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Direct Electrochemical Interaction between a Modified Gold Electrode and a Bacterial Membrane Extract Lars J. C. Jeuken,*,† Simon D. Connell,† Mohammed Nurnabi,‡ John O’Reilly,§ Peter J. F. Henderson,§ Stephen D. Evans,| and Richard J. Bushby‡ Institute of Molecular Biophysics, University of Leeds, Leeds LS2 9JT, United Kingdom, School of Physics and Astronomy, University of Leeds, Leeds LS2 9JT, United Kingdom, Centre for Self-Organising Molecular Systems, University of Leeds, Leeds LS2 9JT, United Kingdom, and School of Biochemistry and Molecular Biology, University of Leeds, Leeds LS2 9JT, United Kingdom Received September 10, 2004. In Final Form: December 1, 2004 A novel electrochemical approach is described for redox-active membrane proteins. A total membrane extract (in the form of vesicles) of Bacillus subtilis is tethered onto gold surfaces modified with cholesterol based thiols. The membrane vesicles remain intact on the surface and do not rupture or fuse to form a planar bilayer. Oxidation/reduction signals are obtained of the natural co-enzyme, menaquinone-7, located in the membrane. The membrane protein, succinate menaquinone oxidoreductase (SQR), remains in the vesicles and is able to reduce fumarate using menaquinone as mediator. The catalysis of the reverse reaction (oxidation of succinate), which is the natural catalytic function of SQR, is almost absent with menaquinone. However, adding the co-enzyme ubiquinone, which has a reduction potential that is about 0.2 V higher, restores the succinate oxidation activity.
Introduction Redox proteins, which are estimated to account for a quarter of all proteins,1 perform a myriad of functions in biology. They shuttle electrons and catalyze redox reactions in many vital processes, including photosynthesis and metabolism. Dynamic electrochemical techniques have proven to be powerful tools to study these proteins. The thermodynamics and kinetics can be studied in detail if they are electrochemically connected or “wired” to the electrode surface.2 Furthermore, these hybrid organicinorganic systems form the basis for the development of biosensors and biofuel cells.3,4 The main challenge is to adsorb proteins in their native state on the electrode while efficiently exchanging electrons. To this purpose, considerable effort has been dedicated to designing electrode surfaces that bind proteins (a) by electrostatic or hydrophobic/hydrophilic type interactions, (b) by selective (e.g., antibody) interactions, and (c) by covalent bonds, either directly or via a cofactor of the target protein.3,5 In some cases the proteins themselves have been (re)designed.1,6 An important and large part of all redox proteins is membrane bound. Indeed, many redox proteins involved in photosynthesis and metabolism are located in the mitochondrial, chloroplast, or plasma7 membranes. However, because membrane proteins are more difficult to * Corresponding author. E-mail: L.J.C.
[email protected]. Fax: 0044-(0)113-3433900. † Institute of Molecular Biophysics. ‡ Centre for Self-Organising Molecular Systems. § School of Biochemistry and Molecular Biology. | School of Physics and Astronomy. (1) Wong, T. S.; Schwaneberg, U. Curr. Opin. Biotechnol. 2003, 14, 590-596. (2) Le´ger, C.; Elliott, S. J.; Hoke, K. R.; Jeuken, L. J. C.; Jones, A. K.; Armstrong, F. A. Biochemistry 2003, 42, 8653-8662. (3) Willner, I.; Katz, E. Angew. Chem., Int. Ed. 2000, 39, 1180-1218. (4) Ikeda, T.; Kano, K. J. Biosci. Bioeng. 2001, 92, 9-18. (5) Schuhmann, W. Rev. Mol. Biotech. 2002, 82, 425-411. (6) Gilardi, G.; Fantuzzi, A.; Sadeghi, S. Curr. Opin. Struct. Biol. 2001, 11, 491-499. (7) Ly, J. D.; Lawen, A. Redox Rep. 2003, 8, 3-21.
