The functionality of the membrane-bound, ubiquinone-dependent pyruvate oxidase from the respiratory chain of Escherichia coli was reconstituted with a supported lipidic structure. The artificial structure was especially designed to allow the electrochemical control of the quinone pool through the lateral mobility of the ubiquinone (Q(8)) molecules. The kinetic coupling of the enzyme bound to the lipid structure with the quinone pool was ensured by the regeneration of the oxidized form of ubiquinone at the electrochemical interface. Such an experimental approach enabled us to carry out an unprecedented determination of the kinetic parameters controlling the reaction between the enzyme bound and the electron carrier under conditions taking rigorously into account the fact that the freedom of motion is restricted to two dimensions. The kinetic constants we found show that the activated enzyme can be efficiently regulated by the oxidation level of the quinone pool in natural membranes.