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

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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.