Electrochemical fingerprints of sequential double-regenerative enzymatic mechanisms in protein-film voltammetry

Protein-film square-wave voltammetry provides a powerful framework for exploring complex redox mechanisms in which surface-confined electron-transfer reactions are coupled to enzymatic regeneration. In this theoretical work, we investigate the voltammetric behavior of a sequential two-step electron-transfer mechanism coupled to two regenerative chemical reactions under square-wave voltammetric conditions. Particular attention is devoted to the effects of the kinetics of both electron-transfer steps and the two regenerative reactions on the shape, position, and magnitude of the resulting voltammetric responses. The simulations reveal characteristic changes in the forward, backward, and net SWV components, providing distinct electrochemical fingerprints for different kinetic regimes of the double-regenerative mechanism. Theoretical analysis further identifies conditions under which the individual kinetic contributions can be distinguished and evaluated experimentally. The presented results establish a mechanistic basis for recognizing complex sequential catalytic pathways in protein-film voltammetry and may assist in the kinetic characterization of surface-confined redox enzymes exhibiting multiple electron-transfer and regenerative steps.