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Control of Human Cytochrome P450 2E1 Electrocatalytic Response as a Result of Unique Orientation on Gold Electrodes Lok Hang Mak,† Sheila J. Sadeghi,‡ Andrea Fantuzzi,† and Gianfranco Gilardi*,†,‡ Division of Molecular Biosciences, Imperial College London, South Kensington, London SW7 2AZ, United Kingdom, and Department of Human and Animal Biology, University of Turin, Italy 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. Cytochromes P450 form a large family of heme-thiolate enzymes that are involved in a wide number of reactions, including hydroxylation, oxygen dealkylation, and nitrogen oxidation.1-3 Their unique chemistry makes them highly promising for use in biocatalysis and their key role in drug metabolism makes the measurement of their activities an essential step in the development of new pharmaceuticals.4 Studies of P450 catalysis in solution require the presence of a NADPH-dependent reductase able to * Author to whom correspondence should be addressed. Phone: (+) 44 207 5945320. Fax: (+) 44 207 5945330. E-mail:
[email protected]. † Division of Molecular Biosciences, Imperial College London. ‡ Department of Human and Animal Biology, University of Turin. (1) Wrighton, S. A.; Stevens, J. C. Crit. Rev. Toxicol. 1992, 22, 1–21. (2) Evans, W. E.; Relling, M. V. Science 1999, 286, 487–491. (3) Rendic, S.; Carlo, F. J. D. Drug Metab. Rev. 1997, 29, 413–580. (4) Guengerich, F. P. Chem. Res. Toxicol. 2001, 14, 611–650. 10.1021/ac101072h 2010 American Chemical Society Published on Web 05/27/2010
transfer the electrons necessary for the enzyme’s turnover. This makes the application of electrochemical methods a very attractive strategy for the construction of a reagentless platform for highthroughput screening of new drug candidates. However, the direct electrochemistry of P450 enzymes on electrodes has proven to be difficult to achieve, because of the deeply buried heme cofactor and instability of the biological matrix that tends to denature upon interaction with the electrode surface.5 Generally, electric communication between the redox center of a protein and the electrode can be achieved in two ways: either using a small electroactive mediator that shuttles the electrons between the protein and the electrode, or by immobilization of the protein for direct communication with the electrode.6,7 In principle, the direct electron supply by the electrode without the use of a mediator is the preferred approach and it is the basis of third-generation biosensors.8 The first reports on direct electron transfer with a redox enzyme were published back in 1977 when Eddows and Hill9 and Yeh and Kuwana10 independently showed reversible cyclic voltammograms of cytochrome c. In 1996, Kazlauskaite et al.11 reported, for the first time, the direct electron transfer using a cytochrome P450. Here, P450cam (CYP101) was immobilized on a bare edge-plane graphite electrode. Following this paper, several studies on direct electron transfer of P450s have been published, most of which used electrode modifiers such as polymers or self-assembled monolayers.5 Despite the many efforts, to date, only a few works on human P450 bioelectrochemistry have shown product formation.12-15 Immobilization is an important aspect when developing an electrochemical sensor based on redox enzymes, as activity and sensitivity strongly depends on both the concentration of the immobilized protein and (5) Sadeghi, S. J.; Fantuzzi, A.; Gilardi; G. Biochim. Biophys. Acta 2010, in press. (6) Wilson, G. S.; Hu, Y. Chem. Rev. 2000, 100, 2693–2704. (7) Anderson, J. L.; Coury, L. A.; Leddy, J. Anal. Chem. 2000, 72, 4497–4520. (8) Zhang, W.; Li, G. Anal. Sci. 2004, 20, 603–609. (9) Eddowes, M. J.; Hill, H. A. O. J. Chem. Soc.-Chem. Commun. 1977, 771– 772. (10) Yeh, P.; Kuwana, T. Chem. Lett. 1977, 1145–1148. (11) Kazlauskaite, J.; Westlake, A. C. G.; Wong, L. L.; Hill, H. A. O. Chem. Commun. 1996, 2189–2190. (12) Fantuzzi, A.; Fairhead, M.; Gilardi, G. J. Am. Chem. Soc. 2004, 126, 5040– 5041. (13) Yang, M. L.; Kabulski, J. L.; Wollenberg, L.; Chen, X. Q.; Subramanian, M.; Tracy, T. S.; Lederman, D.; Gannett, P. M.; Wu, N. Q. Drug Metab. Dispos. 2009, 37, 892–899. (14) Mie, Y.; Suzuki, M.; Komatsu, Y. J. Am. Chem. Soc. 2009, 131, 6646–6647. (15) Dodhia, V. R.; Sassone, C.; Fantuzzi, A.; Di Nardo, G.; Sadeghi, S. J.; Gilardi, G. Electrochem. Commun. 2008, 10, 1744–1747.
