SUBSTRATE DOCKING IN CYTOCHROME C OXIDASE

项目 IV-细胞色素 c 氧化酶中的底物对接（Ferguson-Miller， 库恩、加拉维托、罗伯茨) 细胞色素c氧化酶是一种内在的膜蛋白，其复杂的能量 转导功能涉及电子转移、氧还原 水和质子跨膜易位。这个提议是 旨在确定蛋白质与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，决定了速率和 氧化酶中电子传递的效率。将开发模型 通过强大的计算能力将细胞色素 c 对接在 CcOX 上 使用蛋白质静电场和范德华的算法 分子间能量系统定向搜索中的表面 两种蛋白质。该模型的预测将通过 球形红杆菌 CcOX 的突变、动力学比较 细胞色素c与突变型和野生型酶的结合，并且通过 红细菌 CcOX 的天然和突变形式的结晶和 CcOX/细胞色素 c 复合物，通过 X 射线确定其结构 分析。牛红细菌序列的同源建模 氧化酶坐标（可从我们的合作者 S. Yoshikawa 处获得） 将允许对与更接近的模型的对接进行计算分析 是细菌的酶。相反，嵌合细菌的产生 用哺乳动物 coxII 基因取代红杆菌基因的酶， 将允许在更好的系统中进行结合分析 对应于哺乳动物氧化酶结构并且更好地匹配 哺乳动物细胞色素 C.氧化酶/细胞色素 c 处的地表水 还将分析界面以预测保留的水分子 可能会影响相互作用的化学性质。定义角色 这种蛋白质-蛋白质相互作用中的静电力和其他力将 增加我们对电子机制和力学的理解 呼吸链中的转移。

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.