SUBSTRATE DOCKING IN CYTOCHROME C OXIDASE

项目IV-细胞色素c氧化酶的底物对接(Ferguson-Miller， 库恩、加拉维托、罗伯茨) 细胞色素c氧化酶是一种固有的膜蛋白，其复合能 转导功能包括电子转移，氧还原为 水和质子在膜上的转移。这项建议是 旨在确定蛋白质之间相互作用的性质 细胞色素c和细胞色素c氧化酶，这决定了细胞色素c和 酶中电子传递的效率。将开发模型 对于细胞色素c在CcOX上的对接，通过强大的计算 使用蛋白质静电场和范德瓦尔斯的算法 分子间能的系统定向搜索中的表面 这两种蛋白质中。该模型的预测将通过以下方式进行检验 球形红杆菌CcOX的诱变、动力学比较和 细胞色素c与突变型和野生型酶的结合，并通过 结晶红杆菌CcOX和突变的天然和突变形式 CcOX/细胞色素c络合物，用X射线确定其结构 分析。红细菌序列在牛中的同源模建 氧化酶坐标(我们的合作者S·吉川提供给我们) 将允许计算分析与模型更接近的对接 细菌酶的。相反，嵌合细菌的产生 用哺乳动物的coxII基因取代红细菌基因的酶， 将允许在一个更好的系统中完成绑定分析 与哺乳动物的氧化酶结构相对应，更好地匹配 哺乳动物细胞色素c.表面水中的氧化酶/细胞色素c 还将分析界面以预测滞留的水分子 可能会影响相互作用的化学作用。定义的角色 这种蛋白质-蛋白质相互作用中的静电学和其他力将 增进我们对电子的机理和力学的理解 在呼吸链中转移。

Project IV- Substrate Docking in Cytochrome c Oxidase (Ferguson-Miller, Kuhn, Garavito, Roberts) Cytochrome c oxidase is an intrinsic membrane protein whose complex energy transducing function involves electron transfer, reduction of oxygen to water and translocation of protons across the membrane. This proposal is aimed at determining the nature of the protein-protein interaction between cytochrome c and cytochrome c oxidase, which determines the rate and efficiency of electron delivery in the oxidase. Models will be developed for the docking sties for cytochrome c on CcOX by a powerful computational algorithm that uses protein electrostatic fields and van der Waals surfaces in a systematic orientation search of the intermolecular energies of the two proteins. The predictions of this model will be tested by mutation of Rhodobacter sphaeroides CcOX, comparison of the kinetics and binding of cytochrome c with the mutant and wild-type enzymes, and by crystallizing native and mutant forms of Rhodobacter CcOX and CcOX/cytochrome c complexes, to determine their structure by X-ray analysis. Homology modeling of the Rhodobacter sequence into the bovine oxidase coordinates (available to us from our collaborator, S. Yoshikawa) will permit computational analysis of the docking with a model closer to that of the bacterial enzyme. Conversely, creation of a chimeric bacterial enzyme with a mammalian coxII gene substituted for the Rhodobacter gene, will allow assays of binding to be done in a system that better corresponds to the mammalian oxidase structure and better matches the mammalian cytochrome c. Surface water at the oxidase/cytochrome c interface will also be analyzed to predict retained water molecules that may influence the chemistry of the interaction. Defining the role of electrostatics and other forces in this protein-protein interaction will increase our understanding of the mechanism and mechanics of electron transfer in the respiratory chain.