Characterization of superoxide-producing sites in isolated brain mitochondria
Characterization of superoxide-producing sites in isolated brain mitochondria
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DOI：
10.1074/jbc.m310341200
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发表时间：
2004-02-06
影响因子：
4.8
通讯作者：
Kunz, WS
中科院分区：
文献类型：
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作者：
Kudin, AP;Bimpong-Buta, NYB;Kunz, WS
线粒体呼吸链复合体I和III已被证明能产生超氧化物，但个别位点的确切作用和定位尚不清楚。我们探讨了氧、底物、抑制剂和NAD(+)/NADH氧化还原对大鼠和人脑线粒体过氧化氢和超氧阴离子产生的影响。虽然在谷氨酸+苹果酸单独存在的情况下，大鼠脑线粒体只产生少量的H_2O_2(0.04+/-0.02 nmoL H_2O_2/min/mg)，但在添加复合I抑制剂鱼藤酮(0.68+/-0.25 nmol H_2O_2/min/mg)或单独存在呼吸底物琥珀酸(0.80+/-0.27 nmol H_2O_2/min/mg)后，观察到大量的H_2O_2产生。在抗霉素A存在下，呼吸链复合体III产生H_2O_2的最大速率要低得多(0.14+/-0.07 nmol H_2O_2/min/mg)。从人的海马旁回分离的线粒体也进行了类似的观察。这表明，大多数超氧自由基是在络合物I产生的，高速率产生的活性氧物种分别是呼吸链抑制的线粒体和反向电子流的特征。我们测定了络合物I处超氧化物产生中心的氧化还原电位等于-295 mV。这一点和对抑制剂的敏感性表明，在复合体I产生超氧化物的位置最有可能是黄素单核苷酸部分。由于大鼠脑线粒体与H_2O_2短期孵育后，该部位的H_2O_2生成量增加，因此我们认为，活性氧可以激活一种自我加速的恶性循环，导致线粒体损伤和神经细胞死亡。
Mitochondrial respiratory chain complexes I and III have been shown to produce superoxide but the exact contribution and localization of individual sites have remained unclear. We approached this question investigating the effects of oxygen, substrates, inhibitors, and of the NAD(+)/NADH redox couple on H2O2 and superoxide production of isolated mitochondria from rat and human brain. Although rat brain mitochondria in the presence of glutamate+malate alone do generate only small amounts of H2O2 (0.04+/-0.02 nmol H2O2/min/mg), a substantial production is observed after the addition of the complex I inhibitor rotenone (0.68+/-0.25 nmol H2O2/min/mg) or in the presence of the respiratory substrate succinate alone (0.80+/-0.27 nmol H2O2/min/mg). The maximal rate of H2O2 generation by respiratory chain complex III observed in the presence of antimycin A was considerably lower (0.14+/-0.07 nmol H2O2/min/mg). Similar observations were made for mitochondria isolated from human parahippocampal gyrus. This is an indication that most of the superoxide radicals are produced at complex I and that high rates of production of reactive oxygen species are features of respiratory chain-inhibited mitochondria and of reversed electron flow, respectively. We determined the redox potential of the superoxide production site at complex I to be equal to -295 mV. This and the sensitivity to inhibitors suggest that the site of superoxide generation at complex I is most likely the flavine mononucleotide moiety. Because short-term incubation of rat brain mitochondria with H2O2 induced increased H2O2 production at this site we propose that reactive oxygen species can activate a self-accelerating vicious cycle causing mitochondrial damage and neuronal cell death.