Mechanisms of Respiration

描述：（由申请人提供）来自嗜热栖热菌的细胞色素ba 3， 与其他酶的序列同一性小于20%，是 血红素-铜氧化酶的最大分歧，但它执行的核心功能 血红素铜氧化酶超家族的成员这是一个自然的仓库。 突变赋予蛋白质新的化学行为， 评估能量转换机制。我们的两大成就 实验室支持了一个新的研究方向：（a）开发E. 天然底物栖热菌细胞色素C552的大肠杆菌表达系统 产生具有结构和功能的重组蛋白， 性能与天然C552相同。(b)航天飞机的研制 在栖热菌细胞中过表达细胞色素ba 3 - 4倍的载体， 可用于操纵E.杆菌这个“系统” 将允许我们选择性地改变栖热菌DNA序列， 突变技术，然后在其天然的表达突变蛋白质。 环境因为我们也知道细胞色素C552和 细胞色素ba 3，这些新的工具使我们能够开始研究酶的机制。 本文提出了四个具体目标：（1）研究ba 3的作用机制 在接近生理的条件下， 吸收和MCD光谱的变化发生在氧化还原钡3 通过O2，瞬态共振拉曼光谱来鉴定Fe 3-氧 中间体，并与质子吸收的时间过程的相关性。（2）我们 将使用EXAFS和FTIR来检查在CuB中的配位的潜在变化， CuA缺陷-ba 3和探索可能的氯离子结合CUB。的结果 这些研究将确定中间体的数量和化学性质， 它们在再氧化过程中出现和消失的时间。x射线 吸收研究将提供有关协调的基本新知识 CUB时酶功能的变化。(3)We建议发起一项比较 斯克里普斯研究所的邓肯麦克里博士在晶体学方面的努力 目的是获得完全还原和羰基化形式的结构 细胞色素ba 3，突变的CuA def -蛋白和氧化的复合物 细胞色素C552和ba 3。(4)We提出了提炼我们ba 3的技术目标， - 表达系统包括容易地制备位点选择性表达的能力， 细胞色素ba 3的亚基II和I中的突变。其意义 对人类健康的贡献在于提供有关 生物系统的能量守恒。

DESCRIPTION: (provided by applicant) Cytochrome ba3 from Thermus thernzophilus, with a sequence identity to other enzymes of less than 20 percent, is among the most divergent of the heme-copper oxidases, yet it performs the core functions of the heme-copper oxidase super family. It is thus a storehouse of natural mutations that endow the protein with novel chemical behaviors and can be used to evaluate energy transduction mechanisms. Two major accomplishments in our laboratory support a new direction for the research: (a) Development of an E. coli-based expression system for Thermus cytochrome C552, the natural substrate of ba3, that yields recombinant protein having structural and functional properties identical to those of native C552. (b) Development of a shuttle vector that over expresses cytochrome ba3 -4 fold in Thermus cells and is useful for the manipulation of cytochrome ba3 genes in E. coli. This 'system' will permit us to selectively alter Thermus DNA sequences using standard mutagenesis techniques, then to express the mutant proteins in their natural environment. Because we also know the structures of cytochrome C552 and of cytochrome ba3, these new tools permit us to begin studies of enzyme mechanism. Four Specific Aims are suggested: (1) We will examine the mechanism of ba3 under near-physiological conditions to include recording of transient optical absorption and MCD spectral changes that occur during oxidation of reduced ba3 by O2, transient resonance Raman spectroscopy to identify Fea3-oxygen intermediates, and correlation with the time course of proton uptake. (2)We will use EXAFS and FTIR to examine potential changes in coordination at CuB in CuA deficient- ba3 and explore possible chloride binding to CUB. The results of these studies will define the number and chemical nature of intermediates and the times of their appearance and disappearance during reoxidation. The X-ray absorption studies will provide fundamental new knowledge about coordination changes at CUB in enzyme function.(3)We propose to initiate a comparative crystallographic effort with Dr. Duncan McRee at the Scripps Research institute with the goal of obtaining structures of fully reduced and carbonylated forms of cytochrome ba3, the mutant CuA def -protein and a complex of oxidized cytochromes C552 and ba3. (4)We propose the technical goal of refining our ba3 -expression system to include the capability of easily preparing site-selective mutations in both subunits II and I of cytochrome ba3. The significance of this work to human health lies in providing fundamental new information about conservation of energy by biological systems.