manipulate experimentally than globular proteins, less work has been reported on the electrochemistry of these proteins.8-17 One of the foregoing successful strategies is to reconstitute purified and detergent-solubilized proteins in a biomimicking membrane on the surface.12-17 In some of these cases, direct electron transfer between the electrode and the membrane protein is achieved,12-14 but more often mediators need to be used. For instance, Kinnear and Monbouquette have used membrane-bound ubiquinone-6 (UQ-6) as a mediator between the electrode and fructose hydrogenase.16 Marchal et al. mediated electrons to the peripheral membrane protein pyruvate oxidase using its physiological co-enzyme, UQ-8.17 Here, we report a novel approach in which a total membrane extract is used, with no other purification step than separating the membrane fraction from the cytoplasm. Membrane extracts of Bacillus subtilis, still containing all proteins and co-enzymes, are specifically bound to a gold electrode using cholesterol “tethers” which are separated from the Au-thiol bond via a ethyleneoxy chain (EO3, Figure 1). These cholesterol tethers were initially designed, and successfully used, to prepare supported lipid bilayers,18-24 which are formed by self(8) Elliott, S. J.; Hoke, K. R.; Heffron, K.; Palak, M.; Rothery, R. A.; Weiner, J. H.; Armstrong, F. A. Biochemistry 2004, 43, 799-807. (9) Ikeda, T.; Miyaoka, S.; Matsushita, F. Chem. Lett. 1992, 847850. (10) Razumiene, J.; Niculescu, M.; Ramanavicius, A.; Laurinavicius, V.; Cso¨regi, E. Electroanalysis 2002, 14, 43-49. (11) Haas, A. S.; Pilloud, D. L.; Reddy, K. S.; Babcock, G. T.; Moser, C. C.; Blasie, J. K.; Dutton, P. L. J. Phys. Chem. B 2001, 105, 1135111362. (12) Kinnear, K. T.; Monbouquette, H. G. Langmuir 1993, 9, 22552257. (13) Burgess, B. K.; Rhoten, M. C.; Hawkridge, F. M. Langmuir 1998, 14, 2467-2475. (14) Rhoten, M. C.; Burgess, J. D.; Hawkridge, F. M. Electrochim. Acta 2000, 45, 2855-2860. (15) Devadoss, A.; Burgess, J. D. Langmuir 2002, 18, 9617-9621. (16) Kinnear, K. T.; Monbouquette, H. G. Anal. Chem. 1997, 69, 17711775. (17) Marchal, D.; Pantigny, J.; Laval, J. M.; Moiroux, J.; Bourdillon, C. Biochemistry 2001, 40, 1248-1256.
10.1021/la047732f CCC: $30.25 © 2005 American Chemical Society Published on Web 01/20/2005
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Materials and Methods
Figure 1. Chemical structures of (from left to right) 2-mercaptoethanol (spacer), 6-mercaptohexanol (spacer), and the cholesterol tether molecule (EO3-cholesteryl). On the right is a graphical representation of a supported lipid bilayer on a mixed self-assembled monolayer of EO3-cholesteryl and 2-mercaptoethanol.
