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的线粒体电子转移链中发现的耐氰化物抗性，呼吸道有关。 这种能量浪费的呼吸道在植物能量代谢中的作用尚不清楚，但是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集中在与替代氧化酶的调节和结构相关的三个领域：1）将进一步阐明最近确定的硫磺二酰基 - 硫化物氧化还原氧化还原系统参与调节替代途径活性的研究。 具体而言，在分离的线粒体中，将对替代氧化酶从较小的，氧化状态到更活跃的状态的替代氧化酶相互转化的生化机制进行表征。 此外，将进行对这种调节系统在体内行为的研究，以确定哪些生理和/或发育状态会影响该硫依赖二硫化物系统，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，其特定目标是表征在氧还原催化位点中发现的金属物种和金属位点的特定性质。 31在功能上表达大肠菌菌中替代氧化酶的能力将被利用，以选择对作用在酶上喹诺尔氧化位点作用于抑制剂的替代氧化酶的突变体。 这些耐抑制剂突变的映射将允许在氧化酶上改进当前的奎诺醇氧化位点模型，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lternative氧化酶蛋白水平和底物（即泛醇）浓度的简单函数。 亚硫酸二硫化物氧化还原系统的出现为体内调节这种浪费的途径提供了一个主要的新途径。 该提案中概述的工作将使人们更好地了解该氧化还原系统在替代途径LN体内代谢调节中的机理和作用。 另外，替代氧化酶的结构似乎比任何其他已知的产水氧化酶都要简单。 鉴于其独特的性质，重要的是要更好地理解该酶的活性位点，尤其是与在泛素氧化水平上调节酶活性有关的情况。 该提案中概述的研究应有助于阐明与该神秘氧化酶相关的结构特征。 %%上级植物具有呼吸道途径，即“替代途径”，该途径对氰化物具有抗性，氰化物是植物和动物常见的标准呼吸途径的抑制剂。 替代途径绕过标准求解途径上ATP合成的几个位点，这意味着通过耗能浪费的替代途径的运行，可能会失去大量潜在的收获作物产量。 尚不清楚替代途径在植物代谢中的作用，但可能性包括：1）作为植物对环境应激的反应的组成部分，例如寒冷和2）在发生光合作用时保持呼吸在叶片组织中保持呼吸。 对于了解替代途径的作用至关重要的是了解与途径相关的耐氰化物氧化酶的结构特征和调节机制。 该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IT中发现的M eTals的性质以及蛋白质反应所涉及的氨基酸的性质，以及2）可以调节替代氧化酶的活性，以增强替代氧化酶的活性，以增强替代级别的替代级别的情况。 了解替代氧化酶的性质和调节的更多信息最终可能导致替代途径和更有效的作物产生的潜在产量较小。 ***