Detection of a tryptophan radical as an intermediate species in the reaction of horseradish peroxidase mutant (Phe-221→Trp) and hydrogen peroxide
Detection of a tryptophan radical as an intermediate species in the reaction of horseradish peroxidase mutant (Phe-221→Trp) and hydrogen peroxide
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DOI：
10.1074/jbc.273.24.14753
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发表时间：
1998-06-12
影响因子：
4.8
通讯作者：
Morishima, I
中科院分区：
文献类型：
--
作者：
Morimoto, A;Tanaka, M;Morishima, I
过氧化物酶与过氧化氢（H2 O2）反应中的关键反应中间体化合物I在辣根过氧化物酶（HRP）中含有卟啉π阳离子自由基，其催化小的有机和无机化合物的氧化，而细胞色素c过氧化物酶（CcP）在色氨酸残基（Trp-191）上具有自由基中心，并氧化氧化还原伙伴细胞色素c。为了研究血红素活性中心附近的氨基酸残基在区分这两种酶中过氧化物酶功能中的作用，我们制备了一个类似于CcP的HRP突变体F221 W（Phe-221 -> Trp）。尽管快速光谱扫描和停流实验证实F221 W突变体与H2 O2反应以与野生型酶相同的速率形成卟啉π-阳离子自由基，但卟啉π-阳离子自由基的特征光谱特征迅速消失，并转化为化合物II-型光谱。然而，通过还原卟啉π-阳离子自由基产生的所得物质的EPR谱与HRP中的化合物II的EPR谱完全不同，显示出如对于CcP所发现的来自Trp自由基的典型信号。从卟啉环到Trp残基的连续自由基形成表明，近端Trp是自由基从卟啉环转移过程中的关键残基，这区分了过氧化物酶的功能。
The crucial reaction intermediate in the reaction of peroxidase with hydrogen peroxide (H2O2), compound I, contains a porphyrin pi-cation radical in horseradish peroxidase (HRP), which catalyzes oxidation of small organic and inorganic compounds, whereas cytochrome c peroxidase (CcP) has a radical center on the tryptophan residue (Trp-191) and oxidizes the redox partner, cytochrome c. To investigate the roles of the amino acid residue near the heme active center in discriminating the function of the peroxidases in these two enzymes, we prepared a CcP-like HRP mutant, F221W (Phe-221 --> Trp). Although the rapid spectral scanning and stopped-flow experiments confirmed that the F221W mutant reacts with H2O2 to form the porphyrin pi-cation radical at the same rate as for the wild-type enzyme, the characteristic spectral features of the porphyrin pi-cation radical disappeared rapidly, and were converted to the compound II-type spectrum. The EPR spectrum of the resultant species produced by reduction of the porphyrin pi-cation radical, however, was quite different from that of compound II in HRP, showing typical signals from a Trp radical as found for CcP. The sequential radical formation from the porphyrin ring to the Trp residue implies that the proximal Trp is a key residue in the process of the radical transfer from the porphyrin ring, which differentiates the function of peroxidases.