BIOPHYSICAL STUDIES OF OXYGEN ACTIVATION

拟议的研究将集中在激活的生物物理研究上。 氧，特别强调以细胞色素氧化酶为原型 氧气活化系统。我们希望了解这种蛋白质是如何影响 O2的活化、电子转移和偶联过程 呼吸作用中的电子转移和质子转移。特定的 将开展的项目包括： 1.光解离的配体/金属/蛋白质动力学 将研究不同配体与细胞色素氧化酶的重新结合。 光谱活性配体结合探针包括02，CO，NO，CN-， 和异氰化物的衍生物将使用多种技术进行研究 光谱和动力学方法，包括FTIR，瞬时UV-Vis， 时间分辨磁性圆二色谱、EPR和X射线吸收 从纳秒到秒的时间尺度上的光谱学。分子内 下面将研究细胞色素氧化酶中的电子转移 部分还原配体结合酶复合体的光解。这些 研究将为控制反应性的因素提供新的见解 这种蛋白质的活性部位以及它的物理特性 配体的性质(自旋态、电荷和大小)影响反应活性。 2.02与还原的未连接细胞色素的直接反应 将在不需要CO光解的情况下研究氧化物酶 采用一种新的超高速直接混合方法--脉冲加速流。一个 研究该反应的另一种新方法，其中 在任何相关的时间尺度上原位生产氧气是通过 将继续进行合成氧气载体的光解离。 选定的多胺的双钴过氧和钼超氧配合物将 被探索为光影制作的02。这两种方法的结果 将与之前的流动闪蒸结果(CO存在)进行比较。3. 新发现的细胞色素氧化酶、锌、镁和铜中的非血红素金属， 将对它们的结构和机械结构进行调查 多种化学物质和蛋白质在酶的生理功能中的作用 光谱技术。移除金属并代之以 光谱活性金属(Co2+)和发光的稀土元素将 试图获取有关活动的性质的信息 网站。镁结合部位的位置及其可能性 作为核苷酸结合位点的研究将使用新的 三磷酸腺苷的荧光光亲和类似物。一个全面的研究， 包括EPR的金属分析和表征，将进行 建立准确的化学计量学和Cux的性质。

The proposed research will focus on biophysical studies of activation of oxygen with special emphasis on cytochrome oxidase as the archetypal oxygen activating system. We wish to understand how this protein affects the processes of activation of 02, electron transfer, and the coupling of electron transfer and proton translocation in respiration. Specific projects to be undertaken include the following: 1. The ligand/metal/protein dynamics of the photodissociation and rebinding of various ligands to cytochrome oxidase will be investigated. Spectroscopically active ligand binding probes including 02, CO, NO, CN-, and isocyanide derivatives will be investigated using a multitechnique spectroscopic and kinetics approach, including FTIR, transient UV-Vis, time-resolved magnetic circular dichroism, EPR, and X-ray absorption spectroscopy on timescales of nanoseconds to seconds. The intramolecular electron transfer in cytochrome oxidase will be studied following photolysis of partially reduced ligand-bound enzyme complexes. These studies will provide new insight into what factors control the reactivity of the active site of this protein and how the physical characteristics of the ligand (spin state, charge, and size) affect reactivity. 2. The direct reaction of 02 with the reduced unliganded cytochrome oxidase will be studied without the need for photodissociation of CO using a new superfast direct-mixing method, pulsed-accelerated-flow. An alternative novel approach to the study of this reaction, wherein dioxygen is produced in situ on any relevant timescale by photodissociation of synthetic dioxygen carriers, will be pursued. Dicobalt mu-peroxo and mu-superoxo complexes of selected polyamines will be explored for photoproduction of 02. The results from both approaches will be compared to previous flow-flash results (CO present). 3. The newly discovered nonheme metals in cytochrome oxidase, Zn, Mg, and Cux, will be investigated with respect to their structural and mechanistic roles in the physiological function of the enzyme by various chemical and spectroscopic techniques. Removal of the metals and replacement with spectroscopically active metals (CO2+) and luminescent lanthanides will be attempted to obtain information regarding the nature of the active sites. The location of the Mg binding site and the possibility of it serving as a nucleotide binding site will be explored using novel fluorescent photoaffinity analogs of ATP. A comprehensive study, including metal analyses and characterization by EPR, will be carried out to establish the exact stoichiometry and the nature of Cux.