Characterization of the Cyanide-resistant Oxidase in Plant Mitochondria

这项建议中概述的研究旨在继续提纯和鉴定大多数植物、许多真菌和一些原生动物中与线粒体电子转移链相关的抗氰化物的“替代”氧化酶。这种能量浪费的酶在植物能量代谢中的作用尚不清楚，直到最近才在鉴定与该酶相关的蛋白质种类方面取得了进展。PI已成功地从开花臭菜的高度抗氰化物的花梗中部分纯化了交替氧化酶。该制剂将进一步提纯，并将鉴定与该酶催化的氧还原反应相关的辅因子。更具体地说，将确定与酶有关的可自动氧化的苯二酚的性质和数量，以及与纯化的酶结合的任何金属的性质。还将进行研究，以确定立体因子、氧化还原电位和对苯二酚的亲脂性对某一对苯二酚物种作为替代氧化酶的电子供体的能力的相对贡献。这些研究将有助于更好地了解这种不寻常的氧化酶及其与线粒体电子转移链相互作用的性质。与人类等动物不同，植物的线粒体中有两条呼吸途径。一种类似于在动物身上发现的，对氰化物(中毒)敏感。第二条途径与第一条途径的最初步骤相同，在动物身上不存在，而且是抗氰化物的。在能源生产方面，第二种替代途径的效率低于第一种途径。植物可以调节这两个途径之间的电子流动，这一事实提高了人们对这两个途径的兴趣。因此，环境和生物条件有利于使用一种或另一种途径。另一种途径的效率较低，这一事实从能量上提出了一个问题，即为什么第二种途径存在，并且在某些条件下是首选的。本提案中概述的研究替代氧化酶的实验将有助于表征替代氧化酶及其功能。

The research outlined in this proposal is designed to continue the purification and characterization of the cyanide-resistant, "alternative" oxidase associated with the electron transfer chain of mitochondria in most plants, many fungi and some protozoa. The role of this energetically wasteful oxidase in plant energy metabolism is not known and only recently has progress been made identifying protein species associated with the oxidase. The PI has successfully carried out a partial purification of the alternative oxidase from the highly cyanide-resistant spadices of flowering skunk cabbage. This preparation will be further purified and the cofactors associated with the oxygen reduction reaction catalyzed by the enzyme will be identified. More specifically, the nature and amount of an auto-oxidizable quinone found to be associated with the enzyme will be determined as will the nature of any metals bound to the purified enzyme. Studies will also be carried out to determine the relative contributions of steric factors, redox potentials and quinone lipophilicity to the ability of a given quinone species to serve as an electron donor to the alternative oxidase. These studies will lead to a better under understanding of this unusual oxidase and the nature of its interaction with the mitochondrial electron transfer chain. Unlike animals, such as humans, plants have two respiratory pathways in their mitochondria. One is similar to that found in animals and is sensitive to (poisoned by) cyanide. The second pathway, which shares the initial steps of the first pathway, does not occur in animals and is cyanide resistant. This second, alternative pathway is less efficient than the first in energy production. Interest in the two pathways is heightened by the fact that plants can regulate the flow of electrons between the two pathways. Hence there are environmental and biological conditions that favor the use of one pathway or the other. The fact that the alternative pathway is less efficient energetically raises the question of why the second pathway exists and is preferred under certain conditions. The experiments outlined in this proposal to study the alternative oxidase will help to characterize the alternative oxidase and how it functions.