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 Siedow 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，但最近在表征氧化酶的调节、结构和动力学特征方面取得了进展。 该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）最近确定的硫氢基-二硫化物氧化还原系统在替代途径活性调节中的参与将得到进一步阐明。 具体而言，将在分离的线粒体中表征替代氧化酶从活性较低的氧化态向活性较高的还原态相互转化所涉及的生化机制。 此外，将对该调节系统的体内行为进行研究，以确定哪些生理和/或发育状态影响该硫氢基-二硫化物系统，并确定该调节如何影响体内替代途径的整体能力。 2) 将继续纯化洗涤剂溶解的替代氧化酶，具体目标是表征氧还原催化位点中发现的金属种类和金属位点的具体性质。 31 将利用在大肠杆菌中功能性表达替代氧化酶的能力来选择替代氧化酶的突变体，这些突变体对作用于酶上对苯二酚氧化位点的抑制剂具有抗性。 这些抑制剂抗性突变的图谱将允许改进氧化酶上对醌氧化位点的当前模型，并提供对参与催化的功能上重要的氨基酸残基的见解。 仅在一年前，替代途径活性的调节似乎是替代氧化酶蛋白水平和底物（即泛醇）浓度的简单函数。 巯基二硫化物氧化还原系统的出现为体内调节这种能量浪费途径提供了一条重要的新途径。 该提案中概述的工作将有助于更好地理解该氧化还原系统在体内替代途径代谢调节中的机制和作用。 此外，替代氧化酶的不同寻常之处在于其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，更好地了解这种酶的活性位点非常重要，特别是因为它与泛醇氧化水平上酶活性的调节有关。 该提案中概述的研究应有助于阐明与这种神秘氧化酶相关的结构特征。 %%% 高等植物拥有呼吸途径，即“替代途径”，它对氰化物有抵抗力，氰化物是植物和动物共有的标准呼吸途径的抑制剂。 替代途径绕过了标准呼吸途径上 ATP 合成的多个位点，这意味着大量潜在的收获作物产量可能会因能量浪费的替代途径的运行而损失。 替代途径在植物代谢中的作用尚不清楚，但可能性包括：1）作为植物对环境胁迫（例如寒冷）做出反应的组成部分；2）在光合作用发生时保持叶组织的呼吸作用。 了解替代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尝试更好地表征 1) 替代氧化酶的催化活性位点（相对于其中发现的金属的性质以及参与蛋白质进行的反应的氨基酸）和 2)可以调节替代氧化酶的活性以增强或降低在任何给定条件下发生的抗氰呼吸水平的机制。 更多地了解替代氧化酶的性质和调节可能最终会导致替代途径损失的潜在产量减少，并提高作物生产效率。 ***