Crystal Structure of Leishmania major Peroxidase and Characterization of the Compound I Tryptophan Radical
Crystal Structure of Leishmania major Peroxidase and Characterization of the Compound I Tryptophan Radical
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DOI：
10.1074/jbc.m111.230524
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发表时间：
2011-07-15
影响因子：
4.8
通讯作者：
Poulos, Thomas L.
中科院分区：
文献类型：
--
作者：
Jasion, Victoria S.;Polanco, Julio A.;Poulos, Thomas L.
寄生原生动物大型利什曼原虫产生一种过氧化物酶（大型利什曼原虫过氧化物酶；LmP），该酶表现出酵母细胞色素 c 过氧化物酶 (CCP) 和植物胞质抗坏血酸过氧化物酶 (APX) 的活性特征。一个共同特征是关键的色氨酸残基，即 LmP 中的 Trp(208) 和 CCP 中的 Trp(191)，其位于 CCP、APX 和 LmP 中的近端组氨酸血红素配体附近。在CCP中，Trp(191)与H2O2反应后形成稳定的阳离子自由基，形成化合物I；在APX中，自由基位于卟啉环上。为了阐明Trp(208)在LmP中的作用并进一步探讨过氧化物酶结构-功能关系，我们确定了LmP的晶体结构，并利用电子顺磁共振波谱(EPR)、诱变和酶动力学研究了Trp(208)的作用。 CCP 和 LmP 分别在 Trp(191) 和 Trp(208) 附近都有延伸的 β 结构部分，而 APX 中不存在这一点。该区域为 CCP 中的 Trp(191) 自由基提供稳定性。 LmP 化合物 I 的 EPR 表现出与 CCP 化合物 I 类似的强烈且稳定的信号。在 LmP W208F 突变体中，该信号消失，表明 Trp(208) 形成稳定的阳离子自由基。在 LmP 中，Cys(197) 向 Thr 的转化显着削弱了化合物 I EPR 信号并显着降低了酶活性。这些结果进一步支持了这样的观点：局部静电环境的调节控制着过氧化物酶中色氨酸自由基的稳定性。我们的结果还表明，LmP 的生物学作用是充当细胞色素 c 过氧化物酶。
The parasitic protozoa Leishmania major produces a peroxidase (L. major peroxidase; LmP) that exhibits activities characteristic of both yeast cytochrome c peroxidase (CCP) and plant cytosolic ascorbate peroxidase (APX). One common feature is a key Trp residue, Trp(208) in LmP and Trp(191) in CCP, that is situated adjacent to the proximal His heme ligand in CCP, APX, and LmP. In CCP, Trp(191) forms a stable cationic radical after reaction with H2O2 to form Compound I; in APX, the radical is located on the porphyrin ring. In order to clarify the role of Trp(208) in LmP and to further probe peroxidase structure-function relationships, we have determined the crystal structure of LmP and have studied the role of Trp(208) using electron paramagnetic resonance spectroscopy (EPR), mutagenesis, and enzyme kinetics. Both CCP and LmP have an extended section of beta structure near Trp(191) and Trp(208), respectively, which is absent in APX. This region provides stability to the Trp(191) radical in CCP. EPR of LmP Compound I exhibits an intense and stable signal similar to CCP Compound I. In the LmP W208F mutant, this signal disappears, indicating that Trp(208) forms a stable cationic radical. In LmP conversion of the Cys(197) to Thr significantly weakens the Compound I EPR signal and dramatically lowers enzyme activity. These results further support the view that modulation of the local electrostatic environment controls the stability of the Trp radical in peroxidases. Our results also suggest that the biological role of LmP is to function as a cytochrome c peroxidase.