Spectroscopic investigation of iron(III) cysteamine dioxygenase in the presence of substrate (analogs): implications for the nature of substrate-bound reaction intermediates.
Spectroscopic investigation of iron(III) cysteamine dioxygenase in the presence of substrate (analogs): implications for the nature of substrate-bound reaction intermediates.
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DOI：
10.1007/s00775-021-01904-5
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发表时间：
2021-12
期刊：
影响因子：
--
通讯作者：
Brunold TC
中科院分区：
文献类型：
--
作者：
Fernandez RL;Juntunen ND;Fox BG;Brunold TC
硫醇双加氧酶(TDO)是一类金属酶，能将各种硫醇底物氧化成相应的亚磺酸。最初由X射线结晶学建立的半胱氨酸双加氧酶(CDO)，所有的TDO被认为含有一个3-组氨酸面部三联体，它协调必要的Fe(II)辅助因子。然而，关于半胱胺双加氧酶(ADO)的额外信息很少，ADO是除CDO外唯一的其他哺乳动物TDO。以前的光谱表征表明，ADO可能以单齿方式与底物半胱胺结合，而质谱学研究提供的证据表明，Cys206和Tyr208之间可以形成硫醚交联(小鼠ADO编号)。在本研究中，我们用电子吸收光谱和电子顺磁共振(EPR)光谱研究了Fe(III)ADO与含硫基底物以及超氧化物取代物叠氮和氰化物反应生成的物种。我们的数据显示叠氮不能与半胱胺结合的Fe(III)ADO配位，这表明Fe(III)中心缺乏开放的配位部位，或者叠氮化物与半胱胺竞争相同的结合部位。或者，氰化物与半胱胺或半胱氨酸结合的Fe(III)ADO产生低自旋(S=1/2)的EPR信号，这与氰化物/半胱氨酸结合的Fe(III)CDO不同，揭示了ADO和CDO在活性中心口袋上的不同。最后，野生型Fe(III)ADO及其Tyr208Phe变体的氰化物/半胱胺加合物的EPR谱是可叠加的，这意味着要么是分离的野生型酶的一小部分是交联的，要么是硫醚键的形成对铁辅助因子的电子结构影响很小。
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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