Quantum chemical analysis of different states of Cytochrome c Oxidase from Serial Femtosecond X-ray crystallographic coordinates
Title: Quantum chemical analysis of different states of Cytochrome c Oxidase from Serial Femtosecond X-ray crystallographic coordinates
DNr: NAISS 2023/22-546
Project Type: NAISS Small Compute
Principal Investigator: Jonatan Johannesson <jonatan.johannesson@gu.se>
Affiliation: Göteborgs universitet
Duration: 2023-07-01 – 2024-07-01
Classification: 10407


The electrochemical gradient is the difference in charge and concentration across the inner mitochondrial membrane. It serves as the link between energy acquisition and utilization by the cell and is therefore vital to all cellular life as we know it. In aerobic organisms, it is generated by the reduction of oxygen, which serves as the terminal electron acceptor of the electron transport chain. The enzyme responsible for the reduction of oxygen is Cytochrome c Oxidase, which utilizes the free energy available in the reduction of oxygen to water to translocate protons across the membrane, further contributing to the gradient. The mechanism of this proton pump has been studied extensively over many decades, and a lot is known today. However, there are several important pieces essential to its function which are unknown despite the wealth of structural information from protein crystallography as well as time-resolved spectroscopic techniques currently available. Quantum chemical investigations of its function, starting from crystallographic coordinates of the enzyme, has in recent years contributed greatly to the advancement of the field by different research groups. The starting coordinates are however limited to certain states of the enzyme, mainly the resting state, and the quantum chemical calculations have to extrapolate to other states. Many structures are also plagued by X-ray induced radiation damage of the sample and by the X-ray induced reduction of the metal centers, which form the active site of the enzyme, and therefore altering its state. The attempts by our research group of capturing time-resolved structural information from Time-Resolve Serial Femtosecond X-ray crystallography (TR-SFX), utilizing femtosecond X-ray pulses from X-ray Free-Electron Laser (XFEL) light sources, provide us with unique structural information related to the enzymatic proton pumping mechanism. In addition to this, the technique avoids the X-ray induced radiation damage to the sample, as the diffraction pattern outruns the radiation damage and each crystal is only illuminated once. This provides us with previously inaccessible coordinates of different enzymatic states to a higher resolution (~1.7 Å) than most previous structures, eliminating many uncertainties compared to lower resolution resting state structures. These coordinates need to be interpreted and quantum chemical calculations can provide much additional information from these unique coordinates with regards to the identity of the active site intermediates and protonation states for elucidating the mechanism. Considering that protons are not resolved at the resolution of the crystallographic structures, but bond lengths between other types of atoms can be clearly differentiated between different enzymatic states, quantum chemistry can provide a great toolbox for clarifying the experimental data. The acquisition of intermediate coordinates from TR-SFX leaves a lot of uncertainties out of the equation and allows the site of interest to be treated to a high level of theory. Our aim is that quantum chemical calculations with these unique coordinates will allow us to gain further insight to the mechanism of the proton pumping of this enigmatic enzyme.