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Figure 1. Scheme for surface immobilization of P450 2E1 wild-type and unique cysteine mutants MUT261 and MUT268 used in this study. The covalent linkage is achieved via a dithio-bismaleimidoethane (DTME) spacer. C261 and C268 are positioned on the proximal side of the protein to allow efficient communication between the protein and the electrode. This orientation exposes the distal side to the bulk solution for easy access of the substrate.
its specific orientation. The role that protein orientation exerts on electron transfer to the electrode has been addressed by specific covalent linkage of proteins to derivatized electrodes.16-19 Our laboratory previously demonstrated that direct immobilization of P450 2E1 cysteines on gold led to an inactive enzyme; on the other hand, the covalent binding through a cystaminemaleimide spacer improved the P450 2E1 catalytic activity on gold electrodes when compared to other methods of immobilization.12 We also showed that the combined engineering of spacers as well as unique cysteines is important to achieve a stable electrochemical response of the heme domain of bacterial cytochrome P450 BM3.20 Here, we demonstrate, for the first time, the influence that the choice of a unique cysteine makes in achieving electrocatalysis, by measurement of product formation. A rational protein engineering approach allows the covalent binding of the protein using the proximal electron transfer side of the heme close to the electrode, at the same time orienting the distal substratebinding side toward the bulk solution where the freely diffusible substrate can have access to the enzyme (see Figure 1). The results are compared with data obtained for the enzyme free in solution where orientation effects are not present. MATERIALS AND METHODS Materials. All chemicals were from Sigma-Aldrich (St. Louis, MO) in the highest purity available. The dithio-bismaleimidoethane (DTME) was from Pierce Biotechnology (Rockford, IL). Restriction enzymes were purchased from New England Biolabs (Ipswich, MA), the diethylaminoethyl resin for chromatography was from GE Healthcare (USA). Site-Directed Mutagenesis, Expression, and Purification. Cysteines C268, C480, C488 for mutant MUT261 and cysteines C261, C480, and C488 for MUT268 were replaced by PCR(16) Trammell, S. A.; Wang, L. Y.; Zullo, J. M.; Shashidhar, R.; Lebedev, N. Biosens. Bioelectron. 2004, 19, 1649–1655. (17) Trammell, S. A.; Spano, A.; Price, R.; Lebedev, N. Biosens. Bioelectron. 2006, 21, 1023–1028. (18) Lebedev, N.; Trammell, S. A.; Spano, A.; Lukashev, E.; Griva, I.; Schnur, J. J. Am. Chem. Soc. 2006, 128, 12044–12045. (19) Trammell, S. A.; Griva, I.; Spano, A.; Tsoi, S.; Tender, L. M.; Schnur, J.; Lebedev, N. J. Phys. Chem. C 2007, 111, 17122–17130. (20) Ferrero, V. E. V.; Andolfi, L.; Di Nardo, G.; Sadeghi, S. J.; Fantuzzi, A.; Cannistraro, S.; Gilardi, G. Anal. Chem. 2008, 80, 8438–8446.
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mediated site-directed mutagenesis in the 2E1 gene cloned in the pCW plasmid.21 The following two primers were used for MUT261: (1) C268: 5′-ACTGTCCCCGGGACCTCACCGACTCCCTGCTCGTG-3′ (2) C480/C488: 5′-ATCGATAAGCTTTCAATGGTGATGGTGTGAGCGGGGAATGACAGAGAGTTTGTAACGTGGTGGGATAGAGCCAAACCC-3′ The unique restriction sites for XmaI and HindIII were used to incorporate the mutated fragment back into the pCW vector. For MUT268, two fragments were made with the mutations and both fragments were then ligated into the pCW expression vector. C261 was mutated into serine in fragment 1 with restriction sites NdeI and XmaI, using the following two primers: (1) 5′- GGAGGTCATATGGCTAGGCAGGTGCA-3′ (2) 5′- GGTCCCGGGGAGAGTTGGGGTCC-3′. C480 and C488 were mutated into serines in fragment 2 with restriction sites XmaI and HindIII with the following two primers: (1) 5′-ACTGTCCCCGGGACCTCACCGACT-3′ (2) 5′-ATCGATAAGCTTTCAATGGTGATGGTGTGAGCGGGGAATGACAGAGAGTTTGTAACGTGGTGGGATAGAGCCAAACCC-3′. All mutations were verified by DNA sequencing. To facilitate the purification procedure, a 4xHis-Tag was inserted before the stop codon and the Hind III restriction site. The proteins were then expressed in E. coli strain DH5R, as described previously.21 The harvested cells were resuspended in lysis buffer containing 100 