Proton linkage in formation of the cytochrome c-cytochrome c peroxidase complex: electrostatic properties of the high- and low-affinity cytochrome binding sites on the peroxidase.
Proton linkage in formation of the cytochrome c-cytochrome c peroxidase complex: electrostatic properties of the high- and low-affinity cytochrome binding sites on the peroxidase.
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细胞色素 c-细胞色素 c 过氧化物酶复合物形成中的质子连接：过氧化物酶上高亲和力和低亲和力细胞色素结合位点的静电特性。
DOI：
10.1021/bi00208a011
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发表时间：
1994
期刊：
影响因子：
2.9
通讯作者：
Mauk,AG
中科院分区：
文献类型：
--
作者：
Mauk,MR;Ferrer,JC;Mauk,AG
1994年7月25日收到的修订版Mandalpt ®摘要：通过电位滴定法研究了pH 5.5和7.75之间细胞色素c-细胞色素c过氧化物酶复合物形成的静电特性。在离子强度> 100 mM下获得的电位数据在1：1复合物形成方面进行了充分分析，而能够拟合在较低离子强度下获得的类似数据的最简单模型涉及过氧化物酶上细胞色素的两个不等价结合位点的假设。细胞色素c在高亲和力位点结合的稳定性约为100%。三个数量级大于低亲和力位点观察到的，是最佳的pH 6.75和7之间。这两个结合位点的静电性质是明显不同的，因为在大多数pH值下，细胞色素c与高亲和力位点的结合导致质子释放，而细胞色素与低亲和力位点的结合导致质子吸收。此外，结合的细胞色素的低亲和力网站似乎是最不稳定的pH值范围内，结合的高亲和力网站是最佳的。有趣的是，来自这些测量的结合参数是独立的温度，符合大量的熵复杂的稳定性的贡献。铁细胞色素c结合的过氧化物酶的亲和力略大于铁细胞色素c，并没有证据表明，复杂的稳定性的特定阴离子的影响进行了观察。在低离子强度（< 50 mM）和高pH公式7.75中，两种蛋白质的相互作用更为复杂，不能用双位点模型进行充分分析。（Mochan & Nichols，1971; Mochan，1970）和Yonetani及其同事（Yonetani和伦纳德，1973; Gupta和Yonetani，1973），由细胞色素c和细胞色素c过氧化物酶形成的复合物已经充当由其他电子转移蛋白形成的类似复合物的范例。Poulos和Kraut（1980）基于两种组分蛋白质的三维结构提出了该复合物的假设结构，从而特别关注该复合物。虽然最近对细胞色素c-细胞色素c过氧化物酶复合物的结晶形式的三维结构的测定（Pelletier和Kraut，1992）构成了我们对该复合物的理解的重大进展，但是各种功能研究提供了对细胞色素c-细胞色素c过氧化物酶复合物的三维结构的测定。
Revised Manuscript Received July 25, 1994® abstract: The electrostatic character of cytochrome c-cytochrome c peroxidase complex formation has been studied by potentiometric titration between pH 5.5 and 7.75. Potentiometric data obtained at ionic strength> 100 mM were adequately analyzed in terms of 1: 1 complex formation while the simplest model capable of fitting similar data obtained at lower ionic strength involves the assumption of two inequivalent binding sites for the cytochrome on the peroxidase. The stability of cytochrome c binding at the high-affinity site is ca. three orders of magnitude greater than that observed for the low-affinity site and is optimal between pH 6.75 and 7. The electrostatic properties of the two binding sites are distinctly different because, at most values of pH, binding of cytochrome c to the high-affinity site results in proton release while binding of the cytochrome to the low-affinity site results in proton uptake. Furthermore, binding of the cytochrome to the low-affinity site appears to be least stable in the pH range where binding to the high-affinity site is optimal. Interestingly, the binding parameters derived from these measurements were independent of temperature, consistent with a substantial entropic contribution to complex stability. Ferricytochrome c binds to the peroxidase with a slightly greater affinity than does ferrocytochrome c, and no evidence for specific anion effects on complex stability was observed. At low ionic strength (< 50 mM) and highpH (7.75), the interaction of the two proteins is more complex and cannot be adequately analyzed in terms of the two-site model.Since the pioneering work by Mochan and Nichols (Mochan & Nichols, 1971; Mochan, 1970) and Yonetani and co-workers (Yonetani & Leonard, 1973; Gupta & Yonetani, 1973), the complex formed by cytochrome c and cytochrome c peroxidase has served as a paradigm for similar complexes formed by other electron transfer proteins. Particular attention was focused on this complex by development of the hypothetical structure for this complex proposed by Poulos and Kraut (1980) on the basis of the three-dimensional structures of the two component proteins. While the recent determination of three-dimensional structures for crystallized forms of the cytochrome c-cytochrome c peroxidase complex (Pelletier & Kraut, 1992) constitutes a major advance in our understanding of this complex, a variety of functional studies provide