Direct un-mediated electrochemistry of the enzyme P-cresolmethylhydroxylase

Abstract The enzyme p -cresolmethylhydroxylase, from Pseudomonas putida , is involved in the degradative pathway of p -cresol. The direct, un-mediated reversible electron transfer between the heme of the flavocytochrome enzyme and an edge-plane graphite electrode in the presence of redox-inactive cationic species as promoters of the enzyme electrochemistry is reported. Diffusion controlled heterogeneous electron transfer is modulated by the levels and type of promoter used, which range from simple and complex cations to polyamines and aminoglycosides. The promoter-enzyme assembly affords a degree of macromolecular recognition at the electrode as both the cationic charge and the flexible nature of the promoter are important to achieve direct electron transfer. A quasi-reversible cyclic voltammetric response was observed in the absence of substrate from which the heterogeneous rate of electron transfer between the enzyme and the electrode was determined as k s ~ 1.8 × 10 −4 cm 2 s −1 . An enzyme titration with optimal concentrations of promoter and substrate p -cresol yielded a linear relationship between catalytic current and enzyme concentration up to 1.2 μ M . Similarly, relationships were obtained to p -cresol and p -hydroxybenzylalcohol concentrations with fixed amounts of enzyme and promoter, both yielding a linear current vs. substrate response up to 0.5 m M . Furthermore, the magnitude of the catalytic current of p -cresol is greater than that obtained for mediation with the enzyme's natural redox partner, azurin, but comparable to those obtained with a ferrocene mediator. Catalytic response was also obtained at a peptide-modified gold electrode in the presence of a promoter, spermine.