mM potassium phosphate pH 7.4, 20% glycerol, 5 mM β-mercaptoethanol and 50 µM 4-methylpyrazole. Lysozyme was added to the suspension to a final concentration of 2 mg/mL, and the cell suspension was stirred for 1 h at 4 °C.22 Lysis was performed by ultrasonication of the cell suspension on ice. The suspension was centrifuged at 45 000 rpm at 4 °C for 1 h. The supernatant was discarded and the pellet was resuspended in 100 mM potassium phosphate buffer pH 7.4 containing 20% glycerol, 5 mM β-mercaptoethanol, 50 µM 4-methylpyrazole, and 2% IGEPAL. The suspension was stirred for 2 h at 4 °C and then centrifuged at 45 000 rpm for 1 h. The protein was purified from the supernatant using a diethylaminoethyl (DEAE) column, followed by a His-affinity nickel column. The protein was eluted from the nickel column with increasing concentrations of histidine (1-40 mM). The functionality of purified P450 2E1 wild type and mutants were verified spectrophotometrically using the method of Omura and Sato.23 Proteins were stored in 50 mM potassium phosphate pH 7.4, 0.05% IGEPAL, 0.5 mM DTT, 25 µM 4-methylpyrazol, 50% glycerol, 250 mM KCl. Electrode Preparation. Evaporated gold provided by the Nanotechnology Centre Rutherford Appleton Laboratory, (Chilton, U.K.), with a surface area of ∼17.5 mm2, was cleaned by immersion in piranha solution (a 3:1 mixture of concentrated sulfuric acid and 30% hydrogen peroxide) at room temperature for 30 min. Piranha-treated surfaces were rinsed thoroughly with deionized water and then dried with nitrogen gas. The self-assembled monolayer of dithio-bismaleimidoethane (DTME) (21) Fairhead, M.; Giannini, S.; Gillam, E. M. J.; Gilardi, G. J. Biol. Inorg. Chem. 2005, 10, 842–853. (22) Gillam, E. M. J.; Guo, Z. Y.; Guengerich, F. P. Arch. Biochem. Biophys. 1994, 312, 59–66. (23) Omura, T.; Sato, R. J. Biol. Chem. 1964, 239, 2370–2385.
Table 1. Solvent Accessibility and Location of the Cysteine Residues Present in Wild-Type Human P450 2E1 and MUT261 and MUT268 Mutants Used in This Study cysteine residue
solvent accessibility (%)
P450 2E1 wild type
location, relative to the heme
Mut 261
Mut 268
C174 C177 C261 C268 C437 C452 C480 C488
0.0 0.1 48.2 17.2 43.7 0.4 4.5 3.8
present present present present ligated to heme present present present
distal distal proximal proximal proximal proximal distal distal
present present present mutated into serine ligated to heme present mutated into serine mutated into serine
present present mutated into serine present ligated to heme present mutated into serine mutated into serine
was formed by immersion of the gold electrodes into an acetonitrile solution containing 2 mM DTME for 2 h. Samples were then thoroughly rinsed with acetonitrile and deionized water and dried by blowing nitrogen gas over the surfaces. The DTME modified gold electrodes were then functionalized with P450 2E1 wild type, MUT261 and MUT268. Prior to immobilization onto the gold electrode, the dithiothreitol (DTT) and also glycerol contained in the storage buffer were removed by gel filtration using a PD 10 column (GE Healthcare, UK). The protein was bound to the gold surface by incubating the DTMEmodified gold surface with 5 µM of P450 2E1 for 2 h at 4 °C. The samples were rinsed with 500 mM potassium phosphate pH 7.4 and stored in the same buffer. Fourier Transform Infrared (FT-IR) Measurements. The Fourier transform infrared (FT-IR) measurements were acquired using a Bruker Model Tensor 27 FT-IR spectrometer. Spectra were collected using p-polarized light incident at a grazing angle of 75° from the surface normal, with a grazing angle accessory (Harrick, USA). The resolution was 4 cm-1 with 400 scans collected to improve the signal-to-noise ratio. All spectra are reported as absorbance. Peak assignments were made on the basis of published literature data.26-30 Electrochemical Measurements. A three-electrode setup was used for the electrochemical measurements with a silver/ silver chloride and a platinum wire as the reference and counter electrodes, respectively. All values of potential are referred to 3 M Ag/AgCl (+207 mV versus NHE). Cyclic voltammetry was performed under anaerobic conditions in a Clear View glovebox from Belle Technology UK, Ltd. (Dorset, U.K.), in which the saturated nitrogen atmosphere contained
