Compound ES of dehaloperoxidase decays via two alternative pathways depending on the conformation of the distal histidine.
Compound ES of dehaloperoxidase decays via two alternative pathways depending on the conformation of the distal histidine.
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脱卤过氧化物酶的复合 ES 通过两种替代途径衰减，具体取决于远端组氨酸的构象。
DOI：
10.1021/ja106620q
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发表时间：
2010
影响因子：
15
通讯作者：
Thompson MK
中科院分区：
文献类型：
--
作者：
Thompson MK
脱氢过氧化物酶(DHP)是一种呼吸性血红蛋白(Hb)，在过氧化氢存在下能催化三卤酚转化为二卤代苯二酚。用低温电子顺磁共振波谱技术研究了在3种不同pH值下，50ms~2min的反应时间范围内，静止的DHP和过氧化氢作用下生成的自由基中间体的亚铁血红素状态。在休眠酶中鉴定出两种高自旋铁血红素形式，并将其归类于远端组氨酸His55的开放和闭合构象。在过氧化氢活化的DHP中发现了两个自由基：与化合物ES(血红素处于氧铁基态的酶和多肽链上的一个自由基)相关的自由基被指定为Tyr34，另一个自由基被指定为Tyr38。Tyr34自由基的形成具有非常高的相对产率(几乎100%的血红素)，这与其他球蛋白不同。对反应产物的高效液相色谱分析表明，反应产物形成了与pH有关的共价血红素到蛋白质的交联键。稳定的DHP化合物RH在没有三卤酚底物的情况下，在H_2O_2下形成，被认为是一种铁的血红素与Tyr34共价交联的状态。实验数据的动力学模型表明，化合物RH的形成和Tyr38自由基的形成是化合物ES衰变的两条可供选择的途径。选择哪种途径取决于His55的构象：在密度较低的闭合构象中，形成Tyr38自由基，但在主要的开放构象中，化合物ES衰变，生成化合物RH，这是在没有底物可用时，两个过氧化氢的氧化等价物安全终止的产物。
Dehaloperoxidase (DHP) is a respiratory hemoglobin (Hb) that has been shown to catalyze the conversion of trihalophenols to dihaloquinones in the presence of hydrogen peroxide. Ferric heme states of the resting DHP and the free radical intermediates formed under H2O2treatment were studied by low-temperature electron paramagnetic resonance spectroscopy in the range of reaction times from 50 ms to 2 min at three different pH values. Two high-spin ferric heme forms were identified in the resting enzyme and assigned to the open and closed conformations of the distal histidine, His55. Two free radicals were found in DHP activated by H2O2: the radical associated with Compound ES (the enzyme with the heme in the oxoferryl state and a radical on the polypeptide chain) has been assigned to Tyr34, and the other radical has been assigned to Tyr38. The Tyr34 radical is formed with a very high relative yield (almost 100% of heme), atypical of other globins. High-performance liquid chromatography analysis of the reaction products showed a pH-dependent formation of covalent heme-to-protein cross-links. The stable DHP Compound RH, formed under H2O2in the absence of the trihalophenol substrates, is proposed to be a state with the ferric heme covalently cross-linked to Tyr34. A kinetic model of the experimental data suggests that formation of Compound RH and formation of the Tyr38 radical are two alternative routes of Compound ES decay. Which route is taken depends on the conformation of His55: in the less populated closed conformation, the Tyr38 radical is formed, but in the major open conformation, Compound ES decays, yielding Compound RH, a product of safe termination of the two oxidizing equivalents of H2O2when no substrate is available.
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