Structure and Function of Cytochrome c Oxidase

Sammanfattning: Aerobic organisms, ranging from bacteria to humans, obtain their energy through the process of respiration. Electrons from the breakdown of food are transported through the membrane-bound enzyme complexes of the respiratory chain, to the terminal acceptor molecule, oxygen. The energy released in the process is used to transport protons across the cell membrane, thereby establishing an electrochemical gradient that can be utilised e.g. to synthesise ATP. The last respiratory complex in mitochondria and many bacteria is cytochrome c oxidase (CcO). CcO catalyses the reduction of oxygen to water, and at the same time pumps protons from the more negative to the more positive side of the membrane. In spite of extended and intensive research efforts, the detailed mechanism by which the exergonic electron-transfer reactions are coupled to the endergonic proton pumping is still not understood.The focus of this thesis is the structure and function of CcO from the bacterium Rhodobacter sphaeroides. The structures of the wild-type enzyme and two mutant enzymes have been investigated, providing insights into structural changes taking place upon replacing a glutamate residue (E286) in one of the proton-transfer pathways. E286 is a key element in directing protons either to the catalytic site for the oxygen chemistry or towards the outside for pumping. Results from time-resolved optical absorption spectroscopy show that the pKa of E286 has to be close to 9.5 for the proton pump to function, and that the pKa can be modulated by mutations at a distance of ~25 Å from E286. From another study we conclude that the D-propionate of haem a3 is most likely the acceptor site for pumped protons. Finally, we have identified the structural element which conformational changes determine the reaction rate of each proton-pumping step.The results are summarised in a molecular model for proton pumping by CcO, in which deprotonation of E286 drives proton translocation across the membrane.