Characterization of the non-heme iron center of cysteamine dioxygenase and its interaction with substrates
Characterization of the non-heme iron center of cysteamine dioxygenase and its interaction with substrates
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半胱胺双加氧酶非血红素铁中心的表征及其与底物的相互作用
DOI：
10.1074/jbc.ra120.013915
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发表时间：
2020
影响因子：
4.8
通讯作者：
Liu, Aimin
中科院分区：
文献类型：
--
作者：
Wang, Yifan;Davis, Ian;Chen, Yan;Naik, Sunil G.;Griffith, Wendell P.;Liu, Aimin
半胱胺双加氧酶（ADO）具有两种不同的生物学功能。它催化硫代谢中的半胱胺和N-末端含半胱氨酸的蛋白质或肽的氧化，例如G蛋白信号传导调节因子5（RGS 5）。因此，它在各种生理过程中保持氧稳态。然而，很少有人知道它的催化中心，以及它如何与这两种类型的主要底物，除了O2。在这里，使用电子顺磁共振（EPR），穆斯堡尔和紫外可见光谱，我们探讨了半胱胺和RGS 5的结合模式，人类和小鼠ADO蛋白在其生理相关的亚铁形式。这种表征表明，在一氧化氮作为自旋探针和氧替代物的存在下，小分子和肽底物都以单齿结合模式与其游离巯基协调铁中心，与在其他巯基双加氧酶中观察到的结合行为形成鲜明对比。我们观察到一个底物结合的B-型二亚硝酰铁中心复合物在ADO，这表明的可能性，双氧结合的铁离子在侧上的模式。此外，我们观察到基板介导的还原铁中心从三价铁的亚铁氧化态。随后的MS分析表明底物形成了相应的二硫键，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了解ADO中的底物相互作用，并填补了我们对硫醇双加氧酶底物特异性知识的空白。
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.