Direct Electrochemistry of Drug Metabolizing Human Flavin-Containing Monooxygenase: Electrochemical Turnover of Benzydamine and Tamoxifen Sheila J. Sadeghi, † Rita Meirinhos, † Gianluca Catucci, † Vikash R. Dodhia, ‡ Giovanna Di Nardo, † and Gianfranco Gilardi* ,†,‡ Department of Human and Animal Biology, UniVersity of Turin, Italy, and DiVision of Molecular Biosciences, Imperial College London, U.K. Received October 31, 2009; E-mail: g.gilardi@imperial.ac.uk Human flavin-containing monooxygenase 3 (hFMO3) is a microsomal phase I drug metabolizing enzyme that catalyzes the oxygenation of a wide range of nitrogen- and sulfur-containing drugs as shown in Scheme 1 (where S is the substrate). 1 In general, research on electrochemical catalysis of drug-metabolizing mo- nooxygenases has been mainly confined to cytochrome P450 enzymes with FMO largely neglected. FMO studies have been limited to Clarke-type dissolved-oxygen electrode settings. 2 To date there are no reports on direct immobilization strategies of FMO enzymes with electrocatalytic drug turnover. Herein we describe the first direct electrochemistry of hFMO3 immobilized on both glassy carbon (GC) and gold electrodes. The enzyme was cloned, expressed, and purified in a soluble, active form in bacteria. GC electrodes were derivatized with the cationic surfactant didodecylammonium bromide (DDAB). 3 The protein film was prepared by mixing equal volumes of the protein and surfactant before drop-coating onto the electrode surface. A typical cyclic voltammogram (CV) of hFMO3 is shown in Figure 1A. At room temperature and under anaerobic conditions, the immobilized enzyme showed a single redox couple with a midpoint potential (E m ) of -445 ( 8 mV (vs Ag/AgCl). Integration of the reduction peak from the baseline corrected CV allowed for the calculation of the charge transferred upon reduction of the protein and for the determination of the quantity of the immobilized electroactive protein. The coverage was 2.6 × 10 13 molecules per cm 2 indicating a multilayer formation corresponding to roughly 3 layers. Volta- mmograms of hFMO3 were taken at different scan rates ranging from 10 to 150 mV s -1 . The peak-to-peak separation (ΔE p ) of 61 mV did not vary significantly within the mentioned range. Instrumental limitations did not allow for higher scan rates, therefore preventing k ET measurements. Furthermore, peak currents (i pc and i pa ) were linearly dependent on the scan rate, suggesting that the quasi-reversible reaction is a surface-controlled process, as expected for an immobilized electroactive species. The electrochemically determined E m value for hFMO3 immobilized on the GC electrode is in the range of the literature values for other flavin-containing monooxygenases. 4 The electrochemical response on the gold electrode could only be observed after modification of the surface. Functionalization of the Au surface with dithio-bismaleimidoethane (DTME) 5 led to the formation of maleimide-terminated groups which can covalently link to hFMO3 via surface exposed cysteine residues. The resulting covalently immobilized hFMO3 gave two waves with an E m of -280 ( 12 mV (Figure 1B). This value is ∼165 mV more positive than the E m measured for the noncovalently immobilized protein on the GC electrode and could be attributed to the electrode surface and its modifications which have been shown to affect the reduction potentials of other monooxygenases. 3a,6 The coverage was calcu- lated to be 3.4 × 10 12 molecules per cm 2 (Table 1), indicating a submonolayer formation. The ability of the immobilized hFMO3 to oxygenate two of its known substrates was investigated and compared with solution studies. The first substrate, benzydamine, is a nonsteroidal anti- inflammatory drug shown to be extensively metabolized to its N-oxide by hFMO3. 7,8 The second substrate, tamoxifen, is a widely used antiestrogenic drug for the treatment of breast cancer and has † University of Turin. ‡ Imperial College London. Scheme 1 Figure 1. Anaerobic cyclic voltammograms of hFMO3 immobilized on different electrode surfaces: (A) GC/DDAB/FMO3, (B) Au/DTME/FMO3. Scan rate 50 mV/s (A) and 2 mV/s (B) in 100 mM phosphate buffer with 100 mM KCl pH 7.4 at 25 °C. Shown are the original (black) and baseline corrected CVs (red, intensities multiplied by 2 for clarity). (C) Benzydamine titration of GC/DDAB/hFMO3. The error bars represent the estimated standard deviation for the mean of three separate electrode measurements. Published on Web 12/22/2009 10.1021/ja909261p  2010 American Chemical Society 458 9 J. AM. CHEM. SOC. 2010, 132, 458–459