Peroxynitrite isomerization catalyzed by His64 myoglobin mutants
Peroxynitrite isomerization catalyzed by His64 myoglobin mutants
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DOI：
10.1021/ja010111d
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发表时间：
2001-05-02
影响因子：
15
通讯作者：
Watanabe, Y
中科院分区：
文献类型：
--
作者：
Herold, S;Matsui, T;Watanabe, Y
过氧亚硝酸盐是一种强氧化剂和硝化剂，可以在体内由一氧化氮与超氧化物的几乎扩散控制的反应形成，在过去十年中引起了越来越多的关注。2，3由于过氧亚硝酸盐在生理条件下的不稳定性，3-硝基酪氨酸（NO2-Tyr）4的检测已成为病理生理过程中过氧亚硝酸盐存在的生化标志物。酪氨酸硝化的生物学意义是一个非常感兴趣的主题，因为大量的证据支持在不同的病理条件下在体内形成NO2-Tyr。这些观察结果促使人们寻找一种药物来抑制这种强大的氧化剂和硝化剂。事实上，拦截和分解过氧亚硝酸盐的能力可能代表了与一氧化氮和超氧化物过量产生相关的疾病的治疗干预的一个新的和关键的点。最近，它已被证明，一系列的水溶性铁（III）卟啉配合物催化异构化过氧亚硝酸盐硝酸盐在生理相关的pH值和温度。6特别是，[FeIII（TMPS）] 7-保护培养物中的细胞免受外源性添加的过氧亚硝酸盐的影响，并且细胞保护与释放的胞质蛋白的NO2-Tyr含量的降低密切相关。7此外，据报道，[FeIII（TMPS）] 7-通过直接清除和/或减少再灌注期间过氧亚硝酸盐的产生来减少缺血/再灌注损伤。8.尽管有这些有希望的结果，但最近的研究表明，铁（III）-卟啉络合物还催化硝化以及氧化添加的酚类化合物。[9]可以想象，由铁（III）对过氧亚硝酸根的单电子还原产生的二氧化氮和氧合铁（IV）可能是负责硝化的物质。有趣的是，肌红蛋白的铁（III）形式（metMb）是迄今为止研究的唯一含血红素的蛋白质，其中血红素中心似乎不与过氧亚硝酸盐反应。10在目前的工作中，我们表明metMb对过氧亚硝酸根的反应性受到远端组氨酸的存在的调节，组氨酸通过强氢键部分阻断活性位点并稳定，水配体配位到铁。我们已经研究了通过停止-在三种不同的抹香鲸肌红蛋白突变体的存在下，过亚硝酸盐的分解速率，其中远端组氨酸具有被丙氨酸（H64 A）、亮氨酸（H64 L）或天冬氨酸（H64 D）取代。12通过跟踪在302 nm处的吸光度变化来研究反应，302 nm是过氧亚硝酸根的特征吸光度最大值。13如图1A所示，在0.05 M磷酸盐缓冲液中，在pH 7.0和20 ℃下，过氧亚硝酸盐在约10秒内衰变。在0.016当量的野生型马心铁（III）肌红蛋白（相对于过氧亚硝酸盐）的存在下，过氧亚硝酸盐的寿命几乎不变，而添加0.015当量的H64 L突变体稍微加速其分解速率。相比之下，在仅存在0.013当量的H64 A或H64 D的情况下，过氧亚硝酸根在小于500 ms内消失（图1B）。
Peroxynitrite, 1 a strong oxidizing and nitrating agent that can be formed in vivo from the nearly diffusion-controlled reaction of nitrogen monoxide with superoxide, has attracted increasing interest over the past decade. 2, 3 Because of the instability of peroxynitrite under physiological conditions, 3 the detection of 3-nitrotyrosine (NO2-Tyr) 4 has become a biochemical marker for the presence of peroxynitrite in pathophysiological processes. The biological significance of tyrosine nitration is a subject of great interest, because extensive evidence supports the formation of NO2-Tyr in vivo in diverse pathological conditions. 5 These observations have led to the search for a drug that can scavenge this powerful oxidizing and nitrating agent. Indeed, the ability to intercept and decompose peroxynitrite may represent a novel and critical point of therapeutic intervention in diseases associated with the overproduction of nitrogen monoxide and superoxide. Recently, it has been shown that a series of water-soluble iron (III) porphyrin complexes catalyze the isomerization of peroxynitrite to nitrate at physiological relevant pH and temperature. 6 In particular,[FeIII (TMPS)] 7-protects cells in culture from exogenously added peroxynitrite and cytoprotection correlates well with a reduction in the NO2-Tyr content of released cytosolic proteins. 7 Moreover, it has been reported that [FeIII (TMPS)] 7-reduces ischemia/reperfusion injury via direct scavenging and/or reduction of peroxynitrite production during reperfusion. 8 Despite these promising results, recent studies have shown that iron (III)-porphyrin complexes also catalyze the nitration as well as the oxidation of added phenolic compounds. 9 It is conceivable that nitrogen dioxide and oxoiron (IV), generated from the one-electron reduction of peroxynitrite by iron (III), may be the species responsible for nitration. 9 Interestingly, the iron (III) form of myoglobin (metMb) is the only heme-containing protein studied up to now in which the heme-center does not appear to react with peroxynitrite. 10 In the present work we show that the reactivity of metMb toward peroxynitrite is regulated by the presence of the distal histidine, which partly blocks the active site and stabilizes, via a strong hydrogen bond, the water ligand coordinated to the iron.We have studied by stopped-flow spectroscopy11 the decomposition rate of peroxynitrite in the presence of three different sperm whale myoglobin mutants in which the distal histidine has been replaced with alanine (H64A), leucine (H64L), or aspartic acid (H64D). 12 The reaction was studied by following the absorbance changes at 302 nm, the characteristic absorbance maximum of peroxynitrite. 13 As depicted in Figure 1A, in 0.05 M phosphate buffer at pH 7.0 and 20 C peroxynitrite decays in about 10 s. In the presence of 0.016 equiv of wild-type horse heart iron (III) myoglobin (relative to peroxynitrite), the lifetime of peroxynitrite is almost unchanged, whereas addition of 0.015 equiv of the H64L mutant slightly accelerates its decomposition rate. In contrast, in the presence of only 0.013 equiv of either H64A or H64D, peroxynitrite disappears in less than 500 ms (Figure 1B).