Electrochemically-driven and dynamic enhancement of drug metabolism viacytochrome P450 microsomes on colloidal gold/graphene nanocomposites

Abstract

Cytochrome P450 enzymes (cyt P450s) are iron-heme proteins involved in the metabolism of both endogenous and exogenous compounds. Accurate and rapid in vitro mimicking of the natural metabolic pathways has attracted significant interest from enzyme engineers. Herein, we describe the electrochemically driven and dynamic enhancement of drug metabolism, which is based on the cyt P450 3A4 (CYP3A4) isozyme microsomes with cyt P450 reductase (CPR) immobilized on colloidal gold/graphene nanocomposites via electrostatic interactions. Direct and reversible electron transfer between the electrode and CYP3A4/CPR-microsomes was observed with a formal potential of −0.482 ± 0.003 V. The bioelectrocatalytic response of the P450-electrode has been validated by the catalysis of nifedipine. Through the application of the rotating disk electrode, the corresponding kinetic parameters, namely the Michaelis–Menten constant and the heterogeneous reaction rate constant, were calculated to be 1.30 μM and 7.12 cm s⁻¹, respectively. The inhibition effect of ketoconazole on the oxidation of nifedipine was also investigated and the IC₅₀ value was calculated to be 0.23 μM. The bioelectrocatalytic products were analyzed through high performance liquid chromatography-mass spectrometry (HPLC-MS) measurements. This new format of the cyt P450s system could probe the catalytic pathway with voltammetric kinetic analyses, and facilitated electronically-driven bioreactor and biosensor applications.