Structural elements involved in protein-mediated proton transfer : Implications from studies of cytochrome c oxidase

Proton transfer is one of the most common reactions in biological systems. During energy conversion inside a cell, proton transfer is crucial to maintain an electrochemical proton gradient across the cell membrane. This gradient is in turn used to e.g. produce ATP, the energy currency of the cell. One of the key components of the build-up of this gradient is cytochrome c oxidase. This membrane-bound enzyme catalyzes the reduction of molecular oxygen to water, using protons and electrons, and in the process protons are pumped across the membrane. All protons used during oxygen reduction and those that are pumped, are transferred via hydrophilic pathways inside the hydrophobic interior of the enzyme. One of these pathways, called the D pathway, is used to transfer protons both to the catalytic site and towards a pump site. It is yet not fully understood how these proton-transfer reactions are timed, coupled and controlled. This thesis is focused on studies of proton-transfer reactions through the D pathway in variants of cytochrome c oxidase that lack the ability to pump protons. The results suggest that changes in pKa values of key residues, as well as structural changes inside the pathway, can explain the non-pumping phenotypes. The results have led us to propose that an internal proton shuttle (Glu286I) can adopt two different conformations that are in equilibrium with each other, and that this equilibrium is altered in non-pumping variants of cytochrome c oxidase. We also observed that proton transfer through the D pathway could occur with the same rate as in the wild-type enzyme even when one of the key residues (Asp132I) is absent. This result contradicts previous assumptions that acidic residues must be present at an orifice of proton pathways. We therefore suggest that this specific residue could have an additional role, e.g. as a selectivity filter that excludes all ions except protons from entering the pathway.At the time of doctoral defence the following papers were unpublished and had a status as follows: Paper 2: Accepted; Paper 3: Manuscript