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.

Rev-Erbβ 是一种结合昼夜节律、新陈代谢和炎症的核受体。与蛋白质结合的血红素可调节其作为阻遏物的功能、稳定性、与其他蛋白质结合的能力以及其在气体感应中的活性。Rev-Erbβ 与 Fe ³⁺ -血红素的结合比 Fe ²⁺ -血红素更紧密，表明其活性可能受血红素氧化还原状态的调节。然而，血红素氧化还原化学在定义蛋白质的静息状态和功能方面的这一关键作用尚不清楚。我们通过电化学和全细胞电子顺磁共振实验证明 Rev-Erbβ 以 Fe ³⁺形式存在于细胞内，即使在细胞核中不稳定血红素浓度较低的情况下，蛋白质也能充满血红素。然而，在 Fe ³⁺氧化还原状态与 Rev-Erb 作为气体传感器的已知功能相矛盾，教条断言必须是 Fe ²⁺。本文解释了为什么静止的 Fe ³⁺状态与血红素结合和细胞气体感应是一致的。我们表明，CO/NO 的结合引起 Fe ³⁺ /Fe ²⁺对的氧化还原电位显着增加，这是一种 EC 机制的特征，其中血红素的不利电化学还原与高度有利的化学还原相结合。气体结合反应，使还原自发。因此，Fe ³⁺-Rev-Erbβ 仍然载有血红素，对其阻遏物活性至关重要，并且当双原子气体存在时会减少。这项工作对配体触发的氧化还原变化导致构象变化影响其功能或蛋白质间相互作用（例如，NCoR1 和 Rev-Erbβ 之间）的蛋白质具有广泛的意义。这项研究开辟了通过 Rev-Erbβ 的氧化还原变化来调节 CO/NO 介导的昼夜节律的可能性。