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

Electrochemical transformations of many lipophilic redox proteins and enzymes proceed through sequential two-step electron-transfer pathways that are frequently coupled with regenerative catalytic reactions accompanying both electron-transfer steps. Although protein-film voltammetry (PFV) provides a powerful experimental platform for direct investigation of such systems, theoretical descriptions of surface-confined double-regenerative mechanisms remain largely unexplored. In this work, we present, for the first time, a comprehensive theoretical framework describing a two-step surface-confined double-regenerative mechanism under square-wave protein-film voltammetry conditions. Systematic theoretical analysis demonstrates how the interplay between heterogeneous electron-transfer kinetics and successive regenerative catalytic reactions governs the shape, position, intensity, and kinetic characteristics of the corresponding square-wave voltammetric responses. Simulations reveal a broad spectrum of characteristic electrochemical fingerprints that can be directly associated with variations in the intrinsic kinetic parameters of both electron-transfer and catalytic steps. The developed framework provides a robust basis for mechanistic interpretation of experimentally recorded protein-film voltammograms and establishes practical diagnostic criteria for distinguishing different kinetic regimes. Furthermore, the proposed approach offers a reliable platform for developing experimental protocols aimed at quantitative extraction of electron-transfer and catalytic rate constants, thereby enabling evaluation of enzymatic activity and reaction pathways of various redox proteins and enzymes. The presented model considerably expands the theoretical foundation of protein-film square-wave voltammetry applied to surface-confined redox proteins and enzymes.