Thermodynamic, Kinetic, and UV–Vis/CD Spectroelectrochemical Studies on Interaction and Electron Transfer Between Glucose Oxidase and Ferrocene Carboxylic Acid

In this research, we investigate the interaction between the redox mediator ferrocene carboxylic acid (Fc-COOH) and glucose oxidase (GOD) in order to determine the thermodynamics parameters K_int, ΔG_int, ΔH_int, and ΔS_int using simple UV-visible experiments at different temperatures. Positive values of ΔH_int, ΔS_int, together with a negative value of ΔG_int indicate an entropy-driven hydrophobic interaction typical of spontaneous association processes. The homogeneous electron transfer rate constants between the oxidized organometallic mediator and the reduced enzyme (k_s), along with their activation parameters (ΔG_ET^≠, ΔH_ET^≠ and ΔS_ET^≠), were calculated using data obtained from foot of the wave analysis (FOWA) of cyclic voltammetry experiments performed at variable temperature. According to transition state theory, the obtained parameters indicate a low activation enthalpy that reflects minimal energetic requirements for electron transfer, while the large negative activation entropy suggests the formation of an ordered transition state. The positive activation free energy falls within the expected range for biological electron transfer processes. Variable temperature cyclic voltammetry experiments of ferrocene carboxylic acid (Fc-COOH) were also performed. The obtained ΔG°, ΔH°, and ΔS° parameters indicate strong stabilization of the redox pair, consistent with a small difference in solvation energy. Circular dichroism, UV-vis spectroscopy, and combined CD and UV-Vis Spectroelectrochemistry measurements performed during redox mediation demonstrate that no significant structural alterations occur in either the enzyme or the redox mediator before or during the electron transfer processes.