Redox-dependent modulation of aconitase activity in intact mitochondria
Redox-dependent modulation of aconitase activity in intact mitochondria
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DOI：
10.1021/bi0353979
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发表时间：
2003-12-23
期刊：
影响因子：
2.9
通讯作者：
Szweda, LI
中科院分区：
文献类型：
--
作者：
Bulteau, AL;Ikeda-Saito, M;Szweda, LI
先前有报道称，纯化的线粒体或细胞质乌头酸酶暴露于超氧化物（O-2-）或过氧化氢（H2O2）中会导致酶的[4Fe-4S]（2+）簇中fe - α的释放和失活。然而，关于乌头酸酶对完整线粒体内促氧化剂的反应，我们所知甚少。在本研究中，我们提供的证据表明，当分离的心脏线粒体用H2O2处理时，乌头酸酶迅速失活并随后重新激活。酶的再激活依赖于酶的底物柠檬酸盐的存在。EPR光谱分析表明，酶失活先于不稳定的fe - α从酶的[4Fe-4S]（2+）簇中释放。此外，通过等电聚焦凝胶电泳判断，fe - α释放和簇分解的相对水平并不能反映酶失活的程度。这些观察结果表明，除了铁的释放外，乌头酶的翻译后修饰的某种形式是导致酶失活的原因。为了支持这一结论，H2O2并不是通过直接作用于乌头酸酶来发挥其抑制作用，而是由于乌头酸酶与响应H2O2的线粒体膜组分相互作用而失活。然而，将线粒体长期暴露于稳态水平的H2O2或O-2-(.)会导致[4Fe-4S]（2+）簇的分解、羰基化和蛋白质降解。因此，根据促氧化剂的种类、氧化应激的水平和持续时间以及线粒体的代谢状态，乌头酸酶的活性或进展可能会发生可逆的调节，以[4Fe-4S]（2+）簇分解和蛋白水解降解。
It has previously been reported that exposure of purified mitochondrial or cytoplasmic aconitase to superoxide (O-2-(.)) or hydrogen peroxide (H2O2) leads to release of the Fe-alpha from the enzyme's [4Fe-4S](2+) cluster and to inactivation. Nevertheless, little is known regarding the response of aconitase to pro-oxidants within intact mitochondria. In the present study, we provide evidence that aconitase is rapidly inactivated and subsequently reactivated when isolated cardiac mitochondria are treated with H2O2. Reactivation of the enzyme is dependent on the presence of the enzyme's substrate, citrate. EPR spectroscopic analysis indicates that enzyme inactivation precedes release of the labile Fe-alpha from the enzyme's [4Fe-4S](2+) cluster. In addition, as judged by isoelectric focusing gel electrophoresis, the relative level of Fe-alpha release and cluster disassembly does not reflect the magnitude of enzyme inactivation. These observations suggest that some form of posttranslational modification of aconitase other than release of iron is responsible for enzyme inactivation. In support of this conclusion, H2O2 does not exert its inhibitory effects by acting directly on the enzyme, rather inactivation appears to result from interaction(s) between aconitase and a mitochondrial membrane component responsive to H2O2. Nevertheless, prolonged exposure of mitochondria to steady-state levels of H2O2 or O-2-(.) results in disassembly of the [4Fe-4S](2+) cluster, carbonylation, and protein degradation. Thus, depending on the pro-oxidant species, the level and duration of the oxidative stress, and the metabolic state of the mitochondria, aconitase may undergo reversible modulation in activity or progress to [4Fe-4S](2+) cluster disassembly and proteolytic degradation.