Study by Reflection IR spectroelectrochemistry and Raman spectrometry of the electron transfer process (binding and kinetics) implicating ba3-oxidase and cytochrome c552 from Thermus thermophilus

细胞色素c氧化酶是呼吸链的末端膜蛋白，催化电子从细胞色素c转移到氧气，产生水和ATP。电子从细胞色素c的血红素铁原子转移到细胞色素c氧化酶的CuA氧化还原中心是通过一个复杂的过程发生的，这意味着双方的一些特定氨基酸为途径，以正确的方向结合，最终在它们之间转移电子。参与氧化还原过程的蛋白质通常通过经典电化学来研究其功能。然而，这种技术无法获得它们的结构和功能之间的狭窄联系的信息。为了更好地了解嗜热热菌的祖先细菌ba3氧化酶，我们打算在本项目中使用最新开发的反射光谱电化学电池将IR和拉曼光谱与电化学相结合。金工作电极将被一层细胞色素C552 （ba3氧化酶的氧化还原伙伴）化学修饰。我们的兴趣将集中在这个单分子层和溶液中b分子之间的相互作用。利用FTIR和时间分辨FTIR差分光谱对参与氧化还原过程的ba3氧化酶氨基酸进行鉴定。精心挑选的细胞色素C552突变体也将被研究。电子转移的动力学方面与血红素结构的构象变化在两种蛋白质将研究使用时间分辨拉曼光谱。

Cytochrome c oxidase is the terminal membrane protein of the respiratory chain and catalyses electron transfer from cytochrome c to oxygen for the production of water and ATP. Electron transfer from the heme iron atom of cytochrome c to the CuA redox centre of cytochrome c oxidase occurs through a complex process which implies some specific amino acids of both partners for the approach, binding in the correct orientation and finally electron transfer between them. Proteins involved in redox processes are generally studied by classical electrochemistry to explore their function. However, no information by this technique can be obtained about the narrow link between their structure and their function. For a better understanding of the ancestral bacterial ba3-oxidase from Thermus thermophilus, we intend in this project to combine IR and Raman spectrometry with electrochemistry using a recently developed reflection spectroelectrochemical cell. The gold working electrode will be chemically modified with a monolayer of cytochrome C552, the redox partner of ba3-oxidase. Our interest will be focused on the interaction between this monolayer and b molecules in solution. Amino acids of ba3-oxidase involved in the redox process will be identified with FTIR and time-resolved FTIR difference spectroscopy. Carefully chosen mutants of cytochrome C552 will also be studied. Kinetic aspects of electron transfer with the conformational changes of heme structures in both proteins will be investigated using timeresolved Raman spectroscopy.