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: 2872706
• 负责人: OLOF EINARSDOTTIR
• 金额: $ 14.72万
• 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/2872706
• 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：拟议的研究将集中在电子的机制 呼吸中的质子传递和泵送。 将特别强调 细胞色素氧化酶，这两个过程之间的耦合的主要网站。 我们的目标包括：1）减少的机制， 将在各种条件下研究细胞色素氧化酶对水的氧化作用。 条件下的流动闪光法，它使用的光的能力， CO络合物以引发与O2的氧化还原活性。 时间分辨 多通道光学吸收光谱将用于跟踪 电子和质子在纳秒到 毫秒 奇异值分解与全局指数拟合 方法将被应用于分析动力学和测定紫外-可见光谱 瞬态中间体的光谱。 这些研究将提供新的 深入了解细胞色素氧化酶还原分子氧的机理。 2)CO光解的替代物将用于研究 O2与细胞色素氧化酶的反应。 非配体还原的反应 细胞色素氧化酶与氧将研究使用超快直接 混合方法，脉冲加速流（PAF）。 氧气与 还将使用产生的O2研究未配体的酶， 在任何相关的时间尺度上原位通过光解合成的分子氧 载体如二钴U-过氧多酸络合物。 PAF方法 以及二钴的u-过氧和u-超氧多胺的光解 配合物代表了研究快速双氧反应的新方法， 细胞色素氧化酶，两者都避免了相关的机械模糊性， 与过渡流闪光实验中光解CO的命运。 3)细胞色素氧化酶中的氧化还原质子泵机制将 追究 电子转移和质子泵动力学 闪光诱导的细胞色素氧化酶氧化重组为 磷脂囊泡将使用时间分辨光学 吸收光谱学 质子泵的反应将探测pH值 位于囊外空间的指示器，被困在 囊泡，或共价结合到脂质或蛋白质。 这些研究将 使我们能够将质子泵送事件与 双氧/细胞色素氧化酶氧化还原循环，并将提供基础， 细胞色素氧化酶能量传递机制的结构模型。