Roles of distal asp in heme oxygenase from Corynebacterium diphtheriae, HmuO -: A water-driven oxygen activation mechanism
Roles of distal asp in heme oxygenase from Corynebacterium diphtheriae, HmuO -: A water-driven oxygen activation mechanism
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DOI：
10.1074/jbc.m410263200
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发表时间：
2005-01-28
影响因子：
4.8
通讯作者：
Ikeda-Saito, M
中科院分区：
文献类型：
--
作者：
Matsui, T;Furukawa, M;Ikeda-Saito, M
在哺乳动物、植物和细菌中发现的血红素加氧酶以相同的机制催化血红素的降解。用定点突变、酶动力学、共振拉曼光谱和X射线结晶学等方法研究了远端天冬氨酸(Asp-136)残基在白喉棒状杆菌血红素加氧酶HmuO中的作用。Ala和Phe取代Asp-136导致血红素降解活性降低，这是因为形成了铁基血红素，表明远端的Asp在HmuO血红素加氧酶活性中起关键作用。D136N HmuO催化血红素降解的效率与野生型和D136E HmuO相似，这意味着HmuO分解血红素不需要羧酸部分。共振拉曼结果表明，HmuO突变体中不活跃的铁基血红素的形成是由一个活性Fe-OOH物种与邻近的远端口袋水分子之间的相互作用中断引起的。对HmuO突变体的晶体结构分析证实，附近的水在D136A HmuO中部分消失。我们的结果首次为附近的水分子的催化作用提供了实验证据，该分子在血红素加氧酶催化中可能是普遍关键的，并表明远端的Asp有助于将关键的水分子定位在适合Fe-OH物种有效激活的位置。
Heme oxygenases found in mammals, plants, and bacteria catalyze degradation of heme using the same mechanism. Roles of distal Asp (Asp-136) residue in HmuO, a heme oxygenase of Corynebacterium diphtheriae, have been investigated by site-directed mutagenesis, enzyme kinetics, resonance Raman spectroscopy, and x-ray crystallography. Replacements of the Asp-136 by Ala and Phe resulted in reduced heme degradation activity due to the formation of ferryl heme, showing that the distal Asp is critical in HmuO heme oxygenase activity. D136N HmuO catalyzed heme degradation at a similar efficiency to wild type and D136E HmuO, implying that the carboxylate moiety is not required for the heme catabolism by HmuO. Resonance Raman results suggest that the inactive ferryl heme formation in the HmuO mutants is induced by disruption of the interaction between a reactive Fe-OOH species and an adjacent distal pocket water molecule. Crystal structural analysis of the HmuO mutants confirms partial disappearance of this nearby water in D136A HmuO. Our results provide the first experimental evidence for the catalytic importance of the nearby water molecule that can be universally critical in heme oxygenase catalysis and propose that the distal Asp helps in positioning the key water molecule at a position suitable for efficient activation of the Fe-OH species.