ELECTRON TRANSFER/PROTON PUMPING IN CYTOCHROME OXIDATION

细胞色素氧化中的电子转移/质子泵浦

• 批准号: 2655009
• 负责人: OLOF EINARSDOTTIR
• 金额: $ 14.34万
• 依托单位: UNIVERSITY OF CALIFORNIA SANTA CRUZ
• 依托单位国家: 美国
• 财政年份: 1997
• 资助国家: 美国
• 起止时间: 1997-02-01 至 2001-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2655009
• 关键词: bioenergetics; carbon monoxide; cytochrome oxidase; electron transport; enzyme mechanism; enzyme reconstitution; flash photolysis; membrane model; membrane transport proteins; oxidation reduction reaction; oxygen; photochemistry; spectrometry; time resolved data

DESCRIPTION: The proposed research will focus on the mechanisms of electron transfer and proton pumping in respiration. Special emphasis will be on cytochrome oxidase, the primary site of coupling between the two processes. Our goals include the following: 1) The mechanism of the reduction of dixoygen to water by cytochrome oxidase will be studied under a variety of conditions with the flow-flash method, which uses the photolability of the CO complex to initiate the redox activity with O2. Time-resolved multichannel optical absorption spectroscopy will be used to follow the kinetics of electron and proton transfer on time scales of nanoseconds to milliseconds. Singular value decomposition and global exponential fitting methods will be applied to analyze the kinetics and determine the UV-Vis spectra of the transient intermediates. These studies should provide new insight into the mechanism of the dioxygen reduction by cytochrome oxidase. 2) Alternatives to CO photodissociation will be used to investigate the reaction of O2 with cytochrome oxidase. The reaction of unliganded reduced cytochrome oxidase with oxygen will be studied using a superfast direct mixing method, pulsed-accelerated-flow (PAF). The reaction of oxygen with the unliganded enzyme will also be investigated using O2 which is produced in situ on any relevant time scale by photodissociating synthetic dioxygen carriers such as dicobalt u-peroxo polyaine complexes. Both the PAF method and the photodissociation of the dicobalt u-peroxo and u-superoxo polyamine complexes represent new approaches to study the fast dioxygen reactions of cytochrome oxidase and both avoid the mechanistic ambiguities associated with the fate of photodissociated CO in transitional flow-flash experiments. 3) The mechanism of the redox-linked proton pump in cytochrome oxidase will be investigated. The kinetics of electron transfer and proton pumping upon flash-induced oxidation of cytochrome oxidase reconstituted into phospholipid vesicles will be monitored using time-resolved optical absorption spectroscopy. The proton pumping reactions will be probed by pH indicators located int he extra-vesicular space, trapped inside the vesicles, or covalently bound to the lipid or protein. These studies will allow us to correlate proton pumping events with individual steps in the dioxygen/cytochrome oxidase redox cycle and will provide a foundation for a structural model of the energy transduction mechanism in cytochrome oxidase.

描述：建议的研究将集中在电子的机制上。 呼吸作用中的转移和质子泵送。特别强调的是 细胞色素氧化酶，两个过程之间的主要偶联部位。 我们的目标包括以下几个方面：1)减少排放的机制 二氧嘧啶通过细胞色素氧化酶对水进行多种条件下的研究 条件与流动-闪光法，它使用的光解性 CO络合物启动与O2的氧化还原活性。时间分辨率 将使用多通道光学吸收光谱来跟踪 电子和质子在纳秒时间尺度上的转移动力学 毫秒。奇异值分解与全局指数拟合 采用动力学分析和紫外-可见光谱分析的方法 瞬变中间体的光谱。这些研究应该提供新的 深入探讨细胞色素氧化酶还原氧气的机理。 2)CO光解的替代物将被用于研究 氧与细胞色素氧化酶的反应。未配位的还原反应 细胞色素氧化酶与氧气的研究将使用超快直接 混合法，脉冲加速流(PAF)。氧与水的反应 还将使用产生的O2来研究未连接的酶 在任何相关时间尺度上通过光解合成二氧化氧进行原位 载体如二钴u-过氧基多胺络合物。这两种PAF方法 U-过氧基和U-超氧基多胺双钴的光解离 络合物代表了研究氧的快速反应的新途径 细胞色素氧化酶和两者都避免了机制上的歧义 过渡流动闪蒸实验中光解CO的去向。 3)细胞色素氧化酶氧化还原连接质子泵的作用机制 被调查。电子转移和质子泵浦的动力学 闪光诱导细胞色素氧化酶氧化重组为 将使用时间分辨光学技术监测磷脂小泡 吸收光谱学质子泵浦反应将通过pH来探测 位于囊外间隙的指示器，困在 囊泡，或共价结合到脂质或蛋白质上。这些研究将 使我们能够将质子泵浦事件与 氧/细胞色素氧化酶氧化还原循环，将提供一个基础 细胞色素氧化酶能量传递机制的结构模型。