Single Turnover of Substrate-Bound Ferric Cysteine Dioxygenase with Superoxide Anion: Enzymatic Reactivation, Product Formation, and a Transient Intermediate
Single Turnover of Substrate-Bound Ferric Cysteine Dioxygenase with Superoxide Anion: Enzymatic Reactivation, Product Formation, and a Transient Intermediate
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DOI：
10.1021/bi2011724
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发表时间：
2011-11-29
期刊：
影响因子：
2.9
通讯作者：
Pierce, Brad S.
中科院分区：
文献类型：
--
作者：
Crawford, Joshua A.;Li, Wei;Pierce, Brad S.
半胱氨酸双加氧酶（CDO）是一种非血红素单核铁酶，催化L-半胱氨酸（Cys）的O-2依赖性氧化产生半胱氨酸亚磺酸（CSA）。在这项研究中，我们证明，CDO的催化循环可以“引发”的一个电子通过化学氧化，以产生CDO与三价铁在活性位点（铁III-CDO，称为2）。虽然无催化活性，但Fe-III-CDO的底物结合形式（2a）比“分离的”Fe-II-CDO酶（1）更适合于通过UV-vis和EPR光谱进行询问。进行化学救援实验，其中超氧阴离子（O-2（中心点-））被引入到2a中，以探索Fe-III-超氧物种代表CDO催化途径内的第一中间体的可能性。原则上，O-2（中心点-）作为CSA形成和活性Fe-II-CDO酶再生所需的剩余3-电子的合适受体（1）。事实上，添加O-2（中心点-）。至2a导致通过UV-vis光谱在565 nm处可观察到的瞬态物质（称为3a）的快速形成。随后的衰变3a的动力学匹配CSA的形成。此外，还使用平行模式X波段EPR光谱鉴定了归因于3a的信号（g类似于11）。光谱模拟、观察到的温度依赖性和3a的微波功率饱和行为与铁磁耦合（J类似于-8 cm（-1））高自旋三价铁（S-A = 5/2）与结合自由基（S-B = 1/2）的基态S = 3一致，推测为O-2（中心点-）。在用O-2（中心点-）处理后，回收的CDO的比活性相对于未处理的酶增加至接近60%。
Cysteine dioxygenase (CDO) is a non-heme mononuclear iron enzyme that catalyzes the O-2-dependent oxidation of L-cysteine (Cys) to produce cysteine sulfinic acid (CSA). In this study we demonstrate that the catalytic cycle of CDO can be "primed" by one electron through chemical oxidation to produce CDO with ferric iron in the active site (Fe-III-CDO, termed 2). While catalytically inactive, the substrate-bound form of Fe-III-CDO (2a) is more amenable to interrogation by UV-vis and EPR spectroscopy than the 'asisolated' Fe-II-CDO enzyme (1). Chemical-rescue experiments were performed in which superoxide (O-2(center dot-)) anions were introduced to 2a to explore the possibility that a Fe-III-superoxide species represents the first intermediate within the catalytic pathway of CDO. In principle, O-2(center dot-) serve as a suitable acceptor for the remaining 3-electrons necessary for CSA formation and regeneration of the active Fe-II-CDO enzyme (1). Indeed, addition of O-2(center dot-). to 2a resulted in the rapid formation of a transient species (termed 3a) observable at 565 nm by UV-vis spectroscopy. The subsequent decay of 3a is kinetically matched to CSA formation. Moreover, a signal attributed to 3a was also identified using parallel mode X-band EPR spectroscopy (g similar to 11). Spectroscopic simulations, observed temperature dependence, and the microwave power saturation behavior of 3a are consistent with a ground state S = 3 from a ferromagnetically coupled (J similar to -8 cm(-1)) high-spin ferric iron (S-A = 5/2) with a bound radical (S-B = 1/2), presumably O-2(center dot-). Following treatment with O-2(center dot-), the specific activity of recovered CDO increased to similar to 60% relative to untreated enzyme.