Spectroscopic investigation of iron(III) cysteamine dioxygenase in the presence of substrate (analogs): implications for the nature of substrate-bound reaction intermediates,JBIC Journal of Biological Inorganic Chemistry

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Spectroscopic investigation of iron(III) cysteamine dioxygenase in the presence of substrate (analogs): implications for the nature of substrate-bound reaction intermediates
JBIC Journal of Biological Inorganic Chemistry ( IF 2.7 ) Pub Date : 2021-09-27 , DOI: 10.1007/s00775-021-01904-5
Rebeca L Fernandez ₁ , Nicholas D Juntunen ₁ , Brian G Fox ₂ , Thomas C Brunold ₁

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Thiol dioxygenases (TDOs) are a class of metalloenzymes that oxidize various thiol-containing substrates to their corresponding sulfinic acids. Originally established by X-ray crystallography for cysteine dioxygenase (CDO), all TDOs are believed to contain a 3-histidine facial triad that coordinates the necessary Fe(II) cofactor. However, very little additional information is available for cysteamine dioxygenase (ADO), the only other mammalian TDO besides CDO. Previous spectroscopic characterizations revealed that ADO likely binds substrate cysteamine in a monodentate fashion, while a mass spectrometry study provided evidence that a thioether crosslink can form between Cys206 and Tyr208 (mouse ADO numbering). In the present study, we have used electronic absorption and electron paramagnetic resonance (EPR) spectroscopies to investigate the species formed upon incubation of Fe(III)ADO with sulfhydryl-containing substrates and the superoxide surrogates azide and cyanide. Our data reveal that azide is unable to coordinate to cysteamine-bound Fe(III)ADO, suggesting that the Fe(III) center lacks an open coordination site or azide competes with cysteamine for the same binding site. Alternatively, cyanide binds to either cysteamine- or Cys-bound Fe(III)ADO to yield a low-spin (S = 1/2) EPR signal that is distinct from that observed for cyanide/Cys-bound Fe(III)CDO, revealing differences in the active-site pockets between ADO and CDO. Finally, EPR spectra obtained for cyanide/cysteamine adducts of wild-type Fe(III)ADO and its Tyr208Phe variant are superimposable, implying that either an insignificant fraction of as-isolated wild-type enzyme is crosslinked or that formation of the thioether bond has minimal effects on the electronic structure of the iron cofactor.

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中文翻译：

底物（类似物）存在下铁（III）半胱胺双加氧酶的光谱研究：对底物结合反应中间体性质的影响

硫醇双加氧酶 (TDO) 是一类金属酶，可将各种含硫醇底物氧化为其相应的亚磺酸。最初通过半胱氨酸双加氧酶 (CDO) 的 X 射线晶体学建立，所有 TDO 都被认为含有协调必要的 Fe(II) 辅因子的 3-组氨酸面部三联体。然而，关于半胱胺双加氧酶 (ADO) 的额外信息非常少，ADO 是除 CDO 之外的唯一一种哺乳动物 TDO。先前的光谱表征表明，ADO 可能以单齿方式结合底物半胱胺，而质谱研究提供的证据表明，Cys206 和 Tyr208（小鼠 ADO 编号）之间可以形成硫醚交联。在本研究中，我们使用电子吸收和电子顺磁共振 (EPR) 光谱来研究 Fe(III)ADO 与含巯基底物以及超氧化物替代品叠氮化物和氰化物孵育时形成的物质。我们的数据表明，叠氮化物无法与半胱胺结合的 Fe(III)ADO 配位，这表明 Fe(III) 中心缺乏开放的配位位点，或者叠氮化物与半胱胺竞争相同的结合位点。或者，氰化物与半胱胺或 Cys 结合的 Fe(III)ADO 结合，产生低自旋 ( S = 1/2) EPR 信号，该信号与氰化物/Cys 结合的 Fe(III)CDO 观察到的信号不同，揭示了 ADO 和 CDO 活性位点口袋的差异。最后，野生型 Fe(III)ADO 及其 Tyr208Phe 变体的氰化物/半胱胺加合物获得的 EPR 光谱是重叠的，这意味着分离的野生型酶的一小部分发生了交联，或者硫醚键的形成已发生对铁辅助因子的电子结构影响最小。

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更新日期：2021-09-28

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