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：拟议的研究将重点关注电子的机制 转移和质子抽水。 特别强调 细胞色素氧化酶，这两个过程之间耦合的主要位点。 我们的目标包括：1）减少的机制 将在各种 使用流闪 - 方法的条件，该方法使用 CO复合物可以用O2启动氧化还原活性。 时间分辨 多通道光吸收光谱将用于遵循 电子和质子转移的动力学在纳米秒的时间尺度上转移到 毫秒。 奇异价值分解和全球指数拟合 方法将用于分析动力学并确定UV-VIS 瞬态中间体的光谱。 这些研究应该提供新的 深入了解细胞色素氧化酶还原二氧化物的机制。 2）CO光解离的替代方法将用于研究 O2与细胞色素氧化酶的反应。 无配置的反应减少了 将使用超快速直接研究对氧的细胞色素氧化酶进行研究 混合方法，脉冲加速流（PAF）。 氧与 也将使用产生的O2研究非配体的酶 在任何相关时间尺度上原位通过光解离合成二氧化物 携带者，例如二氧化甲苯烷u-peroxo polyaine络合物。 两种PAF方法 以及二氧化甲虫和u-superoxo多胺的光解离 复合物代表了研究的新方法来研究快速的二氧化反应 细胞色素氧化酶，并且都避免了与机械歧义相关的机械歧义 在过渡流动闪存实验中的光解散CO的命运。 3）细胞色素氧化酶中氧化还原连接的质子泵的机制将 被调查。 电子传递和质子泵送的动力学 闪光诱导的细胞色素氧化酶的氧化被重构 磷脂囊泡将使用时间分辨的光学监测 吸收光谱。 质子抽水反应将通过pH探测 指示器位于int静脉外空间，被困在内部 囊泡，或共价结合到脂质或蛋白质上。 这些研究会 允许我们将质子抽水事件与单个步骤相关联 二恶英/细胞色素氧化酶氧化还原周期，将为A提供基础 细胞色素氧化酶中能量转导机制的结构模型。