Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, 117997 Moscow, Russia.
A.N. Belozersky Institute of Physico-Chemical Biology, M.V. Lomonosov Moscow State University, Leninskie gory 1, Bld. 40, 119992 Moscow, Russia.
Cells. 2023 Sep 19;12(18):2316. doi: 10.3390/cells12182316.
Cytochrome c (CytC) is a single-electron carrier between complex bc1 and cytochrome c-oxidase (CcO) in the electron transport chain (ETC). It is also known as a good radical scavenger but its participation in electron flow through the ETC makes it impossible to use CytC as a radical sensor. To solve this problem, a series of mutants were constructed with substitutions of Lys residues in the universal binding site (UBS) which interact electrostatically with negatively charged Asp and Glu residues at the binding sites of CytC partners, bc1 complex and CcO. The aim of this study was to select a mutant that had lost its function as an electron carrier in the ETC, retaining the structure and ability to quench radicals. It was shown that a mutant CytC with substitutions of five (8Mut) and four (5Mut) Lys residues in the UBS was almost inactive toward CcO. However, all mutant proteins kept their antioxidant activity sufficiently with respect to the superoxide radical. Mutations shifted the dipole moment of the CytC molecule due to seriously changed electrostatics on the surface of the protein. In addition, a decrease in the redox potential of the protein as revealed by the redox titrations of 8Mut was detected. Nevertheless, the CD spectrum and dynamic light scattering suggested no significant changes in the secondary structure or aggregation of the molecules of CytC 8Mut. Thus, a variant 8Mut with multiple mutations in the UBS which lost its ability to electron transfer and saved most of its physico-chemical properties can be effectively used as a detector of superoxide generation both in mitochondria and in other systems.
细胞色素 c（CytC）是电子传递链（ETC）中复合 bc1 和细胞色素 c 氧化酶（CcO）之间的单电子载体。它也被称为良好的自由基清除剂，但它参与电子通过 ETC 的流动使得 CytC 不可能用作自由基传感器。为了解决这个问题，构建了一系列突变体，这些突变体取代了通用结合位点（UBS）中的赖氨酸残基，UBS 与 CytC 结合伙伴、bc1 复合物和 CcO 的结合位点处带负电荷的天冬氨酸和谷氨酸残基静电相互作用。本研究的目的是选择一种在 ETC 中失去电子载体功能的突变体，同时保留结构和淬灭自由基的能力。结果表明，UBS 中取代五个（8Mut）和四个（5Mut）赖氨酸残基的 CytC 突变体几乎对 CcO 无活性。然而，所有突变蛋白都保持了足够的抗氧化活性，相对于超氧自由基。突变由于蛋白质表面的静电发生严重变化，改变了 CytC 分子的偶极矩。此外，通过 8Mut 的氧化还原滴定检测到蛋白质的氧化还原电位降低。尽管如此，CD 光谱和动态光散射表明 CytC 8Mut 分子的二级结构或聚集没有明显变化。因此，在 UBS 中具有多个突变的变体 8Mut 失去了电子转移能力，但保留了其大部分物理化学性质，可以有效地用作线粒体和其他系统中超氧生成的检测器。
