Publication 548

Contribution from the Laboratoire d'Electrochimie Moléculaire de l'Université Denis Diderot (Paris 7), UMR CNRS 7591, 2 place Jussieu, 75251 Paris Cedex 05, France

A precise determination of the complex mechanism of catalysis and inhibition involved in the reaction of HRP with H₂O₂ as substrate and an outersphere single electron donor ([Os(bpy)₂pyCl]⁺) as cosubstrate is made possible by a systematic analysis of the cyclic voltammetric responses as a function of the scan rate and of the substrate and cosubstrate concentrations, complemented by spectrophotometric steady-state and stopped-flow experiments. The bell-shaped calibration curve relating the electrochemical response to the concentration of H₂O₂ is qualitatively and quantitatively explained by taking into account the conversion of the catalytically active forms of the enzyme into the inactive oxyperoxidase in addition to the primary catalytic cycle. These characteristics should be kept in mind in biosensor applications of HRP. The ensuing analysis and data allow one to predict biosensor amperometric responses in all practical cases. From a mechanistic standpoint, conditions may, however, be defined which render inhibition insignificant, thus allowing an electrochemical characterization of the primary catalytic cycle. At very low concentrations of H₂O₂, its diffusion tends to control the electrochemical response, resulting in proportionality with H₂O₂ concentration instead of the square root dependence characteristic of the classical catalytic currents. Intriguing hysteresis and trace crossings behaviors are also quantitatively explained in the framework of the same mechanism. As a consequence of the precise dissection of the rather complex reaction mechanism into its various elementary steps, a strategy may be devised for gaining a better understanding of the mechanism and reactivity patterns of each elementary step.