Cysteamine dioxygenase (ADO) has been reported to exhibit two distinct biological functions with a nonheme iron center. It catalyzes oxidation of both cysteamine in sulfur metabolism and N-terminal cysteine-containing proteins or peptides, such as regulator of G protein signaling 5 (RGS5). It thereby preserves oxygen homeostasis in a variety of physiological processes. However, little is known about its catalytic center and how it interacts with these two types of primary substrates in addition to O2. Here, using electron paramagnetic resonance (EPR), Mössbauer, and UV-visible spectroscopies, we explored the binding mode of cysteamine and RGS5 to human and mouse ADO proteins in their physiologically relevant ferrous form. This characterization revealed that in the presence of nitric oxide as a spin probe and oxygen surrogate, both the small molecule and the peptide substrates coordinate the iron center with their free thiols in a monodentate binding mode, in sharp contrast to binding behaviors observed in other thiol dioxygenases. We observed a substrate-bound B-type dinitrosyl iron center complex in ADO, suggesting the possibility of dioxygen binding to the iron ion in a side-on mode. Moreover, we observed substrate-mediated reduction of the iron center from ferric to the ferrous oxidation state. Subsequent MS analysis indicated corresponding disulfide formation of the substrates, suggesting that the presence of the substrate could reactivate ADO to defend against oxidative stress. The findings of this work contribute to the understanding of the substrate interaction in ADO and fill a gap in our knowledge of the substrate specificity of thiol dioxygenases.

中文翻译：

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半胱胺双加氧酶的非血红素铁中心的表征及其与底物的相互作用。

据报道，半胱胺双加氧酶 (ADO) 表现出两种不同的生物学功能，具有非血红素铁中心。它催化硫代谢中的半胱胺和含 N 端半胱氨酸的蛋白质或肽（例如 G 蛋白信号传导调节剂 5 (RGS5)）的氧化。从而在各种生理过程中保持氧稳态。然而，人们对其催化中心以及它如何与除 O2 之外的这两种主要底物相互作用知之甚少。在这里，我们使用电子顺磁共振 (EPR)、穆斯堡尔和紫外可见光谱，探索了半胱胺和 RGS5 与生理相关亚铁形式的人和小鼠 ADO 蛋白的结合模式。该表征表明，在存在一氧化氮作为自旋探针和氧替代物的情况下，小分子和肽底物均以单齿结合模式将铁中心与其游离硫醇协调，这与在其他硫醇双加氧酶中观察到的结合行为形成鲜明对比。我们在 ADO 中观察到底物结合的 B 型二亚硝酰基铁中心复合物，表明双氧以侧向模式与铁离子结合的可能性。此外，我们观察到底物介导的铁中心从三价铁还原为亚铁氧化态。随后的 MS 分析表明底物形成了相应的二硫化物，表明底物的存在可以重新激活 ADO 以抵御氧化应激。这项工作的发现有助于理解 ADO 中的底物相互作用，并填补我们对硫醇双加氧酶底物特异性知识的空白。