Spectroscopic and Mechanistic Investigations of Dehaloperoxidase B from Amphitrite ornata
Spectroscopic and Mechanistic Investigations of Dehaloperoxidase B from Amphitrite ornata
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DOI：
10.1021/bi100407v
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发表时间：
2010-08-10
期刊：
影响因子：
2.9
通讯作者：
Ghiladi, Reza A.
中科院分区：
文献类型：
--
作者：
D'Antonio, Jennifer;D'Antonio, Edward L.;Ghiladi, Reza A.
脱氢过氧化物酶(DHP)是一种兼具血红蛋白和过氧化物酶活性的双功能酶。在目前已鉴定的两种DHP同工酶中，DHP A是目前研究的热点，而对DHP B的活性、底物专一性、作用机制或光谱性质的研究很少。本文报道了DHP B的重组表达和纯化，以及利用生化分析、停流紫外可见光谱、共振拉曼光谱、快速冷冻猝灭电子顺磁共振光谱和光谱电化学等手段对其催化循环进行研究的详细情况。我们的实验设计揭示了脱氢过氧化物酶的机理和动力学描述，这是以前没有报道过的同工酶A的机理。也就是说，我们证明了一种新的反应途径，在该反应途径中，三卤酚(二卤代酚)氧化脱卤化的产物本身能够诱导生成含氧亚铁DHP B，并提出了一个更新的DHP催化循环。我们进一步证明，与传统的单功能过氧化物酶不同，DHP中的含氧态是一个具有过氧化物酶活性的起始物种，这表明铁氧化状态可能不是该酶的必备起点。本文提供的数据提供了过氧化物酶和氧运输活性之间的联系，这进一步加深了我们对这种双功能酶如何能够将其两种固有功能结合在一个系统中的理解。
Dehaloperoxidase (DHP) from the terebellid polychaete Amphitrite ornata is a bifunctional enzyme that possesses both hemoglobin and peroxidase activities. Of the two DHP isoenzymes identified to date, much of the recent focus has been on DHP A, whereas very little is known pertaining to the activity, substrate specificity, mechanism of function, or spectroscopic properties of DHP B. Herein, we report the recombinant expression and purification of DHP B, as well as the details of our investigations into its catalytic cycle using biochemical assays, stopped-flow UV-visible, resonance Raman, and rapid freeze-quench electron paramagnetic resonance spectroscopies, and spectroelectrochemistry. Our experimental design reveals mechanistic insights and kinetic descriptions of the dehaloperoxidase mechanism which have not been previously reported for isoenzyme A. Namely, we demonstrate a novel reaction pathway in which the products of the oxidative dehalogenation of trihalophenols (dihaloquinones) are themselves capable of inducing formation of oxyferrous DHP B, and an updated catalytic cycle for DHP is proposed. We further demonstrate that, unlike the traditional monofunctional peroxidases, the oxyferrous state in DHP is a peroxidase-competent starting species, which suggests that the ferric oxidation state may not be an obligatory starting point for the enzyme. The data presented herein provide a link between the peroxidase and oxygen transport activities which furthers our understanding of how this bifunctional enzyme is able to unite its two inherent functions in one system.