Mechanistic and structural contributions of critical surface and internal residues to cytochrome c electron transfer reactivity.
Mechanistic and structural contributions of critical surface and internal residues to cytochrome c electron transfer reactivity.
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关键表面和内部残基对细胞色素 c 电子转移反应性的机制和结构贡献。
DOI：
10.1021/bi960430v
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发表时间：
1996
期刊：
影响因子：
--
通讯作者：
Mauk,AG
中科院分区：
文献类型：
--
作者：
Rafferty,SP;Guillemette,JG;Berghuis,AM;Smith,M;Brayer,GD;Mauk,AG
本文研究了酵母异-1-细胞色素两个保守区突变对细胞色素电子转移反应机理的影响。变体Asn 52 Ala，Tyr 67 Phe，Ile 75 Met和Thr 78 Gly涉及与内部水分子（Wat 166）的临界氢键相互作用的扰动，并且已经在它们的电化学性质和动力学方面进行了研究，其中它们被Fe（EDTA）2-还原并被Co（phen）33+氧化。在平行研究中，Co（phen）33+氧化动力学的酪氨酸，亮氨酸，异亮氨酸，丙氨酸，丝氨酸和甘氨酸的变体的遗传保守残基Phe 82已被研究，并与以前的电化学和动力学结果。为了帮助这些结果的机制解释，Asn 52 Ala和Ile 75 Met铁细胞色素变体的三维结构已被确定。在Wat 166区域中修饰的变体的还原电位比野生型蛋白的还原电位低至少33 mV（pH 6，25 °C，μ = 0.1 M）。在氧化和还原反应中的这个家庭的变体的电子转移反应性增加多达10倍以上的野生型细胞色素。另一方面，82位变异体在氧化和还原中的反应性取决于它们与之反应的氧化还原试剂的结构特征，并且这种反应性与该位置残基的性质有关。这些发现已被解释为表明，82位修饰的主要影响来自蛋白质表面反应物-蛋白质相互作用性质的变化以及维持细胞色素的高还原电位，而Wat 166附近内部修饰的主要影响来自氧化态重组能的改变。通过野生型蛋白质的晶体学分析定义的连锁构象变化。
The influence of mutations in two conserved regions of yeast iso-1-cytochromecbelieved to be critical to the mechanism of cytochromecelectron transfer reactions has been investigated. The variants Asn52Ala, Tyr67Phe, Ile75Met, and Thr78Gly involve perturbation of critical hydrogen-bonding interactions with an internal water molecule (Wat166) and have been studied in terms of their electrochemical properties and the kinetics with which they are reduced by Fe(EDTA)2-and oxidized by Co(phen)33+. In parallel studies, the Co(phen)33+oxidation kinetics of Tyr, Leu, Ile, Ala, Ser, and Gly variants of the phylogenetically conserved residue Phe82 have been studied and correlated with previous electrochemical and kinetic results. To assist mechanistic interpretation of these results, the three-dimensional structures of the Asn52Ala and Ile75Met ferrocytochromecvariants have been determined. The reduction potentials of the variants modified in the region of Wat166 were at least 33 mV (pH 6, 25 °C, and μ = 0.1 M) lower than that of the wild-type protein. Electron transfer reactivity of this family of variants in both the oxidation and reduction reactions was increased as much as 10-fold over that of the wild-type cytochrome. On the other hand, the reactivity of the position-82 variants in both oxidation and reduction depended on the structural characteristics of the oxidation−reduction reagent with which they reacted, and this reactivity was related to the nature of the residue at this position. These findings have been interpreted as demonstrating that the principal influence of modification at position-82 arises from changes in the nature of reactant−protein interaction at the surface of the protein and in maintaining the high reduction potential of the cytochrome while the principal influence of internal modifications near Wat166 results from alteration of the reorganization energy for the oxidation state-linked conformational change defined by crystallographic analysis of the wild-type protein.