Time-Resolved X-ray Crystallography and Quantum Chemical Calculations of the Proton Pumping Mechanism in Cytochrome c Oxidase

Abstract

Aerobic respiration and photosynthesis are arguably the two most essential processes for life on earth as known to us. These two processes occur on an immense scale every day and the electron transport chain of aerobic organisms effectively utilizes the energy available from the exergonic reduction of the high potential electron acceptor oxygen. In order to couple the exergonic reaction to energy available for the cell, an electrochemical gradient is generated across the cell membrane which can be used for energy demanding process. The last step of the electron transport chain's generation of the gradient is performed by the enzyme cytochrome c oxidase which catalyses the formation of water and makes use of the free energy by the translocation of protons across the membrane against this gradient. How the enzyme couples this proton translocation to the exergonic redox events of the catalytic mechanism is currently not known in detail, although many of the requirements of the event have been elucidated. Insights of the mechanistic details can help not only to understand this vital process, but also essential aspects of energy transduction efficiency with potential future applicability. Time-Resolved Serial Femtosecond X-ray crystallography enables the 3D visualization of protein structures by their illumination in the crystalline form. By collection of diffraction images at certain time delays in relation to the reaction initiation, structural information of the catalytic mechanism can be retrieved. There are many technical barriers to this however, and those are the topic of the first half of this thesis where overcoming them is the main hurdle. Novel structural information is successfully retrieved in three different papers while one paper investigates and optimizes the chemical barriers to enabling the experiment to as great extent as possible. In the last part of the thesis additional insights of the mechanistic details of the catalytic cycle are gained by applying computational chemistry to study processes within the active site of the enzyme. These methods can give even further detailed information for where temporal and spatial resolution limit the experimental techniques and the insights gained from these are presented in the last two papers.