Covalent linkage of CYP101 with the electrode enhances the electrocatalytic activity of the enzyme: Vectorial electron transport from the electrode

Direct electrochemical transfer of electrons to the enzyme provides an excellent method of driving the catalytic reactions of cytochrome P450 enzymes that form a superfamily of vital heme enzymes involved in biological monooxygenation reactions. Covalent attachment of N-(1-pyrenyl) maleimide (pyrene maleimide) to the bacterial cytochrome P450, CYP101 has been carried out and the conjugated enzyme was shown to be specifically immobilized onto the glassy carbon electrode through the pyrene group. The electrode immobilized pyrene-conjugated enzyme showed quasi-reversible electrochemistry with a midpoint potential at −330 ± 10 mV versus Ag/AgCl. The unconjugated enzyme that did not have specific linkage with the pyrene maleimide was non-specifically adsorbed on the electrode surface and the electrochemical response was much weaker than that observed in case of the conjugated enzyme, though the midpoint potential was almost unchanged. The pyrene maleimide bound CYP101 was found to have surface coverage of 1.35 ± 0.3 × 10−10 mol/cm2 and the heterogeneous rate of electron transfer was found to be 0.21 ± 0.02 s−1, which is larger than that for the unconjugated enzyme. The pyrene maleimide linked immobilized enzyme was oriented to the electrode so that efficient electron transfer takes place from the electrode to the immobilized enzyme. The oxygenase activity of the immobilized conjugated enzyme was assayed from the enhancement of catalytic current in presence of oxygen and the natural substrate camphor. Mass spectrometric studies also showed enhanced formation of hydroxycamphor by electrochemically driven catalysis in the pyrene maleimide linked immobilized CYP101.