Mitochondrial aconitase reaction with nitric oxide, S-nitrosoglutathione, and peroxynitrite:: Mechanisms and relative contributions to aconitase inactivation
Mitochondrial aconitase reaction with nitric oxide, S-nitrosoglutathione, and peroxynitrite:: Mechanisms and relative contributions to aconitase inactivation
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DOI：
10.1016/j.freeradbiomed.2007.01.007
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发表时间：
2007-04-01
影响因子：
7.4
通讯作者：
Castro, Laura
中科院分区：
文献类型：
--
作者：
Tortora, Veronica;Quijano, Celia;Castro, Laura
使用高度纯化的重组线粒体乌头酸酶，我们确定了一氧化氮 ((NO)-N-.)、亚硝基谷胱甘肽 (GSNO) 和过氧亚硝酸盐 (ONOO-) 介导的失活动力学和机制。高(NO)-N-。抑制静息乌头酸酶所需的浓度。简短的。 NO 暴露导致与异柠檬酸盐竞争的可逆抑制（K-I=35 μM）。随后，观察到不可逆失活（0.65 M-1 s(-1)）。无论底物存在还是不存在（0.23 M-1 s(-1)），GSNO 都会介导不可逆失活。过氧亚硝酸盐与 [4Fe-4S] 簇反应，产生无活性的 [3Fe-4S] 酶 (1.1. x 10(5) M-1 s(-1))。二氧化碳通过 CO3.- 与 [4Fe-4S] 簇 (3 x 10(8) s(-1)) 的反应增强了 ONOO 依赖性失活。过氧亚硝酸盐还诱导间乌头酸酶酪氨酸硝化，但该反应不会导致酶失活。 O-2(.-) 和 (NO)-N- 乌头酸酶失活的计算模型。表明，当(NO)-N-时。由于生成并易于消耗，测量活性乌头酸酶的量仍然是检测细胞中 O-2(.-) 生成变化的敏感方法，但是，当细胞暴露于高浓度的“NO”时，乌头酸酶失活并不完全反映 O-2(.-) 生成速率的变化。在后一种情况下，乌头酸酶失活的程度反映了次级活性物质的形成，特别是 ONOO- 和 CO3.-。它还介导间乌头酸酶酪氨酸硝化，这是反应性 (NO)-N-. 衍生物种的足迹。 (c) 2007 Elsevier Inc. 保留所有权利。
Using highly purified recombinant mitochondrial aconitase, we determined the kinetics and mechanisms of inactivation mediated by nitric oxide ((NO)-N-.), nitrosoglutathione (GSNO), and peroxynitrite (ONOO-). High (NO)-N-. concentrations are required to inhibit resting aconitase. Brief. NO exposures led to a reversible inhibition competitive with isocitrate (K-I=35 mu M). Subsequently, an irreversible inactivation (0.65 M-1 s(-1)) was observed. Irreversible inactivation was mediated by GSNO also, both in the absence and in the presence of substrates (0.23 M-1 s(-1)). Peroxynitrite reacted with the [4Fe-4S] cluster, yielding the inactive [3Fe-4S] enzyme (1.1. x 10(5) M-1 s(-1)). Carbon dioxide enhanced ONOO-dependent inactivation via reaction of CO3.- With the [4Fe-4S] cluster (3 x 10(8) s(-1)). Peroxynitrite also induced m-aconitase tyrosine nitration but this reaction did not contribute to enzyme inactivation. Computational modeling of aconitase inactivation by O-2(.-) and (NO)-N-. revealed that, when (NO)-N-. is produced and readily consumed, measuring the amount of active aconitase remains a sensitive method to detect variations in O-2(.-) production in cells but, when cells are exposed to high concentrations of 'NO, aconitase inactivation does not exclusively reflect changes in rates of O-2(.-) production. In the latter case, extents of aconitase inactivation reflect the formation of secondary reactive species, specifically ONOO- and CO3.-. which also mediate m-aconitase tyrosine nitration, a footprint of reactive (NO)-N-.-derived species. (c) 2007 Elsevier Inc. All rights reserved.