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Elucidation of relationship between redox function and Fe-Met bond stability in thermostable cytochrome c

耐热细胞色素 c 中氧化还原功能与 Fe-Met 键稳定性之间关系的阐明

基本信息

批准号：
15550143
负责人：
YAMAMOTO yasuhiko
金额：
$ 2.5万
依托单位：
University of Tsukuba
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2003
资助国家：
日本
起止时间：
2003 至 2004
项目状态：
已结题

项目摘要

Understanding the molecular mechanisms responsible for regulation of the redox potentials (E°') of proteins is a problem of immense fundamental and practical importance. Monoheme Class I cytochromes c (cyts c), in which heme Fe is coordinated to His N and Met S atoms as axial ligands at the redox center, are some of the best characterized redox active proteins. Homologous Class I cyts c, i. e., thermophilic Hydrogenobacter thermophilus cytochrome c_<552> (HT) and mesophilic Pseudomonas aerugiaosa cytochrome c_<5512> (PA), exhibit a unique thermodynamic property, i. e., despite their structural similarity together with their 56 % sequence identity, the oxidized form of HT is significantly more stable than that of PA, as reflected by the large difference in denaturation temperature. Site-directed mutants of PA, for which amino acid substitutions were selected with reference to the corresponding residues in HT, exhibited thermostabilities between those of PA and HT.PA and a series of its mutants exhibiting various thermostabilities have been studied by paramagnetic ^1H NMR and cyclic voltammetry in order to elucidate the molecular mechanisms responsible for control of the E°' value of the proteins. The study revealed that the E°' value of the protein is regulated by two molecular mechanisms operating independently of each other. One is based on the Fe-Met coordination bond strength in the protein, which is determined by the amino acid side-chain packing in the protein, and the other on the pKa value of the heme 17-propionic acid side-chain, which is affected by the electrostatic environment. The former mechanism alters the magnitude of the E°' value throughout the entire pH range and the latter regulates the pK values reflected by the pH profile of the E°' value. These findings provide novel insights into functional regulation of the protein, which could be utilized for tuning the E°' value of the protein by means of protein engineering.

理解负责调节蛋白质氧化还原电位（E ° '）的分子机制是一个具有巨大基础和实际重要性的问题。单血红素I类细胞色素C（cytsC），其中血红素Fe是协调的His N和Met S原子作为轴向配体在氧化还原中心，是一些最好的特征氧化还原活性蛋白质。Homeland I类cyts c，i.例如，嗜热嗜氢菌细胞色素c_<552>（HT）和嗜温铜绿假单胞菌细胞色素c_<5512>（PA）具有独特的热力学性质，即：例如，尽管它们的结构相似性以及它们56%的序列同一性，但是HT的氧化形式比PA的氧化形式明显更稳定，这反映在变性温度的巨大差异上。PA定点突变体的热稳定性介于PA和HT之间。为了阐明控制蛋白质E°'值的分子机制，我们用顺磁^1H NMR和循环伏安法研究了PA及其一系列表现出不同热稳定性的突变体。研究表明，蛋白质的E°'值受两种相互独立运作的分子机制调节。一种是基于蛋白质中的Fe-Met配位键强度，其由蛋白质中的氨基酸侧链堆积决定，另一种是基于血红素17-丙酸侧链的pKa值，其受静电环境的影响。前一种机制在整个pH范围内改变E°'值的大小，后者调节由E°'值的pH曲线反映的pK值。这些发现为蛋白质的功能调控提供了新的见解，这可以用于通过蛋白质工程来调节蛋白质的E°'值。