ENERGY TRANSDUCTION IN CYTOCHROME OXIDASE

细胞色素氧化酶中的能量转导

• 批准号: 7930214
• 负责人: SHELAGH M FERGUSON-MILLER
• 金额: $ 8.5万
• 依托单位: MICHIGAN STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-09-30 至 2011-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7930214
• 关键词: Aging; Biological Models; Cloning; Collaborations; Coupling; Crystallization; Crystallography; Cyanobacterium; Deuterium; Electron Transport; Electrons; Engineering; Enzymes; Freezing; Genes; Grant; Kinetics; Knowledge; Lead; Ligands; Measures; Methods; Modeling; Movement; Nature; Obesity; Oxidases; Oxidation-Reduction; Plasmids; Probability; Property; Proton Pump; Protons; Resolution; Rhodobacter sphaeroides; Site; Site-Directed Mutagenesis; Source; Structure; Synechocystis; Testing; Weight maintenance regimen; base; cytochrome c oxidase; heme a; mutant; respiratory; tool

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.

该提案的总体目标是进一步了解能源转换是如何完成的 细胞色素 c 氧化酶 (CcO) 以及如何控制速率和效率。在此资助期内，我们建议继续我们的 使用我们的模型系统 R. sphaeroides CcO (RsCcO) 分析质子和电子转移的结构基础。我们 将利用 So Iwata 小组在结晶 RsCcO 和几个突变体方面取得的令人兴奋的突破，通过 与乌普萨拉小组合作结晶我们准备的关键突变体。为了方便所有的晶体学 为了对 RsCcO 进行努力，我们建议通过克隆并添加之前的基因来修改我们的过度生产菌株 未检测到的 Rs COX IV，一个似乎参与晶体接触的亚基。另一个模型系统也将是 通过用 .ft 表达来开发。 sphaeroides是蓝藻aa3氧化酶，其独特的性质和真核细胞样 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) 通过定义 CcO 中“外部”的位置来测试质子泵送和呼吸控制的各种模型， 使用停流冷冻淬灭 Mn ESEEM 测量氘的获取率，并识别残留物和 控制访问和活动的构象特征； 2）通过以下方式追求关键突变体的结晶 克隆 COX IV 基因并将其添加到我们的多拷贝质粒中，以提供球形 R. sphaeroides 菌株，用于制备 氧化酶和突变体形式具有高结晶概率； 3) 确定控制的关键因素 通过开发新的钌版 CcO 和 Cc 来更好地分析本征电子，从而研究 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 一个接口； 4) 在球形红藻中表达集胞藻细胞色素c氧化酶(SynCcO)并分析其特性 确定缺失关键残基（例如 Glu286）的重要性、其不寻常的 Cc 相互作用域的性质，以及 其真核样COX IV的调控意义。我们期望通过这些研究获得的知识将引导 更清楚地了解耦合、解耦合的机制以及能量转换效率的控制， 对衰老和肥胖的体重控制具有重要意义。