Kinetics of cytochrome c2+ oxidation by peroxynitrite: implications for superoxide measurements in nitric oxide-producing biological systems.
Kinetics of cytochrome c2+ oxidation by peroxynitrite: implications for superoxide measurements in nitric oxide-producing biological systems.
复制标题
过氧亚硝酸盐氧化细胞色素 c2 的动力学：对产生一氧化氮的生物系统中超氧化物测量的影响。
DOI：
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发表时间：
1995
影响因子：
3.9
通讯作者：
Rafael Radi
中科院分区：
文献类型：
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作者：
Leonor Thomson;M. Trujillo;R. Telleri;Rafael Radi
细胞色素c3+由于其快速的超氧化物还原为细胞色素c2+而被广泛用于检测生物系统中产生的超氧化物。然而，另一种生物分子，有时与超氧化物，一氧化氮，共生成，与超氧化物反应，几乎扩散控制的速率（6.7 × 10（9）M-1 s-1），导致生产的高度氧化物种，过氧亚硝酸根阴离子（ONOO-）。在这项工作中，我们报告过氧亚硝酸盐容易氧化细胞色素C2+细胞色素C3+在抗坏血酸可逆的方式。过氧亚硝酸根和细胞色素c2+之间的反应发生的二级速率常数为2.3 × 10（5）M-1 s-1。的pH依赖性的表观二级速率常数以及不同的清除剂的效果表明，过氧亚硝酸（ONOOH）在基态是负责细胞色素c2+氧化的实际物种。活化焓、自由能和熵分别为+10.8 kcal mol-1、+11.8 kcal mol-1和-3.15 kcal mol-1 K-1，与提出的反应机理一致。此外，我们的研究结果意味着，当定量超氧化物的细胞色素C3+还原法，同时产生的一氧化氮和过氧亚硝酸盐的存在可能会导致低估超氧化物的生产率。
Cytochrome c3+ has been extensively used for the detection of superoxide produced in biological systems due to its fast superoxide-mediated reduction to cytochrome c2+. However, another biomolecule which is sometimes cogenerated with superoxide, nitric oxide, reacts with superoxide at almost diffusion-controlled rates (6.7 x 10(9) M-1 s-1), leading to the production of a highly oxidizing species, peroxynitrite anion (ONOO-). In this work we report that peroxynitrite readily oxidizes cytochrome c2+ to cytochrome c3+ in an ascorbate-reversible manner. The reaction between peroxynitrite and cytochrome c2+ occurs with a second-order rate constant of 2.3 x 10(5) M-1 s-1. The pH dependence of the apparent second-order rate constants as well as the effect of different scavengers indicated that peroxynitrous acid (ONOOH) in the ground state was the actual species responsible of cytochrome c2+ oxidation. The activation enthalpy, free energy, and entropy were +10.8 kcal mol-1, +11.8 kcal mol-1, and -3.15 cal mol-1 K-1, respectively, in agreement with the proposed reaction mechanism. Additionally, our results imply that when quantitating superoxide by the cytochrome c3+ reduction method, the existence of a simultaneous generation of nitric oxide and peroxynitrite may lead to an underestimation of the rates of superoxide production.