Structural and functional insights into ba3-type cytochrome c oxidase using X-ray crystallography and In Crystallo spectroscopy

Abstract

Cellular respiration extracts the energy stored in glucose through a series of electron and proton transfer reactions so as to produce ATP, the primary energy currency of living cells. A key step in this process is oxidative phosphorylation, whereby electrons pass through an electron transport chain while generating a proton concentration gradient across an energy-transducing biological membrane. Cytochrome c oxidase (CcO) is the terminal enzyme of this electron transport chain and facilitates the final electron transfer to molecular oxygen, reducing it to water while simultaneously driving proton translocation across the cellular membrane. Understanding CcO structural and functional properties, including electron and proton transfer and oxygen binding dynamics, is crucial for unraveling the molecular mechanisms of energy conversion. This knowledge provides insights into bioenergetics, metabolic regulation, and potentially has biomedical applications since several diseases are linked to the dysfunction of CcO. This PhD thesis focuses upon the ba3-type CcO from Thermus thermophilus. To gain functional insights into CcO we used X-ray crystallography, a structural biology technique that provides high-resolution three-dimensional snapshots of the enzyme. Since static X-ray structures do not fully capture the enzyme’s dynamic nature, we employed time-resolved serial X-ray crystallography to visualize structural changes in the ba3-type CcO after dioxygen is reduced to water. Additionally, UV-Vis spectroscopy was used to monitor the redox state of the enzyme, allowing greater control over diverse experiments, including during X-ray-induced photoreduction studies. As this work proceeded, numerous experimental limitations had to be addressed, including difficulties in working with photocaged oxygen as a tool for reaction initiation, maintaining an aerobic conditions to ensure a fully reduced enzyme, and the reducing effects of photoelectron. Nevertheless, when taken together our structural results build a consistent picture of conformational changes associated with the ba3-type CcO, as the enzyme is reduced by photoelectron, as the enzyme slowly turns over in the presence of residual dioxygen, as the enzyme rapidly turns over after UV laser pulse releases dioxygen from photocaged oxygen, and as the pH changes. These structural results enhance our understanding of the mechanism by which the enzyme performs its catalysis and translocates protons. These findings are discussed in detail within this thesis.