assembly of lipid vesicles. To form a lipid bilayer instead of a hybrid layer (a monolayer of phospholipids on top of a monolayer of cholesterol), the cholesterol tethers have been mixed with small spacer molecules (e.g., 2-mercaptoethenol or 6-mercaptohexanol), which allow phospholipids to enter both leaflets of the bilayer (Figure 1).18-24 We have used so-called inside-out vesicles of a B. subtilis strain that overexpresses the enzyme succinate menaquinone oxidoreductase (SQR, also known as succinate dehydrogenase).25-29 B. subtilis is an aerobic Grampositive bacterium that catalyzes the oxidation of succinate (using SQR) to fumarate in the respiratory chain while reducing its co-enzyme menaquinone-7 (MQ-7). B. subtilis and some other Gram-positive bacteria are exceptional in that they use menaquinone to oxidize succinate, rather than ubiquinonol (UQ) as do most Gram-negative bacteria and mitochondria.28,29 Contrary to UQ, MQ has a reduction potential that is about 0.1 V lower than that of the succinate/fumarate couple and would therefore be expected to serve as a co-enzyme in the reverse reaction (i.e., as an electron donor for fumarate reduction). Here, we will show that the membrane vesicles adsorb on the cholesterol modified surface but do not rupture/ fuse to form a planar lipid bilayer as has been observed for other lipid vesicles. The MQ in the adsorbed membrane vesicles can be detected electrochemically and is used as a natural electron mediator for SQR which retains its catalytic activity upon adsorption. (18) Boden, N.; Bushby, R. J.; Clarkson, S.; Evans, S. D.; Knowles, P. F.; Marsh, A. Tetrahedron 1997, 53, 10939-10952. (19) Williams, L. M.; Evans, S. D.; Flynn, T. M.; Marsh, A.; Knowles, P. F.; Bushby, R. J.; Boden, N. Langmuir 1997, 13, 751-757. (20) Cheng, Y.; Boden, N.; Bushby, R. J.; Clarkson, S.; Evans, S. D.; Knowles, P. F.; Marsh, A.; Miles, R. E. Langmuir 1998, 14, 839-844. (21) Jenkins, A. T. A.; Bushby, R. J.; Boden, N.; Evans, S. D.; Knowles, P. F.; Liu, Q.; Miles, R. E.; Ogier, S. D. Langmuir 1998, 14, 4675-4678. (22) Jenkins, A. T. A.; Boden, N.; Bushby, R. J.; Evans, S. D.; Knowles, P. F.; Miles, R. E.; Ogier, S. D.; Scho¨nherr, H.; Vancso, G. J. J. Am. Chem. Soc. 1999, 121, 5274-5280. (23) Jenkins, A. T. A.; Bushby, R. J.; Evans, S. D.; Knoll, W.; Offenha¨usser, A.; Ogier, S. D. Langmuir 2002, 18, 3176-3180. (24) Becucci, L.; Guidelli, R.; Liu, Q.; Bushby, R. J.; Evans, S. D. J. Phys. Chem. B 2002, 106, 10410-10416. (25) Ha¨gerha¨ll, C.; Aasa, R.; von Wachenfeldt, C.; Hederstedt, L. Biochemistry 1992, 31, 7411-7421. (26) Lemma, E.; Unden, G.; Kro¨ger, A. Arch. Microbiol. 1990, 155, 62-67. (27) Lemma, E.; Ha¨gerha¨ll, C.; Geisler, V.; Brandt, U.; von Jagow, G.; Kro¨ger, A. Biochim. Biophys. Acta 1991, 1059, 281-285. (28) Lemos, R. S.; Fernandes, A. S.; Pereira, M. M.; Gomes, C. M.; Teixeira, M. Biochim. Biophys. ActasBioenerg. 2002, 1553, 158-170. (29) Schirawski, J.; Unden, G. Eur. J. Biochem. 1998, 257, 210-215.
Materials. EO3-cholesteryl (Figure 1) was made as previously described.18 2-Mercaptoethanol and 6-mercaptohexanol (Sigma) were used without further purification. All solvents (2-propanol, methanol (MeOH), and dichloromethanol (DCM)) were HPLC grade (Fisher) and used as received. Also ubiquinone (co-enzyme Q10) and menaquinone-4 (vitamin K2, Sigma) were used as received. All electrochemical experiments were performed in MOPS buffer (3-(N-morpholino)propanesulfonic acid, Sigma) adjusted to pH 7.4 with NaOH at 20 °C. Control experiments were carried out using vesicles prepared from egg-derived phosphatidylcholine (egg PC) and Escherichia coli derived total lipid extract (“polar”), both from Avanti. Vesicles were prepared by extrusion through 50 or 100 nm Track-Edge nuclepore membrane (Avanti) using standard procedures. Electrodes. Gold (150 nm; Advent, 99.99%) was evaporated through a mask on a 10 nm chromium adhesion layer on cleaned glass microscope slides (Deconex, sonication, methanol) with an Edwards Auto 306 evaporator at
