PDF(Pubmed)
Abstract：
Cytochrome c (CytC), a one-electron carrier, transfers electrons from complex bc1 to cytochrome c oxidase (CcO) in the electron-transport chain. Electrostatic interaction with the partners, complex bc1 and CcO, is ensured by a lysine cluster near the heme forming the Universal Binding Site (UBS). We constructed three mutant variants of mitochondrial CytC with one (2Mut), four (5Mut), and five (8Mut) Lys->Glu substitutions in the UBS and some compensating Glu->Lys substitutions at the periphery of the UBS for charge compensation. All mutants showed a 4-6 times increased peroxidase activity and accelerated binding of cyanide to the ferric heme of CytC. In contrast, decomposition of the cyanide complex with ferrous CytC, as monitored by magnetic circular dichroism spectroscopy, was slower in mutants compared to WT. Molecular dynamic simulations revealed the increase in the fluctuations of Cα atoms of individual residues of mutant CytC compared to WT, especially in the Ω-loop (70-85), which can cause destabilization of the Fe…S(Met80) coordination link, facilitation of the binding of exogenous ligands cyanide and peroxide, and an increase in peroxidase activity. It was found that only one substitution K72E is enough to induce all these changes, indicating the significance of K72 and the Ω-loop (70-85) for the structure and physiology of mitochondrial CytC. In this work, we also propose using a ferro-ferricyanide buffer as a substrate to monitor the peroxidase activity of CytC. This new approach allows us to determine the rate of peroxidase activity at moderate (200 µM) concentrations of H2O2 and avoid complications of radical formation during the reaction.
摘要：
细胞色素c(CytC),一个单电子的载体,将电子从复合物bc1转移到电子传输链中的细胞色素c氧化酶（CcO）。与合作伙伴的静电相互作用，复杂的bc1和CcO，由血红素附近的赖氨酸簇确保形成通用结合位点(UBS)。我们构建了线粒体CytC的三个突变变体，其中一个（2Mut），四（5Mut），以及UBS中的五个（8Mut）Lys->Glu取代，以及UBS外围的一些补偿性Glu->Lys取代，用于电荷补偿。所有突变体均显示过氧化物酶活性增加4-6倍，并加速了氰化物与CytC的三价铁血红素的结合。相比之下,氰化物配合物与亚铁CytC的分解，通过磁圆二色性光谱监测，与WT相比，突变体更慢。分子动力学模拟显示，与WT相比，突变体CytC的单个残基的Cα原子波动增加，尤其是在Ω环(70-85)中，这会导致Fe...S(Met80)配位链的不稳定，促进外源配体氰化物和过氧化物的结合，和过氧化物酶活性的增加。结果发现，只有一个替代K72E就足以引起所有这些变化，表明K72和Ω环（70-85）对线粒体CytC的结构和生理学的意义。在这项工作中,我们还建议使用铁氰化物缓冲液作为底物来监测CytC的过氧化物酶活性。这种新方法使我们能够确定中等浓度（200µM）H2O2下过氧化物酶活性的速率，并避免反应过程中自由基形成的并发症。
