Electrochemically-driven turnover of tamoxifen by immobilised human flavin-containing monooxygenase

Flavin-containing monooxygenases (FMOs) are the second most important family of drug-metabolising enzymes in humans and catalyze the oxygenation of a wide variety of drugs, pesticides and other xenobiotics. They use FAD, NADPH and molecular oxygen for their detoxification reaction. FMO3 appears to be the predominant member in the adult human liver and is associated with the majority of FMO-mediated hepatic metabolism, playing a prominent role in the metabolism of clinically important drugs such as the anticancer tamoxifen. The presence of the cofactor FAD in the active site of the FMO3 makes this protein amenable to electrochemical measurements. The reducing equivalents of NADPH cofactor, necessary for the monooxygenation reaction of hFMO3 in vivo, can be substituted by use of electrodes and application of a controlled potential. The achievement of a direct electron transfer between solid electrodes and the protein on the surface provides a simple and efficient way of coupling enzymatic turnover events with signal transduction. Herein we present two methods for electrochemical characterization of human FMO3: non-covalent immobilization on glassy carbon (GC) together with covalent immobilization on gold electrodes. The ability of the immobilized hFMO3 to catalyse the turnover of tamoxifen was investigated by chronoamperometry methods in fully oxygenated buffer where a potential bias of -400 mV (versus NHE) was applied to the electrochemical cell with constant stirring. The catalysis products were separated by HPLC and the presence of tamoxifen N-oxide confirmed the electrocatalytic behaviour of the immobilized enzyme. Finally, the direct correlation between the catalytic current and drug turnover allows us to use these electrodes in drug sensing.