Inactivation of human Cu,Zn superoxide dismutase by peroxynitrite and formation of histidinyl radical
Inactivation of human Cu,Zn superoxide dismutase by peroxynitrite and formation of histidinyl radical
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DOI：
10.1016/j.freeradbiomed.2004.06.006
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发表时间：
2004-09-15
影响因子：
7.4
通讯作者：
Radi, R
中科院分区：
文献类型：
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作者：
Alvarez, B;Demicheli, V;Radi, R
人重组铜锌超氧化物歧化酶 (CuZnSOD) 被过氧亚硝酸盐（一氧化氮和超氧化物之间的反应产物）灭活。使活性降低 50% 的过氧亚硝酸盐浓度 (IC50) 与 5 μM CuZnSOD 下的 100 μM 相似，并且在碱性 pH 下失活更高。停流测定表明，在pH 7.5和37℃下，过氧亚硝酸盐与CuZnSOD直接反应的二阶速率常数为(9.4+/-1.0)×10(3)M-1s(-1)/单体。在自旋陷阱 2-甲基-2-亚硝基丙烷存在下，将过氧亚硝酸盐 (1 mM) 添加到 CuZnSOD (0.5 mM) 中，导致电子顺磁共振检测到蛋白质自由基加合物典型的各向异性信号。用链霉蛋白酶处理显示出与组氨酸自由基的堡垒一致的几乎各向同性的信号。评估了亚硝酸盐、过氧化氢、碳酸氢盐和甘露醇对灭活的影响。考虑到CuZnSOD与过氧化氢反应的公认机制以及CuZnSOD促进过氧亚硝酸盐对酚类的硝化的事实，我们在此提出过氧亚硝酸盐与CuZnSOD反应产生二氧化氮以及与组氨酸残基反应形成组氨酸基自由基的铜结合羟基自由基。 (C) 2004 Elsevier Inc. 保留所有权利。
Human recombinant copper-zinc superoxide dismutase (CuZnSOD) was inactivated by peroxynitrite, the product of the reaction between nitric oxide and superoxide. The concentration of peroxynitrite that decreased the activity by 50% (IC50) was similar to100 muM at 5 muM CuZnSOD and the inactivation was higher at alkaline pH. Stopped-flow determinations showed that the second-order rate constant for the direct reaction of peroxynitrite with CuZnSOD was (9.4 +/- 1.0) x 10(3) M-1 s(-1) per monomer at pH 7.5 and 37degreesC. Addition of peroxynitrite (1 mM) to CuZnSOD (0.5 mM) in the presence of the spin trap 2-methyl-2-nitrosopropane led to the electron paramagnetic resonance detection of an anisotropic signal typical of a protein radical adduct. Treatment with Pronase revealed a nearly isotropic signal consistent with the fort-nation of histidinyl radical. The effects of nitrite, hydrogen peroxide, bicarbonate, and mannitol on the inactivation were assessed. Considering the mechanism accepted for the reaction of CuZnSOD with hydrogen peroxide and the fact that CuZnSOD promotes the nitration of phenolics by peroxynitrite, we herein propose that peroxynitrite reacts with CuZnSOD leading to nitrogen dioxide plus a copper-bound hydroxyl radical species that reacts with histidine residues, forming histidinyl radical. (C) 2004 Elsevier Inc. All rights reserved.