ENERGY TRANSDUCTION IN CYTOCHROME OXIDASE

这项建议的总体目标是加深我们对能源转换是如何实现的理解 细胞色素c氧化酶(CcO)以及如何控制速度和效率。在此授权期内，我们建议继续我们的 用我们的模型系统R.sphaeroidesCcO(RsCcO)分析了质子和电子转移的结构基础。我们 将利用岩田先生的团队在结晶RsCcO和几个突变体方面的令人兴奋的突破，通过 与Uppsala小组合作，使我们准备的关键突变体具体化。为了促进所有的结晶学 在RsCcO上的努力，我们建议通过克隆和添加以前的基因来修改我们的高产菌株 未检测到的Rs COX IV，一个似乎与晶体接触有关的亚基。另一个示范系统也将是 通过以.ft表示来开发。球状蓝藻，蓝藻Aa3氧化酶，其独特的性质和真核相似 COX IV将使我们能够研究在哺乳动物酶中观察到的一些控制特征。具体目标是： 1)为了测试质子泵浦和呼吸控制的各种模型，通过定义CCOO中的“外部”位置， 使用停流冷冻猝灭锰ESEEM来测量氚的存取率，并识别残留物和 同时控制访问和活性的构象特征；2)通过以下方式追求关键突变体的结晶 克隆COX IV基因并将其克隆到多拷贝表达载体中，获得一株可用于生产的球形葡萄球菌 高概率结晶的氧化酶和突变形式；3)确定影响 通过开发新的Ruthenate版本的CcO和CC来更好地分析本征电子，实现CcO中的电子转移 转移动力学，并利用新方法测量CuA配体和CuA/血红素突变体的氧化还原电位 4)聚球藻细胞色素C氧化酶(SynCcO)在球形拟青霉中的表达及性质分析 确定缺失关键残基(例如Glu286)的重要性、其不寻常的CC相互作用结构域的性质，以及 其类真核细胞环氧合酶IV的调控意义。我们预计这些研究获得的知识将导致 为了更清楚地了解耦合、去耦合的机理和能量传递效率的控制， 对老龄化和肥胖症的体重控制具有重要意义。

The overall objective of this proposal is to further our understanding of how energy conversion is accomplished in cytochrome c oxidase (CcO) and how rate and efficiency is controlled. In this grant period we propose to continue our analysis of the structural basis of proton and electron transfer, using our model system R. sphaeroides CcO (RsCcO). We will take advantage of the exciting breakthrough by So Iwata's group in crystallizing RsCcO and several mutants, through collaboration with the Uppsala group to crystallize key mutants we have prepared. To facilitate all the crystallography efforts on RsCcO, we propose to modify our over-producing strain by cloning and adding in the gene for the previously undetected Rs COX IV, a subunit that appears to be involved in crystal contacts. Another model system will also be developed by expressing in .ft. sphaeroides the cyanobacterial aa3 oxidase, whose unique properties and eukaryotic-like COX IV will allow us to study some of the control features observed in the mammalian enzyme. The specific aims are: 1) To test various models of proton pumping and respiratory control by defining where "outside" is located in CcO, using stopped-flow freeze-quench Mn ESEEM to measure deuterium access rates, and identifying residues and conformational features that control both access and activity; 2) To pursue the crystallization of key mutants by cloning and adding in the COX IV gene to our multi-copy plasmid to provide a strain of R. sphaeroides for making oxidase and mutant forms with a high probability of crystallizing; 3) To determine the critical factors governing electron transfer in CcO by developing new ruthenated versions of CcO and Cc to better analyze intrinsic electron transfer kinetics, and utilizing new methods for measuring redox potentials of mutants of CuA ligands and the CuA/heme a interface; 4) To express Synechocystis cytochrome c oxidase(SynCcO) in R. sphaeroides and analyze its properties to determine the importance of missing key residues (e.g.Glu286), the nature of its unusual Cc interaction domain, and the regulatory significance of its eukaryotic-like COX IV. We expect that the knowledge gained by these studies will lead to a clearer understanding of the mechanism of coupling, uncoupling and the control of efficiency of energy transduction, with important implications for weight control in aging and obesity.