Sites and mechanisms of aconitase inactivation by peroxynitrite: Modulation by citrate and glutathione
Sites and mechanisms of aconitase inactivation by peroxynitrite: Modulation by citrate and glutathione
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DOI：
10.1021/bi0509393
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发表时间：
2005-09-13
期刊：
影响因子：
2.9
通讯作者：
Cadenas, E
中科院分区：
文献类型：
--
作者：
Han, D;Canali, R;Cadenas, E
乌头酶是存在于细胞线粒体和细胞质中的含铁硫簇蛋白；活性位点的立方体铁硫（Fe-S）簇对催化活性至关重要，但它也使乌头碱酶极易受到活性氧和活性氮的影响。本研究探讨了过氧亚硝酸盐（ONOO-）对乌头酶失活的作用位点和机制。过氧亚硝酸盐是一种强氧化剂和硝化剂，容易由线粒体产生的超氧阴离子和一氧化氮形成。ONOO-以剂量依赖的方式灭活乌头酶（半最大抑制作用与3 μ M ONOO-相似）。低温EPR分析证实，低水平的ONOO-导致Fe-S簇从[4Fe-4S]（2+）形态转变为非活性[3Fe-4S]（1+）形态，同时失去了不稳定的铁。在底物柠檬酸盐存在的情况下，需要66倍浓度的ONOO-才能达到半最大抑制。柠檬酸盐的保护作用与其与活性位点的结合是一致的。Western blot分析显示，在柠檬酸盐存在下，乌头酸酶的失活是由于ONOO介导的半胱氨酸硫醇损失和酪氨酸硝化。LC/MS/MS分析表明，ONOO-处理乌头酸酶导致酪氨酸151和472硝化，半胱氨酸126和385氧化为磺酸。后者是乌头酶中结合Fe-S簇的三个半胱氨酸残基之一。所有其他修饰的酪氨酸和半胱氨酸残基都位于结合位点附近，这表明这些修饰引起了构象变化，导致活性位点破坏。乌头酶半胱氨酸硫醇除氧化为硫酸外的其他修饰，如s -谷胱甘肽化，也会降低乌头酶的活性，这表明谷胱甘肽化可能是在氧化和营养胁迫下调节乌头酶活性的重要手段。综上所述，这些结果表明，活性位点上的Fe-S簇、与Fe-S簇结合的半胱氨酸385以及活性位点附近的酪氨酸和半胱氨酸残基是氧化和/或硝化攻击的重要目标，而氧化和/或硝化攻击是由线粒体基质柠檬酸盐水平选择性控制的。从线粒体基质代谢和硫醇氧化还原状态的角度讨论了ONOO-对乌头酶失活的内在机制。
Aconitases are iron-sulfur cluster-containing proteins present both in mitochondria and cytosol of cells; the cubane iron-sulfur (Fe-S) cluster in the active site is essential for catalytic activity, but it also renders aconitase highly vulnerable to reactive oxygen and nitrogen species. This study examined the sites and mechanisms of aconitase inactivation by peroxynitrite (ONOO-), a strong oxidant and nitrating agent readily formed from superoxide anion and nitric oxide generated by mitochondria. ONOO- inactivated aconitase in a dose-dependent manner (half-maximal inhibition was observed with similar to 3 mu M ONOO-). Low levels of ONOO- caused the conversion of the Fe-S cluster from the [4Fe-4S](2+) form to the inactive [3Fe-4S](1+) form with the loss of labile iron, as confirmed by low-temperature EPR analysis. In the presence of the substrate, citrate, 66-fold higher concentrations of ONOO- were required for half-maximal inhibition. The protective effects of citrate corresponded to its binding to the active site. The inactivation of aconitase in the presence of citrate was due to ONOO--mediated cysteine thiol loss and tyrosine nitration in the enzyme as shown by Western blot analyses. LC/MS/MS analyses revealed that ONOO- treatment to aconitase resulted in nitration of tyrosines 151 and 472 and oxidation to sulfonic acid of cysteines 126 and 385. The latter is one of the three cysteine residues in aconitase that binds to the Fe-S cluster. All other modified tyrosine and cysteine residues were adjacent to the binding site, thus suggesting that these modifications caused conformational changes leading to active-site disruption. Aconitase cysteine thiol modifications other than oxidation to sulfortic acid, such as S-glutathionylation, also decreased aconitase activity, thus indicating that glutathionylation may be an important means of modulating aconitase activity under oxidative and nitrative stress. Taken together, these results demonstrate that the Fe-S cluster in the active site, cysteine 385 bound to the Fe-S cluster, and tyrosine and cysteine residues in the vicinity of the active site are important targets of oxidative and/or nitrative attack, which is selectively controlled by the mitochondrial matrix citrate levels. The mechanisms inherent in aconitase inactivation by ONOO- are discussed in terms of the mitochondrial matrix metabolic and thiol redox state.