In a previous study [E.T. Smith, C.A. Davis, M.J. Barber, Anal. Biochem. 323 (2003) 114–121], cyclic voltammograms were simulated using DigiSim software for reaction mechanisms involving multiple electron transfer steps coupled to proton transfer. Specifically, the overall reaction mechanism of the form: FAD+2e−+2H+⇌FADH2 was used to simulate experimental reduction potentials as a function of pH. Experimental observations for free FAD were simulated based on selected reduction potentials and acid dissociation constants for three different reaction mechanisms. In this study, these three reaction mechanisms were examined further using simulations of concentration profiles to identify species that are present in significant concentrations during the electron transfer process. These concentration profiles can then be used as working curves to identify reaction mechanisms and equilibrium constants. For example, two of the three reaction mechanisms, both of which involve a redox reaction coupled to a single proton, indicate significant formation of the semiquinone species at the electrode surface at high pH and low potentials. Previous spectroscopic studies of flavoproteins with an n=2 reaction mechanism have been unable to detect the formation of a semiquinone under any experimental conditions. Thus, the most reasonable pathway for a proton-coupled n=2 reaction mechanism is likely to involve two protons.