The redox properties of ascorbate peroxidase
The redox properties of ascorbate peroxidase
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DOI：
10.1021/bi7006492
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发表时间：
2007-07-10
期刊：
影响因子：
2.9
通讯作者：
Raven, Emma Lloyd
中科院分区：
文献类型：
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作者：
Efimov, Igor;Papadopoulou, Nektaria D.;Raven, Emma Lloyd
已经确定了抗坏血酸过氧化物酶（APX）和一些位点导向变体的催化化合物I/化合物II和化合物II/Fe3+氧化还原对，以及双电子化合物I/Fe3+氧化还原对的还原电位。对于野生型酶，E度（化合物I/化合物II） = 1156 mV， E度（化合物II/Fe3+） = 752 mV， E度（化合物I/Fe3+） = 954 mV。对于这些变体，分析还包括Fe3+/Fe2+电位的测定，这些电位用于计算（实验上无法获得的）E度（化合物II/Fe3+）电位。这些数据为APX催化提供了许多新的见解。野生型蛋白的E度（化合物I/化合物II）和E度（化合物II/Fe3+）的测量值说明了化合物I比化合物II具有更高的氧化反应活性，并且这种相关性适用于APX的许多其他活性位点和底物结合变体。化合物I的高还原潜力也说明了该中间体已知的热力学不稳定性。并且提出这种不稳定性可以解释在抗坏血酸的稳态氧化过程中观察到的大多数APX酶与标准米切里斯动力学的偏差。本研究首次采用多种方法对抗坏血酸过氧化物酶的氧化还原特性进行了系统评价，并为准确测定相关酶中Fe3+、化合物I和化合物II的氧化还原特性提供了实验和理论框架。
Reduction potentials for the catalytic compound I/compound II and compound II/Fe3+ redox couples, and for the two-electron compound I/Fe3+ redox couple, have been determined for ascorbate peroxidase (APX) and for a number of site-directed variants. For the wild type enzyme, the values are E degrees'(compound I/compound II) = 1156 mV, E degrees'(compound II/Fe3+) = 752 mV, and E degrees'(compound I/Fe3+) = 954 mV. For the variants, the analysis also includes determination of Fe3+/Fe2+ potentials which were used to calculate (experimentally inaccessible) E degrees'(compound II/Fe3+) potentials. The data provide a number of new insights into APX catalysis. The measured values for E degrees'(compound I/compound II) and E degrees'(compound II/Fe3+) for the wild type protein account for the much higher oxidative reactivity of compound I compared to compound II, and this correlation holds for a number of other active site and substrate binding variants of APX. The high reduction potential for compound I also accounts for the known thermodynamic instability of this intermediate, and it is proposed that this instability can account for the deviations from standard Michaelis kinetics observed for most APX enzymes during steady-state oxidation of ascorbate. This study provides the first systematic evaluation of the redox properties of any ascorbate peroxidase using a number of methods, and the data provide an experimental and theoretical framework for accurate determination of the redox properties of Fe3+, compound I, and compound II species in related enzymes.