Simple voltammetric approach for characterization of two-step surface electrode mechanism in protein-film voltammetry

Abstract

Many enzymes embedding multivalent metal ions or quinone moieties as redox-active centres undergo electrochemical transformation via two successive electron transfer steps. If electrochemical features of such redox enzymes are analyzed with “protein-film voltammetry”, one frequently meets a challenging reaction scenario where the two electron transfers take place at the same formal potential. Under such conditions, one observes voltammogram with a single oxidation-reduction pattern hiding voltammetric features of both redox reactions. By exploring some aspects of the two-step surface EECrev mechanism one can develop simple methodology under conditions of square-wave voltammetry to enable recognizing and characterizing each electron transfer step. The method relies on the voltammetric features of the second electron transfer, which is coupled to a follow-up chemical reaction. The response of the second electron transfer step shifts to more positive potentials by increasing the rate of the chemical reaction. The proposed methodology can be experimentally applied by modifying the concentration of an electrochemically inactive substrate, which affects the rate of the follow-up chemical reaction. The final voltammetric output is represented by two well-separated square-wave voltammetric peaks that can be further exploited for complete thermodynamic and kinetic analysis of the EECrev mechanism.