An engineered cation site in cytochrome c peroxidase alters the reactivity of the redox active tryptophan
An engineered cation site in cytochrome c peroxidase alters the reactivity of the redox active tryptophan
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DOI：
10.1021/bi960122x
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发表时间：
1996-05-14
期刊：
影响因子：
2.9
通讯作者：
Poulos, TL
中科院分区：
文献类型：
--
作者：
Bonagura, CA;Sundaramoorthy, M;Poulos, TL
细胞色素c过氧化物酶和抗坏血酸过氧化物酶的晶体结构非常相似，包括活性中心的构筑。这两种过氧化物酶都有一个色氨酸残基，命名为近端Trp，直接位于近端组氨酸血红素配体附近。在催化循环中，细胞色素c过氧化物酶近端的色氨酸被氧化成阳离子自由基。然而，在抗坏血酸过氧化物酶中，卟啉被氧化，而不是近端的色氨酸，尽管这两个过氧化物酶活性部位结构非常相似。抗坏血酸过氧化物酶中距色氨酸近端约8埃的阳离子被认为是抗坏血酸过氧化物酶不形成色氨酸自由基的原因之一。利用定点突变技术将抗坏血酸过氧化物酶阳离子结合位点引入细胞色素c过氧化物酶。晶体结构表明，突变体现在结合了一个阳离子。电子顺磁共振波谱分析表明，细胞色素c过氧化物酶的阳离子突变体不再形成稳定的色氨酸自由基。以铁细胞色素c为底物的阳离子突变体的活性为
The crystal structures of cytochrome c peroxidase and ascorbate peroxidase are very similar, including the active site architecture. Both peroxidases have a tryptophan residue, designated the proximal Trp, located directly adjacent to the proximal histidine heme ligand. During the catalytic cycle, the proximal Trp in cytochrome c peroxidase is oxidized to a cation radical. However, in ascorbate peroxidase, the porphyrin is oxidized, not the proximal Trp, despite the close similarity between the two peroxidase active site structures. A cation located approximate to 8 Angstrom from the proximal Trp in ascorbate peroxidase but absent in cytochrome c peroxidase is thought to be one reason why ascorbate peroxidase does not form a Trp radical. Site-directed mutagenesis has been used to introduce the ascorbate peroxidase cation binding site into cytochrome c peroxidase. Crystal structures show that mutants now bind a cation. Electron paramagnetic resonance spectroscopy shows that the cation-containing mutants of cytochrome c peroxidase no longer form a stable Trp radical. The activity of the cation mutants using ferrocytochrome c as a substrate is