Introduction 1 2 3 4 1 c c 5 6 8 9 12 7 c 5 13 19 20 5 21 5 13 13 22 19 23 24 25 26 27 28 Materials and methods Chemicals 6 2 4 2 4 Expression and purification of CYP2D6 Escherichia coli 29 30 g Spectroelectrochemical flow cell 28 1 1 Fig. 1 WE CE SCE RE  Raman spectroscopy instrumentation −1 28 −1 −1 ® Electrochemical instrumentation 2 2+ + Sample preparation 31 32 −2 The MUA-coated Ag electrodes were rinsed with ethanol to eliminate the excess of MUA, dried by gently blowing nitrogen on them and then immersed into 50 μL of a 3 μM solution of protein in potassium phosphate buffer (40 mM, pH 7.4, 10% glycerol) for at least 3 h at 4 °C, to induce adsorption of the protein on the electrode. Then, the coated electrodes with the adsorbed protein were rinsed with buffer to eliminate possible excess of loosely bound protein, and were put into the spectroelectrochemical cell described already. −1 33 −1 Results and discussion The resting state 2 34 35 36 34 20 c 5 13 Fig. 2 A a b c d e f −1 b e a B −1 a b d ν 2 ν 3 Materials and methods  −1 −1 −1 37 33 37 1 3+ 36 37 3+ 35 36 Table 1 − 1 4 ν 3 ν 2 Mode 3+ 2+ 6cLS 5cHS Without substrate/inhibitor With inhibitor (imidazole) With substrate (dextromethorphan) a ν 2 1,582 (0.19) 1,582 (0.14) 1,567 b 1,561 (0.91) ν 3 1,501 (0.08) 1,501 (0.10) 1,486 0.05 1,466 (0.65) ν 4 1,371 (1.00) 1,371 (1.00) 1,371 (1.00) c parentheses 6cLS 5cHS NA a b 33 c −1 33 38 3 34 20 Fig. 3 a b a′ b′ a a′ b b′ b a ν 4 ν 3 ν 2 Materials and methods  Reversible binding of a substrate 36 33 2 ν 3 −1 ν 2 −1 2 1 33 33 37 39 34 2 2 2 Reversible binding of imidazole as an exogenous heme axial ligand 37 40 43 44 2 1 ν 3 ν 2 2 1 41 45 47 37 −1 −1 ν 10 −1 37 −1 48 48 49 50 −1 41 45 47 37 ν 3 ν 2 2 ν 3 ν 2 2 −1 Redox activity 36 3+ 2+ E 36 E 1 36 51 36 36 2 1 2 ν 4 −1 ν 4 −1 ν 4 −1 −1 20 37 52 53 −1 ν 4 ν 4 20 37 52 55 −1 ν 4 −1 ν 4 −1 4 38 Fig. 4 a b  inset Materials and methods  4 −1 −1 E 51 E E 5 14 19 5 56 −1 −1 5 Fig. 5 ν 4 a b c d dotted lines −1 −1 Materials and methods  E E 1 51 E E 1 −1 57 6 Fig. 6 a b ν 4 ν 3 ν 2 Materials and methods  1 3 1 3 22 20 58 22 59 c 13 60 62 20 20 63 64 66 Conclusions Upon immobilization on MUA-coated Ag electrodes, the human enzyme CYP2D6 retains its active-site structure, its ability to reversibly bind a substrate (DX) and an exogenous heme ligand (imidazole), and its capacity to exchange electrons, demonstrating the efficiency of the coating to prevent major protein conformational changes or denaturation. However, despite its success in preserving an intact protein structure, the MUA coating appears to be unsuitable for performing direct electrochemistry experiments on immobilized CYP2D6, since its reduced state could not be adequately achieved by varying the electrode potential. The application of potentials from −0.4 to −1.0 V appears to induce enzyme inactivation instead. Among other factors influencing the redox activity, a more favorable enzyme orientation or distance with respect to the electrode surface might be achieved using other coatings, an approach currently under study in our group. In general, SERRS spectroscopy on coated Ag surfaces was proven to be successful in monitoring the active-site structure of immobilized CYP2D6, enabling the detection of reversible substrate and ligand binding using only very low amounts of protein. Besides its technological relevance as an optical biosensing tool, this technique might have a significant potential for fundamental research on the mechanisms of catalysis for CYPs, as well as for other heme enzymes. Electronic supplementary material Below is the link to the electronic supplementary material.  Supplementary Material (PDF 58 kb)