Langmuir 1998, 14, 6705-6708
6705
Electron Transfer to a Gold Electrode from Cytochrome Oxidase in a Biomembrane via a Polyelectrolyte Film Britta Lindholm-Sethson* and Juan Carrasco Gonzalez Department of Analytical Chemistry, Umeå University, S-901 87 Umeå, Sweden
Gertrud Puu Defence Research Establishment, S-901 82 Umeå, Sweden Received September 10, 1997. In Final Form: August 27, 1998 We have developed an electrode modification, which permits the communication between cytochrome c in solution, cytochrome oxidase residing in its natural environment of a lipid membrane, and a gold electrode. The connection is made via an electroactive polyelectrolyte containing an osmium complex covalently attached to poly(4-vinylpyridine), sandwiched between thin layers of a negatively charged polyelectrolyte, polystyrene sulfonate. The outermost layer is the support for the oxidase-containing lipid film. Electron transfer from a pulse of reduced cytochrome c in the surrounding medium via the enzyme and the polyelectrolyte to the electrode is evident as a transient current during anaerobic conditions. Inhibiting the enzyme with sodium azide results in a temporarily decreased cytochrome c signal. The enzyme-mediated electron transport increased with increasing substrate concentration, resulting in a Michaelis-Menten constant of the same magnitude as for free or liposomal enzyme.
Introduction Electrochemical means, both for studying electrontransferring proteins and for utilizing them in, for example, biosensors, are highly desirable. The redox site in these proteins is usually embedded in the protein structure, which renders direct communication with an electrode difficult. Some elegant solutions have been described for water-soluble enzymes.1-3 Many membranebound proteins also have great potential for biosensing, but their requirements in a lipidic environment implicate unique technical solutions. In the present paper we report on signal transduction from an active membrane protein residing in a biomembrane supported on a polymer cushion containing an artificial electron acceptor. Cytochrome c oxidase (EC 1.9.3.1) is a transmembrane protein, which catalyzes the transfer of electrons from ferrocytochrome to molecular oxygen under aerobic conditions.4 The structures of the metal sites in this large membrane-bound protein were recently published.5 One redox-active, dinuclear copper site, CuA, is located in a part of the molecule extending into the aqueous space on the cytoplasmic side of the membrane, accessible to cytochrome c (cyt c). Heme a, heme a3, and the second copper site CuB are all located in the transmembrane part of the protein, approximately 13 Å below the membrane surface toward the cytosol. The locations of magnesium close to CuA and zinc in the hydrophilic moiety of the protein near the matrix side were also reported. For reconstituting the enzyme in a planar lipid bilayer on a solid support, the dimensions of the enzyme must be * To whom correspondence should be addressed (1) Heller, A. J. Phys. Chem. 1992, 96, 3579. (2) Schuhmann, W. Biosens. Bioelectron. 1995, 10, 181. (3) Riklin, A.; Katz, E.; Willner, I.; Stocker, A.; Bu¨ckmann, A. F. Nature 1995, 376, 672. (4) Malatesta, F.; Antonini, G.; Sarti, P.; Brunori, M. Biophys. Chem. 1995, 54, 1. (5) Tsukihara, T.; Aoyama, H.; Yamashita, E.; Tomizaki, T.; Yamaguchi, H.; Shinzawa-Itoh, K.; Nakashima, R.; Yaono, R.; Yoshikawa, S. Science 1995, 269, 1069.
