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

了解调节蛋白质氧化还原电位(E°‘)的分子机制是一个具有重大基础和实际意义的问题。单血红素I类细胞色素c(Cytsc)是氧化还原活性最好的蛋白质之一，其中血红素Fe与His N配位，Met S作为轴向配体位于氧化还原中心。同源的I类细胞色素C，即嗜热嗜热杆菌细胞色素C_&lt；552&gt；(HT)和中温嗜热铜绿假单胞菌细胞色素C_&lt；5512&gt；(PA)显示出独特的热力学性质，即尽管它们的结构相似且有56%的序列同源性，但从变性温度的巨大差异可以看出，HT的氧化形式明显比PA更稳定。PA及其一系列具有不同热稳定性的突变体经顺磁、~1H核磁共振和循环伏安等方法研究，以阐明控制蛋白质E°‘值的分子机制。研究表明，蛋白质的E°‘值受两种相互独立的分子机制调节。一种是基于蛋白质中Fe-Met配位键的强度，它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，它受静电环境的影响。前者改变整个pH范围内E°‘值的大小，后者调节E°’值的pH剖面所反映的pK值。这些发现为蛋白质的功能调控提供了新的见解，可用于通过蛋白质工程调整蛋白质的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.