ENERGY TRANSDUCTION IN CYTOCHROME OXIDASE

描述：原子分辨晶体结构的最新成就 细胞色素c氧化酶的表达为更深入地分析其 用突变和光谱方法研究能量转导机制。论 红细菌新结构的基础和我们以前的研究 球藻细胞色素c氧化酶(哺乳动物的优秀模型 酶)、CUA位点和一个密切相关的镁被认为具有 在引导电子输入和控制质子输出方面的重要作用。 亚基I和亚基II的界面立即发生突变 在血红素A3幼崽位置上方，在那里这两种金属都被连接和质子 预计将会出现产出。突变体将通过瞬变进行分析 动力学、共振拉曼光谱、电子顺磁共振、傅立叶变换红外光谱、氧化还原电势和质子泵浦 确定特定残基和金属中心作用的技术 在调节泵浦的快速定向电子转移和向外流动中 质子。这个相同的蛋白质区域和亚基中的额外残基 II是预测电子从细胞色素c进入的位置。 与氧化酶突变体的相互作用将通过稳态动力学进行分析， 结合和快速动力学方法来确定电子的路径 输入。我们最近观察到的脂肪酸对化学拯救作用的影响 将进一步研究羧酸盐突变体，目的是 了解影响能量耦合效率的因素，以及 区分泵浦质子和衬底质子的入射位置。 努力使红细菌氧化酶和选定的突变体单晶化 已启动。拟议的研究将对当前的模型进行严格的测试 电子转移、质子转移和能量耦合机制，以及 为能量转换的效率提供了新的见解 控制住了。

DESCRIPTION: The recent achievement of atomic resolution crystal structures of cytochrome c oxidase provides the basis for more incisive analysis of its energy transduction mechanism by mutational and spectral methods. On the basis of the new structures and our previous studies on Rhodobacter sphaeroides cytochrome c oxidase (an excellent model of the mammalian enzyme), the CuA site and a closely associated Mg are proposed to have important roles in directing electron input and controlling proton output. Mutations have been made at the interface of subunits I and II, immediately above the heme a3 CuB site, where both these metals are ligated and proton output is predicted to occur. The mutants will be analyzed by transient kinetics, resonance Raman, EPR, FTIR, redox potential and proton pumping techniques to determine the roles of specific residues and the metal centers in mediating rapid, directed electron transfer and outward flow of pumped protons. This same region of the protein and additional residues in subunit II are predicted entry sites for electrons from cytochrome c. Cytochrome c interactions with oxidase mutants will be analyzed by steady state kinetics, binding, and rapid kinetic methods to determine the pathways of electron input. Our recent observation of chemical rescue by fatty acids of carboxylate mutants will be further investigated with the aim of understanding factors that affect the efficiency of energy coupling, and of distinguishing between entry sites for pumped versus substrate protons. Efforts to cyrstallize the Rhodobacter oxidase and selected mutants will be initiated. The proposed studies will critically test current models of electron transfer, proton transfer, and the energy coupling mechanism, and provide new insight into how efficiency of energy transduction may be controlled.