Mitochondrial cytochrome c oxidase has been studied intensively because of its essential role in energy metabolism. It is the final electron acceptor of the respiratory chain. Additionally, it pumps protons to the outside of the membrane. These protons are used for the production of ATP as an energy source for the cell. However, because the mitochondrial enzyme has structural and genetic complexity it is not very amenable to analysis. The cytochrome c oxidase from Rhodobacter sphaeroides has a high degree of structural and functional homology but it is a simpler system. Using this enzyme it has been possible to determine much of the structure and mechanism of the enzyme using site-directed mutagenesis. New crystal structures of the oxidase cannot show the proton pathways but they suggest likely positions for a proton relay system. The aims of the proposal are to establish sensitive, time-resolved procedures for the analysis of rates of proton pumping with the enzyme reconstituted into defined, artificial vesicles. These methods will be used to study current site-directed mutants that are deficient in proton pumping and any future mutants, in order to define the route and the mechanism of proton translocation. Knowledge of the proton pathway will give insights into the most challenging question of how the reduction of oxygen to water is energetically coupled to proton movement across the membrane.