Catalytic oxygenation of phenols by arthropod hemocyanin, an oxygen carrier protein, from Portunus trituberculatus
Catalytic oxygenation of phenols by arthropod hemocyanin, an oxygen carrier protein, from Portunus trituberculatus
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DOI：
10.1039/c000760a
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发表时间：
2010-01-01
影响因子：
4
通讯作者：
Itoh, Shinobu
中科院分区：
文献类型：
--
作者：
Fujieda, Nobutaka;Yakiyama, Aki;Itoh, Shinobu
本文纯化了梭子蟹三管蟹血青素六聚体（Pt-6Hc）及其一个单体亚基（Pt-1Hc），并经尿素处理转化为高效的苯酚单加氧酶（酚酶）。为了探讨Pt-Hc的二铜中心的内在化学反应活性，对分离蛋白的光谱特征和苯酚单加氧酶活性进行了详细的研究。Pt-6Hc和Pt-1Hc的含(mu-eta（2):eta(2)-过氧）二铜（II）种（氧- hc）的氧形式在0.5 M硼酸缓冲液（pH 9.0）中相对稳定，即使在25℃下高浓度尿素（3 M）存在。单体Pt-1Hc在4-甲基苯酚氧化（多次转化反应）生成4-甲基-1,2-二羟基苯（4-甲基儿茶酚）中的催化活性高于六聚Pt-6Hc。尿素的加入进一步加速了其催化活性。对一系列对取代苯酚衍生物在厌氧条件下进行的动力学氘同位素效应分析和Hammett分析表明，与酪氨酸酶（双核铜单加氧酶）的反应一样，氧- hc的过氧物种通过亲电芳香取代机制将苯酚与儿茶酚进行单加氧反应。在光谱分析和反应性研究的基础上，讨论了尿素对氧- hc氧化还原功能的影响。
The hexamer (Pt-6Hc) of swimming crab Portunus trituberculatus hemocyanin (Pt-Hc) and one of its monomeric subunits (Pt-1Hc) have been purified and converted to an efficient phenol monooxygenase (phenolase) by treatment with urea. To explore the intrinsic chemical reactivity of the dicopper center of Pt-Hc, the spectroscopic features and phenol monooxygenase (phenolase) activity of the isolated proteins have been examined in detail. The oxy-forms involving a (mu-eta(2):eta(2)-peroxo) dicopper(II) species (oxy-Hc) of Pt-6Hc and Pt-1Hc are relatively stable in 0.5 M borate buffer (pH 9.0) even in the presence of a high concentration of urea (3 M) at 25 degrees C. The catalytic activity of monomeric Pt-1Hc in the oxygenation reaction (multi-turnover reaction) of 4-methylphenol to 4-methyl-1,2-dihydroxybenzene (4-methylcatechol) was higher than that of hexameric Pt-6Hc, and its catalytic activity was further accelerated by the addition of urea. Kinetic deuterium isotope effect analysis and Hammett analysis using a series of p-substituted phenol derivatives under anaerobic conditions (single-turnover reaction) have indicated that the monooxygenation reaction of phenols to catechols by the peroxo species of oxy-Hc proceeds via electrophilic aromatic substitution mechanism as in the case of tyrosinase (dinuclear copper monooxygenase). The effect of urea on the redox functions of oxy-Hc is discussed on the basis of spectroscopic analysis and reactivity studies.