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

了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 (cyts c)，其中血红素Fe作为轴向配体与His N和Met S原子在氧化还原中心配位，是一些最具表征的氧化还原活性蛋白。同源的I类细胞c，即嗜热嗜热氢杆菌细胞色素c_<552> (HT)和嗜热嗜绿假单胞菌细胞色素c_<5512> (PA)，表现出独特的热力学性质，即尽管它们的结构相似，且56%的序列相同，但HT的氧化形式明显比PA更稳定，这体现在变性温度的较大差异上。位点导向突变体的热稳定性介于PA和HT之间，其氨基酸替换是参照HT中相应的残基选择的。通过顺磁1H NMR和循环伏安法研究了PA及其一系列具有不同热稳定性的突变体，以阐明控制蛋白质E°值的分子机制。研究表明，该蛋白的E°值是由两种相互独立的分子机制调节的。一种是基于蛋白质中的Fe-Met配位键强度，由蛋白质中氨基酸侧链的填充决定；另一种是基于血红素17-丙酸侧链的pKa值，受静电环境的影响。前者改变了整个pH范围内E°′值的大小，后者调节了E°′值的pH曲线所反映的pK值。这些发现为该蛋白的功能调控提供了新的见解，可用于通过蛋白质工程手段调节该蛋白的E°'值。

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.