Castro L, Rodriguez M, Radi R
Department of Biochemistry, Facultad de Medicina, Universidad de la República, Montevideo, Uruguay.
J Biol Chem. 1994 Nov 25;269(47):29409-15.
Mitochondrial and cytosolic aconitases have been indicated as major targets of .NO- and O2-.-mediated toxicity in cells due to the oxidant-mediated disruption of the [4Fe-4S] prosthetic group. However, under circumstances in which both .NO and O2-. are generated, their almost diffusion-controlled combination reaction (k = 6.7 x 10(9) M-1 s-1), leading to the formation of peroxynitrite anion (ONOO-), can out-compete the direct reactions of .NO and O2-. with aconitase and even the enzymatic dismutation of O2-. by superoxide dismutase. In this work, we report that ONOO- reacts with isolated pig heart mitochondrial aconitase at 1.4 x 10(5) M-1 s-1, resulting in a significant loss of enzymatic activity. Aconitase activity was totally recovered after postincubation with thiols and ferrous iron, indicating that ONOO- reactions with the enzyme involve the perturbation of the labile Fe alpha to yield the inactive [3Fe-4S] cluster, which is also evident by spectral changes. On the other hand, anaerobic exposure of isolated aconitase to high concentrations of .NO (> 100 microM) led to a moderate inhibition of the enzyme, which could be fully overcome by .NO displacement under an argon-saturated atmosphere, in agreement with the formation of a reversible inhibitory complex between .NO and the active site of aconitase. Superoxide inactivated mitochondrial aconitase at (3.5 +/- 2) x 10(6) M-1 s-1, a reaction rate 3 orders of magnitude slower than its reaction rate with .NO. O2-. could represent the main mechanism of inactivation of the enzyme in systems in which it is formed without significant concomitant production of .NO. Our results imply that the mechanisms by which .NO and O2-. inactivate aconitase in cell systems may not be simple due to their direct reactions with the iron-sulfur cluster, but may rely on the formation of ONOO-.
线粒体和胞质乌头酸酶已被指出是细胞中一氧化氮（·NO）和超氧阴离子（O₂⁻·）介导的毒性的主要靶点，这是由于氧化剂介导的[4Fe-4S]辅基的破坏。然而，在同时产生·NO和O₂⁻·的情况下，它们几乎受扩散控制的结合反应（k = 6.7×10⁹ M⁻¹ s⁻¹）会导致过氧亚硝酸根阴离子（ONOO⁻）的形成，这可能会胜过·NO和O₂⁻·与乌头酸酶的直接反应，甚至胜过超氧化物歧化酶对O₂⁻·的酶促歧化反应。在这项研究中，我们报告ONOO⁻与分离的猪心脏线粒体乌头酸酶的反应速率为1.4×10⁵ M⁻¹ s⁻¹，导致酶活性显著丧失。用硫醇和亚铁离子孵育后，乌头酸酶活性完全恢复，这表明ONOO⁻与该酶的反应涉及不稳定的Feα的扰动以产生无活性的[3Fe-4S]簇，光谱变化也证明了这一点。另一方面，将分离的乌头酸酶在厌氧条件下暴露于高浓度的·NO（>100 μM）会导致该酶受到中度抑制，在氩气饱和气氛下通过·NO置换可以完全克服这种抑制，这与·NO和乌头酸酶活性位点之间形成可逆抑制复合物一致。超氧化物使线粒体乌头酸酶失活的反应速率为（3.5±2）×10⁶ M⁻¹ s⁻¹，该反应速率比其与·NO的反应速率慢3个数量级。在没有大量伴随产生·NO的情况下形成O₂⁻·的系统中，O₂⁻·可能是该酶失活的主要机制。我们的结果表明，在细胞系统中，·NO和O₂⁻·使乌头酸酶失活的机制可能并不简单，这并非由于它们与铁硫簇的直接反应，而是可能依赖于ONOO⁻的形成。
