过氧化氢和过氧化物酶活性在羧甲基化细胞色素c中的反应:光谱和动力学研究

Swati Prasad , Nakul C. Maiti , Shyamalava Mazumdar , Samaresh Mitra
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引用次数: 39

摘要

采用光谱学和动力学方法研究了羧甲基化细胞色素c (Cmcytc)过氧化物酶活性,探讨了羧甲基化对天然细胞色素c (cytc)过氧化物酶活性的影响。光谱表明,Cmcytc与H2O2的反应只通过一种中间体化合物i进行,在pH 7.0、5.0和3.5时,反应的表观速率常数kapp分别为17、72和210 M−1 s−1。这些值大约是原生cytc的60倍(pH 7.0时为0.236 M−1 s−1),比经典过氧化物酶低5个数量级。发现Cmcytc催化有机和无机底物的氧化。发现在pH 6.0时,Cmcytc (205 [H2O2] s−1)氧化2,2′-氮基-双(3-乙基苯并噻唑-6-磺酸)(ABTS)的二级速率常数大于天然cytc (50 [H2O2] s−1)的相应值。细胞的羧甲基化破坏了Fe-S (Met 80)键,提高了其与H2O2的反应速率和催化活性。以ABTS为底物,用分光光度法测定了Cmcytc在pH 7.0、5.0和3.5时的比活性,分别为288、473和872 μM min−1 mg−1。共振拉曼研究表明,Cmcytc在中性pH下存在双组氨酸配位形式,在较低pH下存在双组氨酸(HH)、组氨酸-水(HW)和五种坐标(5C)形式的不同连接状态的种群分布。在pH 7.0、4.7和3.1下,Cmcytc中不同物种的相对种群分别为HH(~ 100%、~ 50%、~ 44%)、HW(~ 0%、~ 44%、41%)和5C(~ 0%、~ 6%、15%)。我们试图将Cmcytc与过氧化氢反应的pH依赖性及其过氧化物酶活性与Cmcytc观察到的血红素立体化学结构联系起来。对Cmcytc进行稳态和时间分辨色氨酸荧光研究,以探测Cmcytc血红素袋周围的构象变化。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
Reaction of hydrogen peroxide and peroxidase activity in carboxymethylated cytochrome c: spectroscopic and kinetic studies

The peroxidase activity of carboxymethylated cytochrome c (Cmcytc) has been investigated by spectroscopic and kinetic techniques to examine the effect of carboxymethylation on the peroxidase activity of native cytochrome c (cytc). The optical spectrum suggests that the reaction of Cmcytc with H2O2 proceeds through only one intermediate, compound I. The apparent rate constant (kapp) for the reaction was found to be 17, 72 and 210 M−1 s−1 at pH 7.0, 5.0 and 3.5 respectively. These values are about 60 times larger than those reported for native cytc (0.236 M−1 s−1 at pH 7.0), and about five orders of magnitude lower than those for classical peroxidases. Cmcytc was found to catalyse oxidation of organic and inorganic substrates. The second order rate constant for the oxidation of 2,2′-azino-bis(3-ethylbenzthiazoline-6-sulphonic acid) (ABTS) by Cmcytc (205 [H2O2] s−1) is found to be larger than the corresponding value for native cytc (50 [H2O2] s−1) at pH 6.0. The carboxymethylation of cytc ruptures the Fe-S (Met 80) bond and increases the rate of its reaction with H2O2, and its catalytic activity. The specific activity of Cmcytc was measured spectrophotometrically by the reported method using ABTS as substrate, and was found to be 288, 473 and 872 μM min−1 mg−1 at pH 7.0, 5.0 and 3.5 respectively. Resonance Raman studies indicated the presence of a bis-histidine coordinated form of Cmcytc at neutral pH, and the existence of a population distribution of different ligation states such as bis-histidine (HH), histidine-water (HW) and five coordinate (5C) forms at lower pH. The relative population of different species in Cmcytc was found to be HH (∼100%, ∼50%, ∼44%), HW (∼0%, ∼44%, 41%) and 5C (∼0%, ∼6%, 15%) at pH 7.0, 4.7 and 3.1 respectively. We have attempted to correlate the pH dependence of the reaction of Cmcytc with hydrogen peroxide and its peroxidase activity with the haem stereochemical structures observed for Cmcytc. Steady-state and time-resolved tryptophan fluorescence studies on Cmcytc were done to probe the conformational changes around the haem pocket of Cmcytc.

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