Reactivity of Deoxy- and Oxyferrous Dehaloperoxidase B from Amphitrite ornata: Identification of Compound II and Its Ferrous-Hydroperoxide Precursor
Reactivity of Deoxy- and Oxyferrous Dehaloperoxidase B from Amphitrite ornata: Identification of Compound II and Its Ferrous-Hydroperoxide Precursor
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DOI：
10.1021/bi200311u
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发表时间：
2011-07-12
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;Ghiladi, Reza A.
脱盐过氧化物酶（DHP）是一种具有血红蛋白和过氧化物酶活性的双功能酶。DHP作为珠蛋白过氧化物酶的双功能性质似乎与每个个体活性的传统起始氧化状态不一致。也就是说，可逆氧结合仅通过珠蛋白中的亚铁血红素介导，过氧化物酶活性是从铁中心开始的，并且从过氧化物酶循环中排除了氧化亚铁状态。因此，为了解决看似矛盾的问题，我们在这里报告了我们使用生化分析、停流紫外可见、快速冷冻猝灭电子顺磁共振光谱和厌氧方法研究从脱氧和氧化亚铁状态启动的DHP催化循环的细节。我们证明了脱氧亚铁DHP B在与过氧化氢反应后直接形成化合物II，并表明这在存在和不存在三卤酚的情况下都发生。在化合物II形成之前，我们已经确定了一个新的物种，我们初步将其归因于铁-氢过氧化物前体，该前体经过异裂解生成上述铁基中间体。综上所述，结果表明DHP中的氧化亚铁态是一个过氧化物酶的起始态，并且提出了DHP的更新催化循环，其中铁氧化态不是脱氢过氧化物酶过氧化物酶催化循环的强制性起点。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在一个系统中结合其两种固有功能的理解。
Dehaloperoxidase (DHP) from the terebellid polychaete Amphitrite ornata is a bifunctional enzyme that possesses both hemoglobin and peroxidase activities. The bifunctional nature of DHP as a globin peroxidase appears to be at odds with the traditional starting oxidation state for each individual activity. Namely, reversible oxygen binding is only mediated via a ferrous heme in globins, and peroxidase activity is initiated from ferric centers and to the exclusion of the oxyferrous oxidation state from the peroxidase cycle. Thus, to address what appears to be a paradox, herein we report the details of our investigations into the DHP catalytic cycle when initiated from the deoxy- and oxyferrous states using biochemical assays, stopped-flow UV-visible, and rapid-freeze-quench electron paramagnetic resonance spectroscopies, and anaerobic methods. We demonstrate the formation of Compound II directly from deoxyferrous DHP B upon its reaction with hydrogen peroxide and show that this occurs both in the presence and in the absence of trihalophenol. Prior to the formation of Compound II, we have identified a new species that we have preliminarily attributed to a ferrous-hydroperoxide precursor that undergoes heterolysis to generate the aforementioned ferryl intermediate. Taken together, the results demonstrate that the oxyferrous state in DHP is a peroxidase competent starting species, and an updated catalytic cycle for DHP is proposed in which the ferric oxidation state is not an obligatory starting point for the peroxidase catalytic cycle of dehaloperoxidase. 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.