title: Control of human cytochrome P450 2E1 electrocatalytic response as a result of unique orientation on gold electrodes.
abstract: Oriented immobilization of human cytochrome P450 2E1 and its catalytic activity by direct electrochemistry was achieved by engineering two multisite mutants of P450 2E1: MUT261 (C268S-C480S-C488S) and MUT268 (C261S-C480S-C488S). Here, all the exposed cysteines are mutated into serines, with the exception of one (C261 for MUT261 and C268 for MUT268) that is able to link covalently to a modified gold electrode. The P450 2E1 wild type, as well as the two mutants, were immobilized onto gold electrodes using dithio-bismaleimidoethane as a self-assembled monolayer. The catalytic activity of the wild type and of the two cysteine mutants were determined using p-nitrophenol as a substrate, and the amount of the electrocatalysis product (p-nitrocatechol) was determined spectrophotometrically. The amounts of product formed by the mutants on the electrodes were 2-fold to 3-fold higher than those of the wild type. Control experiments performed in solution using the cytochrome P450 reductase as the electron donor show no significant differences in the level of product formed. The higher level of product formation of the two mutants on the electrode is ascribed to the controlled immobilization on the gold surface: the heme electron transfer proximal side is linked to the electrode, while the substrate binding distal side is exposed to the bulk solution. This is the first evidence that the control over the orientation of the human cytochromes P450 is key to maximize the electrocatalytic efficiency of these enzymes.
authors.affiliation: Division of Molecular Biosciences, Imperial College London, South Kensington, London SW7 2AZ, United Kingdom.
authors.forename: Lok Hang
authors.initials: LH
authors.lastname: Mak
authors.forename: Sheila J
authors.initials: SJ
authors.lastname: Sadeghi
authors.forename: Andrea
authors.initials: A
authors.lastname: Fantuzzi
authors.forename: Gianfranco
authors.initials: G
authors.lastname: Gilardi
mesh_terms: D001483:Base Sequence; D002384:Catalysis; D019392:Cytochrome P-450 CYP2E1 / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D004563:Electrochemistry; D004566:Electrodes; D004800:Enzymes, Immobilized / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D006046:Gold / Q000737:chemistry; D006801:Humans; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation
chemical_list: D004800:Enzymes, Immobilized; D006046:Gold; D019392:Cytochrome P-450 CYP2E1