Abstract: The effect of pH on the kinetics of the electroreduction of H2O2 catalyzed by horseradish peroxidase (HRP) has been studied with LSV in the potential range from 700 to -50 mV versus SCE (under steady-state conditions and with an RDE system) and at -50 mV versus Ag/AgCl on HRP-modified graphite electrodes placed in a wall-jet flow-through electrochemical cell. Increasing [H3O+] was shown to enhance significantly the current of the bioelectroreduction of H2O2 due to direct electron transfer (ET) between graphite and the enzyme over the potential range involved. It is demonstrated that at high overvoltages (E3O+ does not affect the rate of the enzymatic reduction of H2O2, but it increases the rate of direct ET between graphite and HRP. The values of the apparent rate constant of heterogeneous ET between HRP and graphite, ks, changed from a value of 0.54±0.05 s-1 in phosphate buffer solution (PBS) at pH 7.9, to a value of 11.0±1.7 s-1 in PBS at pH 6.0. Analysing the pH rate profile and the variation of the ks with increasing [H3O+] made it possible to consider the reaction mechanism as implying the participation of a proton in the limiting step of charge transfer.

Abstract: The effect of pH on the kinetics of the bioelectrocatalytic reduction of H2O2 catalyzed by horseradish peroxidase (HRP) has been studied at -50 mV vs. Ag/AgCl on HRP-modified Au electrodes placed in a wall-jet flow-through electrochemical cell. Native HRP (nHRP) and a nonglycosylated recombinant form containing a six-histidine tag at the C-terminus, CHisrHRP, produced by genetic engineering of nonglycosylated recombinant HRP using an E. coli expression system, have been used for adsorptive modification of Au electrodes. A favourable adsorption of CHisrHRP on preoxidized Au from a protein solution at pH 6.0 provided a high and stable current response to H2O2 due to its bioelectrocatalytic reduction based on direct (mediatorless) electron transfer (ET) between Au and the active site of HRP. The heterogeneous ET rate constant, ks, calculated from experimental data on direct ET, on mediated ET in the presence of catechol as well as from microbalance data, increased more than 30 times when changing from nHRP to CHisrHRP. For both forms of HRP, the increasing efficiency of bioelectrocatalysis with increasing [H3O+] was observed. The values of the apparent ks between CHisrHRP and Au changed from a value of 12±2 s-1 in PBS at pH 8.0 to a value of 434±62 s-1 at pH 6.0; a similar ks-pH dependence was also observed for nHRP, providing the possibility to consider the reaction mechanism involving the participation of a proton in the rate-determining step of the charge transfer.