Direct electron transfer in the system gold electrode-recombinant horseradish peroxidases

The kinetics of the bioelectrocatalytic reduction of hydrogen peroxide has been studied at gold electrodes modified with different forms of horseradis h peroxidase (HRP). Native HRP, wild type recombinant HRP (rec-HRP) and its two mutant forms containing a six-histidine tag at the C- or N-terminus, C (His)rec-HRP and N(His)rec-HRP, respectively, have been used for an adsorpt ive modification of the gold electrodes. The histidine sequences, i.e, hist idine tags, were introduced into the peroxidase structure by genetic engine ering of non-glycosylated rec-HRP using an Escherichia coli expression syst em. Experiments with a gold rotating disc electrode demonstrated that elect rodes with the adsorbed rec-HRP forms exhibited high and stable current res ponse to H2O2 due to its bioelectrocatalytic reduction based on direct (med iatorless) ET between gold and the active site of HRP. The heterogeneous ET rate constants were evaluated to be in the order of 20 or 33 s(-1) between rec-HRP or its histidine mutants and gold, respectively, in 0.01 M phospha te buffer (pH 7.4) containing 0.15 M NaCl. The increase in the heterogeneou s ET rate found for C(His)rec-HRP and N(His)rec-HRP is probably due to the interaction of the histidine tag with the electrode surface. The kinetic da ta demonstrate that new possibilities for enhancing the catalytic activity of the enzyme at the electrode I solution interface can be achieved by gene tic engineering design of the enzyme molecules. (C) 2001 Elsevier Science B .V. All rights reserved.