Gu Jie, Shin Dong-Woo, Pletneva Ekaterina V
Department of Chemistry, Dartmouth College , Hanover, New Hampshire 03755, United States.
Biochemistry. 2017 Jun 13;56(23):2950-2966. doi: 10.1021/acs.biochem.6b01187. Epub 2017 May 31.
Perturbations in protein structure define the mechanism of allosteric regulation and biological information transfer. In cytochrome c (cyt c), ligation of Met80 to the heme iron is critical for the protein's electron-transfer (ET) function in oxidative phosphorylation and for suppressing its peroxidase activity in apoptosis. The hard base Lys is a better match for the hard ferric iron than the soft base Met is, suggesting the key role of the protein scaffold in favoring Met ligation. To probe the role of the protein structure in the maintenance of Met ligation, mutations T49V and Y67R/M80A were designed to disrupt hydrogen bonding and packing of the heme coordination loop, respectively. Electronic absorption, nuclear magnetic resonance, and electron paramagnetic resonance spectra reveal that ferric forms of both variants are Lys-ligated at neutral pH. A minor change in the tertiary contacts in T49V, away from the heme coordination loop, appears to be sufficient to execute a change in ligation, suggesting a cross-talk between the different regions of the protein structure and a possibility of built-in conformational switches in cyt c. Analyses of thermodynamic stability, kinetics of Lys binding and dissociation, and the pH-dependent changes in ligation provide a detailed characterization of the Lys coordination in these variants and relate these properties to the extent of structural perturbations. The findings emphasize the importance of the hydrogen-bonding network in controlling ligation of the native Met80 to the heme iron.
蛋白质结构的扰动决定了变构调节和生物信息传递的机制。在细胞色素c(cyt c)中,Met80与血红素铁的连接对于该蛋白质在氧化磷酸化中的电子传递(ET)功能以及在细胞凋亡中抑制其过氧化物酶活性至关重要。硬碱赖氨酸比软碱甲硫氨酸更适合与硬铁离子结合,这表明蛋白质支架在促进甲硫氨酸连接方面的关键作用。为了探究蛋白质结构在维持甲硫氨酸连接中的作用,设计了突变T49V和Y67R/M80A,分别破坏血红素配位环的氢键和堆积。电子吸收、核磁共振和电子顺磁共振光谱表明,在中性pH条件下,两种变体的铁离子形式均与赖氨酸连接。T49V中远离血红素配位环的三级接触的微小变化似乎足以导致连接变化,这表明蛋白质结构的不同区域之间存在相互作用,并且cyt c中存在内在构象开关的可能性。对热力学稳定性、赖氨酸结合和解离动力学以及连接的pH依赖性变化的分析详细表征了这些变体中赖氨酸的配位情况,并将这些性质与结构扰动程度相关联。这些发现强调了氢键网络在控制天然Met80与血红素铁连接中的重要性。