Mitochondrial respiratory chain-dependent generation of superoxide anion and its release into the intermembrane space
Mitochondrial respiratory chain-dependent generation of superoxide anion and its release into the intermembrane space
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DOI：
10.1042/0264-6021:3530411
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发表时间：
2001-01-15
影响因子：
4.1
通讯作者：
Cadenas, E
中科院分区：
文献类型：
--
作者：
Han, D;Williams, E;Cadenas, E
目前普遍认为，线粒体呼吸运输链产生的超氧阴离子向外释放到线粒体基质中，通过mn -超氧化物歧化酶的催化作用转化为过氧化氢。超氧阴离子释放到膜间空间是一个有争议的话题，部分原因是超氧阴离子与细胞色素c的反应无法解决，细胞色素c面向膜间空间，并以高浓度存在于膜间空间。本研究旨在评估呼吸链活动过程中超氧阴离子释放的拓扑位点。为了解决这个问题，从分离的线粒体中通过洋地黄苷处理制备丝裂体，去除部分外膜和部分细胞色素c。EPR分析结合抗霉素补充的丝裂体的自旋陷阱揭示了超氧阴离子自旋加合物的形成。EPR信号(i)被超氧化物歧化酶消除，（ii）被外源性铁细胞色素c竞争性地降低，（iii）被膜不渗透的自旋加宽剂三草酸铬加宽。这些结果证实了超氧阴离子在线粒体内膜胞质侧的产生和释放。此外，粘噻唑和抗霉素A共同处理丝分裂体，抑制了泛醇氧化为亚二甲基醌，消除了EPR信号，这表明复合物- iii泛醌库外位点的亚二甲基醌自氧化是超氧阴离子形成并随后释放到膜间空间的途径。向膜间空间产生超氧阴离子需要考虑超氧阴离子和过氧化氢的线粒体稳态值，这些氧化剂在膜间空间的衰变途径以及这些过程对细胞质事件的影响。
It has been generally accepted that superoxide anion generated by the mitochondrial respiratory transport chain are vectorially released into the mitochondrial matrix, where they are converted to hydrogen peroxide through the catalytic action of Mn-superoxide dismutase. Release of superoxide anion into the intermembrane space is a controversial topic, partly unresolved by the reaction of superoxide anion with cytochrome c, which faces the intermembrane space and is present in this compartment at a high concentration. This study was aimed at assessing the topological site(s) of release of superoxide anion during respiratory chain activity. To address this issue, mitoplasts were prepared from isolated mitochondria by digitonin treatment to remove portions of the outer membrane along with portions of cytochrome c. EPR analysis in conjunction with spin traps of antimycin-supplemented mitoplasts revealed the formation of a spin adduct of superoxide anion. The EPR signal was (i) abrogated by superoxide dismutase, (ii) decreased competitively by exogenous ferricytochrome c and (iii) broadened by the membrane-impermeable spin-broadening agent chromium tri-oxalate. These results confirm the production and release of superoxide anion towards the cytosolic side of the inner mitochondrial membrane.. In addition, co-treatment of mitoplasts with myxothiazol and antimycin A, resulting in an inhibition of the oxidation of ubiquinol to ubisemiquinone, abolished the EPR signal, thus suggesting that ubisemiquinone autoxidation at the outer site of the complex-III ubiquinone pool is a pathway for superoxide anion formation and subsequent release into the intermembrane space. The generation of superoxide anion towards the intermembrane space requires consideration of the mitochondrial steady-state values for superoxide anion and hydrogen peroxide, the decay pathways of these oxidants in this compartment and the implications of these processes for cytosolic events.