Fenton-like Inactivation of Tobacco Peroxidase Electrocatalysis at Negative Potentials

Abstract

The effect of the operational potential on the stability of electrochemical biosensors is particularly relevant in the case of peroxidase biosensors, because these enzymes can catalyze the reduction of hydrogen peroxide via either a high-potential redox cycle [involving Compound I, Compound II, and Fe(III)] or a low-potential redox cycle [involving Fe(III) and Fe(II)]. Herein, it is shown that recombinant tobacco peroxidase immobilized on a graphite surface displays two well-separated electrocatalytic waves, associated with each of these two catalytic cycles. While continuous scanning in the highpotential region does not alter significantly the electrocatalytic current, it is shown that just modest incursions into the low-potential region cause an irreversible loss of the electrocatalytic response. A quantitative analysis of the extent of inactivation as a function of time, potential, and hydrogen peroxide concentration is shown to be consistent with a fast inactivation caused by hydroxyl radicals generated by a Fenton-like mechanism. Accordingly, the inactivation process is shown to slow via the addition of radical scavengers to the solution. Preliminary results indicate that the same inactivation process may also be present in horseradish peroxidase-modified electrodes.

Electrocatalysis by Heme Enzymes—Applications in Biosensing

“…The observed catalytic reduction peak for H 2 O 2 is usually close to the reduction potential of the Fe(III)/Fe(II) redox pair, which indicates that an alternative reaction involving direct reduction of Fe(II)-O 2 species at the electrode, equivalent to the low potential redox cycle described for peroxidases (cf. Section 3.1.1) probably takes place [96]. Further evidence for this hypothesis is the catalytic response to O 2 , observed at similar potential values [98].…”

“…In this case, it is likely that the H 2 O 2 detection follows an alternative non-native route, which proceeds via formation of the ferrous [Fe(II)] enzyme. This low potential redox cycle of peroxidase electrocatalysis has been associated with non-native enzyme states and transient formation of inactive catalytic intermediates (e.g., Fe(II)-O 2 ), which can also be generated in the presence of high concentrations of H 2 O 2 [51,91,95,96]. Among the DET mechanisms proposed to rationalize these experimental findings (cf.…”

“…Among the DET mechanisms proposed to rationalize these experimental findings (cf. reference [96] for details), we highlight the one that postulates that the alternative electrocatalytic reaction follows a catalase-like mechanism, in which H 2 O 2 acts as an oxidant and as a reductant. Consequently, the generated compound I (Equation ( 4)) can be reduced by H 2 O 2 , returning the peroxidase to the Fe(III) state and producing O 2 (Equation ( 7)).…”

Highly Sensitive Hydrogen Peroxide Biosensor Based on Tobacco Peroxidase Immobilized on p‐Phenylenediamine Diazonium Cation Grafted Carbon Nanotubes: Preventing Fenton‐like Inactivation at Negative Potential

“…Although HRP and TOP show a similar reaction mechanism, the two enzymes exhibit different parameters, such as glycosylation, stability, isoelectric point and specificity for various electron donor substrates. [57][58][59] The common peroxidase reaction cycle proceeds through the following reaction steps: [60][61][62][63][64][65][66][67][68] Native…”

“…At this point, it might be important to highlight that the continuous production of •OH would result in a drastic reduction of the operating life of the biosensor due to enzyme denaturation. [67,68] Herein, we report on the development of a new electrode platform based on the immobilization of anionic recombinant tobacco peroxidase (r-TOP) onto p-phenylenediamine diazonium cation (p-PD) grafted carbon nanotubes. [69] This approach allowed to enhance the sensitivity, stability and selectivity of H 2 O 2 detection through electrical wiring of TOP by using glutaraldehyde as cross-linking agent between the p-PD diazonium cation grafted carbon nanotubes and the enzyme.…”