Studies of the Cyanide-resistant Respiratory Pathway

9407759 Siedo是一种与所有高等植物线粒体电子转移链中发现的抗氰化物呼吸途径有关的“替代”氧化酶。这种耗能的呼吸途径在植物能量代谢中的作用尚不清楚，但最近在表征该酶的调节、结构和动力学特征方面取得了进展。本提案中概述的研究将集中在与替代氧化酶的调控和结构有关的三个领域：1)最近发现的一个巯基-二硫键氧化还原系统在替代途径活性调节中的参与将进一步阐明。具体地说，替代氧化酶从较不活跃的氧化状态到较活跃的还原状态的相互转换所涉及的生化机制将在分离的线粒体中得到表征。此外，还将对体内这种调节系统的行为进行研究，以确定什么生理和/或发育状态会影响这个巯基-二硫键系统，并确定这种调节如何影响体内替代途径的整体能力。2)将继续对洗涤剂增溶的替代氧化酶进行提纯，具体目标是确定氧还原催化部位中发现的金属物种和金属部位的具体性质。在大肠杆菌中功能性表达交替氧化酶的能力将被用来选择对作用于酶上的喹酚氧化位点的抑制剂具有抵抗力的交替氧化酶的突变体。绘制这些耐抑制剂突变的图谱将有助于改进目前酶上的喹酚氧化位点的模型，并提供对参与催化的重要氨基酸残基的深入了解。就在一年前，对替代途径活性的调节似乎是交替氧化酶蛋白水平和底物(即泛喹酚)浓度的简单函数。巯基-二硫键氧化还原体系的出现为体内调节这一耗能途径提供了一条重要的新途径。这项建议中概述的工作将有助于更好地理解这一氧化还原系统在体内替代途径的代谢调节中的机制和作用。此外，这种替代酶的不同寻常之处在于，它的结构似乎比任何其他已知的产水酶都要简单。鉴于其独特的性质，重要的是要更好地了解这种酶的活性部位，特别是当它与泛喹酚氧化水平上的酶活性调节有关时。这项提案中概述的研究应该有助于澄清与这种神秘的氧化酶有关的结构特征。高等植物有一条呼吸途径，即“替代途径”，它对氰化物具有抵抗力，氰化物是植物和动物共同的标准呼吸途径的抑制剂。替代途径绕过了标准呼吸途径上的几个ATP合成位点，这意味着通过运行耗能的替代途径可能会损失大量潜在的作物产量。另一种途径在植物新陈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(如低温)的反应的组成部分；2)当光合作用发生时，在叶片组织中保持呼吸作用。要了解替代途径的作用，关键是要了解与该途径相关的抗氰化物氧化酶的结构特征和调控机制。这项建议中概述的研究将试图更好地描述1)替代酶的催化活性部位与其所含金属的性质以及蛋白质所进行的反应所涉及的氨基酸；2)调节替代酶的活性以增强或降低在任何给定条件下发生的抗氰化物氧化水平的机制。更多地了解替代氧化酶的性质和调控，最终可能会减少潜在产量损失，从而提高作物生产效率。***

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. ***