Covalently linked films of the ferric heme protein myoglobin and poly-L-lysine on pyrolytic graphite electrodes reacted with tert-butylhydroperoxide (tBuOOH) to form ferryloxy protein species according to Michaelis-Menten enzyme kinetics. Rotating disk voltammetry data obtained in rnicroemulsions. micellar solution. and buffers revealed a strong influence of water phase acidity on kinetic parameters. Microemulsion and surfactant type had a much smaller influence on reaction kinetics, possibly because the reaction takes place entirely in a water environment surrounding Mb in the films in all fluids. A large apparent Michaelis k(cat) in microemulsions with neutral water phases was offset by much weaker binding as shown by larger protein-substrate dissociation constants (K-m). Acidic SDS microemulsions and pH 2 buffer provided the most efficient reaction conditions as judged by the ratio k(cat)/K-m. Apparent kinetic constants are most likely governed by acidity-controlled protein conformations and their binding with tBuOOH in the intermediate protein-substrate complex.