Direct electrochemistry of recombinant tobacco peroxidase on gold

Direct electron transfer (DET) reactions of recombinant tobacco peroxidase (rTOP), namely direct electroreduction of Compound I/Compound II and heme Fe^3+/2+ conversion, were studied on gold electrodes. rTOP of wild type, non-glycosylated, was produced using an Escherichia coli expression system. At pH 5.0, the redox potential for direct electrochemical transformation of the Fe^3+/2+ of the peroxidase heme was -143 mV vs. Ag|AgCl, and 0.26 ± 0.07 pmol of the adsorbed rTOP were in DET contact with the gold electrode. The total amount of the adsorbed rTOP estimated from QCM data was 53 ± 5 pmol/cm² or 1.67 pmol when referred to the surface area of the electrodes used for electrochemical measurements. Of 1.67 pmol of adsorbed rTOP, only 0.76 pmol were catalytically active. DET between Au and the enzyme was also studied in the reaction of the bioelectrocatalytic reduction of H ₂O₂ by cyclic voltammetry and amperometric detection of H₂O₂ at +50 mV with rTOP-modified Au electrodes placed in a wall-jet flow-through electrochemical cell. Maximal bioelectrocatalytic current response of the rTOP-modified gold electrodes to H₂O ₂ was observed at pH 5.0 and stemmed from its bioelectrocatalytic reduction based on DET between Au and the active site of rTOP. Kinetic analysis of the DET reactions gave 52% of the adsorbed rTOP molecules active in DET reactions (0.4 pmol of adsorbed catalytically active rTOP, correspondingly), which correlated well with the non-catalytic-voltammetry data. DET was characterised by a heterogeneous ET rate constant of 13.2 s^-1, if one takes into account the QCM data, and 19.6 s^-1, if the amount of rTOP estimated from the data on DET transformation of Fe^3+/2+ couple of rTOP is considered. The sensitivity for H₂O₂ obtained for the rTOP-modified Au electrodes was 0.7 ± 0.1 A M^-1 cm ^-2. These are the first ever-reported data on DET reactions of anionic plant peroxidases on bare gold electrodes.