ENERGY TRANSDUCTION IN CYTOCHROME OXIDASE

这项建议的总体目标是加深我们对 细胞色素c氧化酶的能量转换是如何完成的 (CCO)以及如何控制速度和效率。在这一授权期内，该协会 建议继续分析质子的结构基础和 电子转移，使用我们的模型系统球形R.sphaeroides CcO(RsCcO)。《少年派》 将利用岩田的团队在 通过与Uppsala合作，结晶RsCcO和几个突变体 小组将她准备的关键突变体具体化。为了方便所有的 关于RsCcO的结晶学努力，PI建议修改过剩的 通过克隆和添加以前未检测到的Rs COX基因来筛选菌株 IV，一种似乎与晶体接触有关的亚基。另一款车型 系统的开发也将通过在球形拟青霉中表达 蓝藻Aa3氧化酶，其独特的性质和类真核环氧合酶IV 将允许申请人研究在 哺乳动物的酶。具体目标是：1)测试各种型号的质子 泵送和呼吸控制--通过定义CCO中“外部”的位置， 使用停止流动的冷冻淬火Mn ESEEM来测量氢的存取率， 以及确定控制这两种途径的残基和构象特征 和活性；2)通过克隆和寻找关键突变体的结晶 将COX IV基因添加到申请人的多拷贝质粒中以提供 球形红假单胞菌产酶菌株及高产酶突变株 结晶概率；3)确定影响结晶的关键因素 通过开发新的Ruthenate版本的CcO和CC TO来实现CcO中的电子转移 更好地分析本征电子转移动力学，并利用新方法 用于测量CuA配体和CuA/血红素a突变体的氧化还原电位 4)表达集胞藻细胞色素C氧化酶(SynCcO)。 并分析其属性以确定丢失的重要性 关键残基(例如，Glu286)，其不寻常的CC相互作用域的性质， 以及其类真核细胞环氧合酶IV的调控意义。 国际和平研究所预计，通过这些研究获得的知识将导致更清晰的 对偶联、解偶联的机理及控制的认识 能量转导效率，对体重有重要影响 控制老龄化和肥胖症。

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 are controlled. In this grant period the PI proposes to continue the analysis of the structural basis of proton and electron transfer, using our model system R. sphaeroides CcO (RsCcO). The PI 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 she has prepared. To facilitate all the crystallography efforts on RsCcO, the PI proposes to modify the 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 R. sphaeroides the cyanobacterial aa3 oxidase, whose unique properties and eukaryotic-like COX IV will allow the applicant 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-flowed 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 the applicant's 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 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. The PI expects that 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.