Studies of the Cyanide-resistant Respiratory Pathway

抗氰化物呼吸途径的研究

• 批准号: 9407759
• 负责人: James Siedow
• 金额: $ 24万
• 依托单位: Duke University
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 1994
• 资助国家: 美国
• 起止时间: 1994-09-01 至 1997-08-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=9407759&HistoricalAwards=false
• 关键词: Studies; Cyanide; resistant; Respiratory; Pathway

9407759 Siedow An "alternative" oxidase is associated with the cyanide-resistant, respiratory pathway found in the mitochondrial electron transfer chain of all higher plants. The role of this energetically wasteful respiratory pathway in plant energy metabolism is unclear, but recent progress has been made in characterizing regulatory, structural and kinetic features of the oxidase. The research outlined in this proposal will focus on three areas that relate to the alternative oxidase's regulation and structure: 1) The involvement of a recently identified sulfhydryl-disulfide redox system in the regulation of alternative pathway activity will be further elucidated. Specifically, the biochemical mechanism involved in the interconversion of the alternative oxidase from the less active, oxidized state to the more active, reduced state will be characterized in isolated mitochondria. In addition, studies of the behavior of this regulatory system in vivo will be undertaken to determine what physiological and/or developmental states affect this sulfhydryl-disulfide system and to establish how this regulation affects the overall capacity of the alternative pathway in vivo. 2) Purification of the detergent-solubilized alternative oxidase will be continued with the specific goals of characterizing both the metal species found in the oxygen reducing catalytic site and the specific nature of the metal site. 31 The ability to functionally express the alternative oxidase in the bacterium Escherichia coli will be exploited to select for mutants of the alternative oxidase that are resistant to inhibitors acting at the quinol oxidation site on the enzyme. Mapping of these inhibitor-resistant mutations will allow refinement of the current model of thequinol oxidation site on the oxidase and provide insights into functionally important amino acid residues involved in catalysis. Only a year ago, regulation of alternative pathway activity appeared to be a simple function of a lternative oxidase protein level and substrate (i.e., ubiquinol) concentration. The appearance of the sulfhydryl-disulfide redox system provides a major new avenue for the in vivo regulation of this energetically wasteful pathway. The work outlined in this proposal will lead to a better understanding of both the mechanism and role of this redox system in the metabolic regulation of the alternative pathway ln vivo. In addition, the alternative oxidase is unusual in that its structure appears to be simpler than that of any other known water-producing oxidase. Given its unique nature, it is important to develop a better understanding of this enzyme's active site, particularly as it relates to the regulation of enzyme activity at the level of ubiquinol oxidation. The studies outlined in this proposal should help to clarify such structural features associated with this enigmatic oxidase. %%% Higher plants possess a respiratory pathway, the "alternative pathway," which is resistant to cyanide, an inhibitor of the standard respiratory pathway common to both plants and animals. The alternative pathway bypasses several sites of ATP synthesis on the standard repiratory pathway, which means that large amounts of potential harvested crop yield may be lost through operation of the energetically wasteful alternative pathway. The role of the alternative pathway in plant metabolism is not known, but possibilities include: 1) serving as a component of the response of plants to environmental stresses such as chilling and 2) acting to keep respiration operating in leaf tissues when photosynthesis is taking place. Crucial to knowing the role of the alternative pathway is an understanding of both the structural features and the regulatory mechanisms of the cyanide-resistant oxidase associated with the pathway. The research outlined in this proposal will attempt to better characterize 1) the catalytic active site of the alternative oxidase with respect to the nature of the m etals found in it and the amino acids involved in the reactions carried out by the protien and 2) the mechanisms by which the activity of the alternative oxidase can be regulated to either enhance or reduce the level of cyanide-resistant repiration taking place under any given set of conditions. Knowing more about the nature and regulation of the alternative oxidase could ultimately lead to less potential yield being lost to the alternative pathway and more efficient crop production. ***

9407759一种“替代”氧化酶与所有高等植物线粒体电子传递链中发现的抗氰化物呼吸途径有关。 这种能量浪费的呼吸途径在植物能量代谢中的作用尚不清楚，但最近的进展已在表征氧化酶的调节，结构和动力学特征。 本提案中概述的研究将重点关注与替代氧化酶的调节和结构相关的三个领域：1）将进一步阐明最近发现的巯基-二硫键氧化还原系统在替代途径活性调节中的参与。 具体而言，涉及交替氧化酶从活性较低的氧化状态到活性较高的还原状态的相互转化的生化机制将在分离的线粒体中表征。 此外，将进行该调节系统在体内的行为研究，以确定什么生理和/或发育状态影响该巯基-二硫化物系统，并确定该调节如何影响体内替代途径的总体能力。 2)洗涤剂溶解的替代氧化酶的纯化将继续进行，具体目标是表征在氧还原催化位点中发现的金属物质和金属位点的特定性质。 将利用在细菌大肠杆菌中功能性表达交替氧化酶的能力来选择交替氧化酶的突变体，所述突变体对作用于酶上的醌醇氧化位点的抑制剂具有抗性。 这些耐药突变的映射将允许目前模型的thequinol氧化酶氧化位点的细化，并提供了重要的功能参与催化的氨基酸残基的见解。 仅在一年前，旁路途径活性的调节似乎是一个简单的功能，一个替代氧化酶蛋白质水平和底物（即，泛醇）浓度。 巯基-二硫化物氧化还原系统的出现为这种能量浪费途径的体内调节提供了一个主要的新途径。 在这个建议中概述的工作将导致更好地理解这一氧化还原系统在体内的替代途径的代谢调节的机制和作用。 此外，替代氧化酶是不寻常的，因为它的结构似乎比任何其他已知的产水氧化酶更简单。 鉴于其独特的性质，重要的是要更好地了解这种酶的活性位点，特别是因为它涉及到泛醇氧化水平上的酶活性调节。 在这项建议中概述的研究应有助于澄清这种神秘的氧化酶相关的结构特征。 %高等植物拥有一条呼吸途径，即“替代途径”，它对氰化物有抗性，氰化物是植物和动物共同的标准呼吸途径的抑制剂。 替代途径绕过标准呼吸途径上的几个ATP合成位点，这意味着大量潜在的收获作物产量可能通过能量浪费的替代途径的操作而损失。 替代途径在植物代谢中的作用尚不清楚，但可能包括：1）作为植物对环境胁迫（如寒冷）的反应的组成部分，2）在光合作用发生时保持叶组织中的呼吸作用。 了解旁路途径的作用的关键是了解与该途径相关的抗氰氧化酶的结构特征和调节机制。 本提案中概述的研究将试图更好地表征：1）替代氧化酶的催化活性位点，与其中发现的金属的性质和参与蛋白质进行的反应的氨基酸有关; 2）替代氧化酶的活性可被调节以增强或降低氰化物水平的机制。在任何特定条件下发生的抵抗性复发。 更多地了解交替氧化酶的性质和调节，最终可能导致较少的潜在产量损失的替代途径和更有效的作物生产。 ***