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.

Understanding the Logistics for the Distribution of Heme in Cells

“…It is worth noting that, while the binding of π-acid ligands like CO is traditionally associated exclusively with heme in its ferrous form, ferric heme has also been shown capable of binding CO/NO. 41 For the purposes of this review, movement of ferric/ferrous heme is presumed to mean heme b .…”

Heme oxygenase-2 (HO-2) binds and buffers labile ferric heme in human embryonic kidney cells

“…For instance, ferric, but not ferrous, heme is required for RNA-binding protein DGCR8 for primary microRNA processing ( 95 ). The nuclear receptor Rev-Erbβ can sense and bind ferric heme, which can then act as a sensor for CO or NO by coupling gas binding to heme reduction ( 96 ). In addition, ferric heme was demonstrated to regulate ATP-dependent potassium channels ( 97 ).…”

“…Although LH is largely oxidized in HEK293 cells, it may vary between different cell types and be highly dynamic and responsive to various stimuli. The Fe(III)/Fe(II) redox couple is linked to a number of factors, including access to certain cellular reductants or oxidants ( 98 ), ligand binding to the heme iron center, for example, CO or NO ( 96 ), and allosteric protein conformational changes ( 99 ). Exactly how these factors conspire to affect steady-state LH oxidation state, its dynamics, and role in metabolism and physiology are not known and may be elucidated by the future development of oxidation-state specific heme sensors or chelators.…”

Heme oxygenase-2 (HO-2) binds and buffers labile heme, which is largely oxidized, in human embryonic kidney cells

“…For instance, ferric, but not ferrous, heme is required for RNA-binding protein DGCR8 for primary microRNA processing (91). The nuclear receptor Rev-Erb can sense and bind ferric heme, which can then act as a sensor for CO or NO by coupling gas binding to heme reduction (92). In addition, ferric heme was demonstrated to regulate ATP dependent potassium channels (93).…”

“…The Fe(III)/Fe(II) redox couple is linked to a number of factors, including access to certain cellular reductants or oxidants (94), ligand binding to the heme iron center, e.g. CO or NO (92), and allosteric protein conformational changes (95). Exactly how these factors conspire to affect steady-state LH oxidation state, its dynamics, and role in metabolism and physiology is not known and may be elucidated by the future development of oxidation-state specific heme sensors or chelators.…”