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: 2023200
• 负责人: OLOF EINARSDOTTIR
• 金额: $ 15.56万
• 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/2023200
• 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：拟议的研究将集中于电子的机制 呼吸中的转移和质子泵送。 将特别强调 细胞色素氧化酶，两个过程之间耦合的主要位点。 我们的目标包括以下内容： 1）减少的机制 将在多种条件下研究通过细胞色素氧化酶将双氧转化为水 流动闪蒸法的条件，该方法利用了光稳定性 CO 与 O2 络合引发氧化还原活性。 时间分辨 多通道光学吸收光谱将用于跟踪 电子和质子在纳秒时间尺度上转移的动力学 毫秒。 奇异值分解和全局指数拟合 方法将用于分析动力学并确定紫外-可见光 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双氧的机制。 2) CO 光解的替代方案将用于研究 O2 与细胞色素氧化酶的反应。 未配体的反应减少 将使用超快速直接技术研究含氧的细胞色素氧化酶 混合方法，脉冲加速流（PAF）。 与氧气反应 还将使用产生的 O2 研究未配体的酶 通过光解合成双氧在任何相关时间尺度上原位进行 载体例如二钴u-过氧聚胺络合物。 两种PAF方法 以及二钴 u-peroxo 和 u-superoxo 多胺的光解 配合物代表了研究快速双氧反应的新方法 细胞色素氧化酶并且两者都避免了相关的机械模糊性 与过渡流动闪蒸实验中光解二氧化碳的命运有关。 3）细胞色素氧化酶中氧化还原连接的质子泵的机制 被调查。 电子转移和质子泵浦的动力学 闪光诱导的细胞色素氧化酶氧化重组为 将使用时间分辨光学来监测磷脂囊泡 吸收光谱。 质子泵浦反应将通过 pH 值进行探测 位于囊外空间的指示器，被困在 囊泡，或共价结合至脂质或蛋白质。 这些研究将 允许我们将质子泵浦事件与各个步骤相关联 双氧/细胞色素氧化酶氧化还原循环，将为 细胞色素氧化酶能量转导机制的结构模型。