Genaro-Mattos Thiago C, Queiroz Raphael F, Cunha Daniela, Appolinario Patricia P, Di Mascio Paolo, Nantes Iseli L, Augusto Ohara, Miyamoto Sayuri
†Departamento de Bioquímica, Instituto de Química, Universidade de São Paulo, São Paulo, SP 05508-000, Brazil.
‡Departamento de Química e Exatas, Universidade Estadual do Sudoeste da Bahia, Jequié, BA 45200-000, Brazil.
Biochemistry. 2015 May 12;54(18):2841-50. doi: 10.1021/bi501409d. Epub 2015 Apr 27.
Lipid peroxidation is a well-known process that has been implicated in many diseases. Recent evidence has shown that mitochondrial cholesterol levels are increased under specific conditions, making it an important target for peroxidation inside the mitochondria. Cholesterol peroxidation generates, as primary products, several hydroperoxides (ChOOH), which can react with transition metals and metalloproteins. In this sense, cytochrome c (CYTC), a heme protein largely found in the mitochondria, becomes a candidate to react with ChOOH. Using CYTC associated with SDS micelles to mimic mitochondrial conditions, we show that ChOOH induces dose-dependent CYTC Soret band bleaching, indicating that it is using ChOOH as a substrate. This reaction leads to protein oligomerization, suggesting the formation of a protein radical that, subsequently, recombines, giving dimers, trimers, and tetramers. EPR experiments confirmed the production of carbon-centered radicals from both protein and lipid in the presence of ChOOH. Similar results were obtained with linoleic acid hydroperoxides (LAOOH). In addition, replacing SDS micelles by cardiolipin-containing liposomes as the mitochondrial mimetic led to similar results with either ChOOH or LAOOH. Importantly, kinetic experiments show that CYTC bleaching is faster with ChOOH than with H2O2, suggesting that these hydroperoxides could be relevant substrates for CYTC peroxidase-like activity in biological media. Altogether, these results show that CYTC induces homolytic cleavage of lipid-derived hydroperoxides, producing lipid and protein radicals.
脂质过氧化是一个众所周知的过程，与许多疾病有关。最近的证据表明，在特定条件下线粒体胆固醇水平会升高，这使其成为线粒体内过氧化的重要靶点。胆固醇过氧化的主要产物是几种氢过氧化物（ChOOH），它们可与过渡金属和金属蛋白发生反应。从这个意义上讲，细胞色素c（CYTC），一种主要存在于线粒体中的血红素蛋白，成为与ChOOH发生反应的候选物。使用与SDS胶束结合的CYTC来模拟线粒体条件，我们发现ChOOH会诱导剂量依赖性的CYTC Soret带漂白，这表明它将ChOOH用作底物。该反应导致蛋白质寡聚化，提示形成了蛋白质自由基，随后这些自由基重新结合，产生二聚体、三聚体和四聚体。电子顺磁共振实验证实在存在ChOOH的情况下会产生来自蛋白质和脂质的碳中心自由基。用亚油酸氢过氧化物（LAOOH）也得到了类似结果。此外，用含心磷脂的脂质体替代SDS胶束作为线粒体模拟物，使用ChOOH或LAOOH时都得到了类似结果。重要的是，动力学实验表明，ChOOH使CYTC漂白的速度比H2O2更快，这表明这些氢过氧化物可能是生物介质中CYTC类过氧化物酶活性的相关底物。总之，这些结果表明CYTC会诱导脂质衍生氢过氧化物的均裂，产生脂质和蛋白质自由基。
