Structural studies of ba3-type cytochrome c oxidase using serial crystallography and X-ray absorption spectroscopy

Sammanfattning: Cytochrome c oxidase (CcO) catalyses the reduction of molecular oxygen to water while the energy released in this process is used transport protons “up-hill” across an energy transducing biological membrane, creating a proton-motive force for ATP synthesis. Given its key role in energy transduction in organisms, proton pumping has been extensively studied across species. Even though many members of the CcO superfamily have been structurally characterized in detail, time-resolved structural details of electron transfer coupled to proton pumping have not been entirely understood. A billion-fold jump in the peak X-ray brilliance delivered by X-ray free electron laser (XFEL) and the development of serial femtosecond crystallography (SFX) allowed the determination of protein structures at room temperature, opening up the opportunities to measure ultrafast reactions in proteins. Moving beyond the study of only light-sensitive systems, our long-term goal is to use time-resolved serial femtosecond crystallography at XFELs for a comprehensive structural study of cytochrome c oxidase activity. First, we employed a standard pump-probe SFX setup in a time-resolved (TR) study of structural events following the photodissociation of carbon monooxide (CO) from a reduced CO-bound ba3-type CcO. In the following study, we performed TR-SFX to track structural changes at the active site of ba3-type CcO upon photoinitiated release of oxygen molecule from cobal-based cage compound. Moreover, to utilize the potential of synchrotron radiation and lower the entry barrier for serial crystallography at synchrotron beamlines, we designed and experimentally validated a flow-cell device for serial synchrotron crystallography (SSX) at room-temperature. Finally, we used X-ray absorption spectroscopy (XAS) to access electronic configuration and coordination geometry of copper and iron co-factors of ba3-type CcO in different redox states. Qualitative analysis of X-ray absorption near-edge structure (XANES) and theory-based model of extended X-ray absorption fine structure (EXAFS) highlighted differences between analyzed samples, leaving room for additional computational input to obtain a detailed fingerprint of ba3-type CcO redox states and better understanding of possible ligands in the heme a3-CuB active site. The work presented in this thesis highlights the importance of utilizing different methods for comprehensive understanding of enzyme reaction dynamics.