Benefits of membrane electrodes in the electrochemistry of metalloproteins: mediated catalysis of Paracoccus pantotrophus cytochrome c peroxidase by horse cytochrome c: a case study

Abstract

A comparative study of direct and mediated electrochemistry of metalloproteins in bulk and membrane-entrapped solutions is presented. This work reports the first electrochemical study of the electron transfer between a bacterial cytochrome c peroxidase and horse heart cytochrome c. The mediated catalysis of the peroxidase was analysed both using the membrane electrode configuration and with all proteins in solution. An apparent Michaelis constant of 66 +/- 4 and 42 +/- 5 microM was determined at pH 7.0 and 0 M NaCl for membrane and bulk solutions, respectively. The data revealed that maximum activity occurs at 50 mM NaCl, pH 7.0, with intermolecular rate constants of (4.4 +/- 0.5) x 10(6) and (1.0 +/- 0.5) x 10(6) M(-1) s(-1) for membrane-entrapped and bulk solutions, respectively. The influence of parameters such as pH or ionic strength on the mediated catalytic activity was analysed using this approach, drawing attention to the fact that careful analysis of the results is needed to ensure that no artefacts are introduced by the use of the membrane configuration and/or promoters, and therefore the dependence truly reflects the influence of these parameters on the (mediated) catalysis. From the pH dependence, a pK of 7.5 was estimated for the mediated enzymatic catalysis.