺-heme–bound state in cells. Under limiting concentrations of intracellular labile heme pools, Fe³⁺, not Fe²⁺, Rev-Erbβ remains in the heme-replete state. Signaling gases such as CO and NO exert a thermodynamic pull that allows the protein to be in the reduced gas-bound form. This intriguing redox chemistry allows the protein to shuffle between Fe³⁺ and Fe²⁺ heme states thereby not limiting Rev-Erb’s function as a gas sensor even when the protein primarily exists in the oxidized form.

Fig. 4.

Effect of CO and NO on the redox state of Rev-Erbβ in cellulo. (A) EPR spectra of E. coli cells overexpressing LBD with and without 20 mM CORM-A1 treatment for variable times (20, 40, and 60 min). Full EPR spectra are shown in SI Appendix, Fig. S7A. EPR conditions were as follows: temperature, 11 °K; microwave power, 20 µW; microwave frequencies, 9.3803, 9.3797, 9.3808, and 9.3795 GHz; modulation frequency, 100 kHz; modulation amplitude, 7 G; two scans; and 327.68 ms time constant. (B) EPR spectra of 1) E. coli cells overexpressing LBD with and without Proline NONOate treatment (10 mM, 6 min), 2) E. coli cells containing empty vector pMCSG9 after Proline NONOate treatment (10 mM, 6 min), and 3) Rev-Erbβ after Proline NONOate addition. EPR conditions were as follows: temperature, 11 °K; microwave power, 20 µW; microwave frequency, 9.3831 and 9.3823 GHz for 1, 9.3840 GHz for 2, and 9.3849 GHz for 3; modulation frequency, 100 kHz; modulation amplitude, 3 G; four scans; and 327.68 ms time constant.