Pepelina T Yu, Chertkova R V, Ostroverkhova T V, Dolgikh D A, Kirpichnikov M P, Grivennikova V G, Vinogradov A D
Shemyakin and Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, Moscow, 117997, Russia.
Biochemistry (Mosc). 2009 Jun;74(6):625-32. doi: 10.1134/s0006297909060066.
Three forms of horse heart cytochrome c with specific substitutions of heme cleft surface located amino acid residues involved in specific interactions with ubiquinol:cytochrome c reductase (complex III) and cytochrome c oxidase (complex IV) were constructed, and their reactions with superoxide radical produced by NADH:ubiquinone reductase (complex I) were studied. The proteins with six (K27E/E69K/K72E/K86E/K87E/E90K and K8E/E62K/E69K/K72E/K86E/K87E) and eight (K8E/K27E/E62K/E69K/K72E/K86E/K87E/E90K) substitutions were inactive in the cytochrome c oxidase reaction, and their reduction rates by complex III were significantly lower than that seen with acetylated cytochrome c. The reduction of these modified cytochromes c under conditions where complex I generates superoxide was almost completely (about 90%) inhibited by superoxide dismutase. The genetically modified cytochromes c are useful analytical reagents for studies on superoxide generation by the mitochondrial respiratory chain. Quantitative comparison of superoxide-mediated cytochrome c reduction with hydrogen peroxide-mediated Amplex Red oxidation suggests that complex I within its native environment (submitochondrial particles) produces both superoxide (50%) and hydrogen peroxide (50%).
构建了三种马心细胞色素c，它们在血红素裂隙表面具有特定的氨基酸残基取代，这些残基参与了与泛醇：细胞色素c还原酶（复合体III）和细胞色素c氧化酶（复合体IV）的特异性相互作用，并研究了它们与NADH：泛醌还原酶（复合体I）产生的超氧自由基的反应。具有六个（K27E/E69K/K72E/K86E/K87E/E90K和K8E/E62K/E69K/K72E/K86E/K87E）和八个（K8E/K27E/E62K/E69K/K72E/K86E/K87E/E90K）取代的蛋白质在细胞色素c氧化酶反应中无活性，并且它们被复合体III还原的速率显著低于乙酰化细胞色素c。在复合体I产生超氧的条件下，这些修饰的细胞色素c的还原几乎完全（约90%）被超氧化物歧化酶抑制。这些基因修饰的细胞色素c是用于研究线粒体呼吸链产生超氧的有用分析试剂。超氧介导的细胞色素c还原与过氧化氢介导的Amplex Red氧化的定量比较表明，在其天然环境（亚线粒体颗粒）中的复合体I产生超氧（约50%）和过氧化氢（约50%）。
