MECHANISMS IN RESPIRATION

这项工作涉及酶的一般特性， 呼吸 受试生物是嗜热菌栖热菌 嗜热菌 在过去的供资期间，我们建立了一个工作 生物学、生物化学和分子生物学的基础， 有机体 有了这些知识，就有可能进行广泛的 一系列的生物化学，生物物理和物理特性，其 呼吸蛋白 最重要的是最近的结论， 基因测序表明这种生物的呼吸蛋白 真核生物的功能和结构同源物。 的确， 细菌酶的类似物似乎形成真核生物的核心， 内切酶 因此，我们的结构-功能研究结果 直接适用于所有其他这类酶，特别是那些 哺乳动物的生殖器 在过去的一段时间里，我们净化了两个 新的蛋白质，细胞色素ba 3和b562;克隆和测序的基因 编码细胞色素c552和caa 3;进行了详细的物理研究 在细胞色素ba 3和caa 3中的CuA和a3/CuB位点;检查了 Rieske铁硫中心使用稳定同位素标记的组合 和ENDOR光谱;并建立了分子的雏形 在我们实验室里为栖热菌做生物学研究。 目前的提案建议 继续广泛的实验方法， 目标：[1]建立细胞色素ba 3与更大的 血红素/铜氧化酶家族。 [2]计数所有疑似组氨酸， 半胱氨酸配体的典型金属的细胞色素氧化酶， 同位素标记和共振光谱的结合。 [3]第一章 获取活动现场的结构细节的过滤型Rieske 蛋白 [4]确定氧化的a3/CuB的电子结构 金属对。 并且，[5]检查跨膜电位对 细胞色素c氧化酶的振动特性。 我们还建议两个 扩大项目业务基础的主要技术目标：[1] 尝试获得几种呼吸蛋白的单晶， [2]构建了栖热菌基因在大肠杆菌中表达的质粒载体， 他们的自然环境。 在过去的几年里， 越来越多的人认识到， 呼吸链在人类疾病中很重要。 的意义 这项对人类健康有益的工作在于更深入地了解酶 为所有细胞提供能量

This work involves general characterization of enzymes involved in respiration. The subject organism is the thermophilic bacterium Thermus thermophilus. During past funding periods we have established a working foundation in the biology, biochemistry, and molecular biology of this organism. With this knowledge it has been possible to carry out a wide range of biochemical, biophysical, and physical characterizations of its respiratory proteins. Of great importance is the recent conclusion from gene sequencing work that the respiratory proteins from this organism are functional and structural homologs of eukaryotic counterparts. Indeed, analogs of the bacterial enzymes appear to form the core of eukaryotic enzymes. For this reason, the results of our structure-function studies are directly applicable to all other such enzymes, notably those of the mammalian mitochondrion. During the past funding period; we purified two new proteins, cytochromes ba3 and b562; cloned and sequenced the genes encoding cytochromes c552 and caa3; carried out detailed physical studies of the CuA and a3/CuB sites in cytochromes ba3 and caa3; examined the Rieske iron-sulfur center using a combination of stable isotope labeling and ENDOR spectroscopy; and established the rudiments of a molecular biology for Thermus in our laboratory. The current proposal suggests a continued wide-ranging experimental approach with five major scientific goals: [1] Establish the relationship of cytochrome ba3 to the greater family of heme/copper oxidases. [2] Count all suspected histidine and cysteine ligands to the canonical metals of cytochrome oxidase by using a combination of isotope labeling and resonance spectroscopies. [3] Obtain active site structural details of the respiratory-type Rieske protein. [4] Determine the electronic structure of the oxidized a3/CuB metal pair. And, [5] examine the effect of transmembrane potential on the vibrational properties of cytochrome c oxidases. We also suggest two major technical goals to expand the operational base of the project: [1] Attempt to obtain single crystals of several respiratory proteins, and [2] establish a plasmid vector for the expression of Thermus genes in their natural environment. Over the past several years, there has been increasing recognition that inherited defects in the mitochondrial respiratory chain are important in human diseases. The significance of this work to human health lies in a deeper understanding of the enzymes that provide energy to all cells.