considered. The length of the transmembrane part is 48 Å, while the protein extends its more hydrophilic parts, about 35 Å, on either side of the membrane.5 Thus, an aqueous space of at least that distance between a solid support and the lipid bilayer is required to accommodate the protein. Such requirements have been considered in general terms for membrane spanning proteins.6-8 A black lipid membrane-like approach, using a Teflon spacer on solid supports, has been used for studies on different membrane proteins including cyt oxidase.9,10 Reports on cyt oxidase in lipidic structures on platinum,11 modified gold,12 and modified gold-silver,13,14 all having limited aqueous spaces, show that the enzyme can still be at least partly and temporarily functional. Polymers are good candidates for providing the required space. The alternating polycationic and polyanionic polymer films described by Decher15 were shown to be suitable substrates for the transfer of multilayers of phosphatidic acid.16 Hence, in the present paper a polyelectrolyte-supported biomembrane is the key feature in our biosensing device. The activity of the redox enzyme residing in a lipidic environment is wired to the gold electrode via an osmium complex bound to the polymer. An idealized scheme of the modified electrode is shown in Figure 1. Electron transport from reduced cyt c in solution (6) Sackmann, E. Science 1996, 271, 43. (7) Beyer, D.; Elender, G.; Knoll, W.; Ku¨hner, M.; Maus, S.; Ringsdorf, H.; Sackmann, E. Angew. Chem., Int. Ed. Engl. 1996, 35, 5, 1682. (8) Heyse, S.; Vogel, H.; Sa¨nger, M.; Sigrist, H. Protein Sci. 1995, 4, 2532. (9) Salamon, Z.; Hazzard, J. T.; Tollin, G. Proc. Natl. Acad. USA 1993, 90, 6420. (10) Salamon, Z.; Tollin, G. Biophys. J. 1996, 71, 858. (11) Puu, G.; Gustafson, I.; Artursson, E.; Ohlsson, P.-A° . Biosens. Bioelectron. 1995, 10, 463. (12) Cullison, J. K.; Hawkridge, F. M.; Nakashima, N.; Yoshikawa, S. Langmuir 1994, 10, 877. (13) Burgess, J. D.; Rhoten, M. C.; Hawkridge, F. M. Langmuir 1998, 14, 2467. (14) Burgess, J. D.; Rhoten, M. C.; Hawkridge, F. M. J. Am. Chem. Soc. 1998, 120, 4488. (15) Decher, G. Science 1997, 277, 1232. (16) Lindholm-Sethson, B. Langmuir 1996, 12, 2, 3305.
10.1021/la9710278 CCC: $15.00 © 1998 American Chemical Society Published on Web 10/24/1998
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Figure 1. Idealized representation of the thin polyelectrolyte lipid film. The thickness of the electroactive polyelectrolyte film was estimated to be 15-20 nm. A lipid membrane with cytochrome c oxidase, with an average thickness of 5-6 nm, was formed by fusion of lecithin proteoliposomes. The second scheme shows energy levels for the components involved. Literature data were used for the formal potentials of the biomolecules. The box for the oxidase indicates the presence of several redox centers as well as the span in reported values for each individual center.
was evident as a transient current. A dual assay, in which the currents were registered in the absence and presence of a cyt oxidase inhibitor, sodium azide, was used to estimate the kinetic parameters of the enzyme. Increased concentrations of cyt c resulted in a characteristic saturation curve. The Michaelis-Menten constant was estimated to be 8.1 ( 4.2 µM, and the turnover rate, to be 4 electrons/s. Experimental Section Cytochrome oxidase was purified from bovine heart following the Hartzell procedure.17 The specific activity was 30 units per mg of protein, and the heme content, 7.2 nmol per mg of protein. Liposomes were prepared from 12 mg of lecithin (Sigma type IV-S), with addition of 1.2 mg of oxidase for proteoliposomes. Liposomes were formed by detergent depletion on Sephadex G-50 with n-octylglucoside as detergent.18 The final phospholipid concentration based on phosphate determination was 1 mM, and the protein concentration, 0.15 mg mL-1. The protein/lipid molar ratio was thus 1:1300, calculated from a molecular weight of 200 000 for the oxidase. Gold film electrodes were deposited with a thickness of 100 nm, by electron gun evaporation in ultrahigh vacuum onto glass slides, using (3-mercaptopropyl)trimethoxysilane as a molecular glue between the glass and the gold.19 The electrode was cleaned in a boiling 1:1 mixture of ethanol and chloroform for 1 min, rinsed with water, dried under a nitrogen stream, and immersed for 30 min in 5 mM 2-aminoethanethiol (Fluka Chemie) in ethanol. A polyelectrolyte sandwich structure was then built up on this positively charged surface. The first and the third layers were obtained by immersion into a 9.8 mM solution of polystyrenesulfonate in 18 mM H2SO4 and 0.5 M Na2SO4 (Na-PSS from Scientific Polymer Products). The middle layer consisted of an electroactive cationic polyelectrolyte, Os(bpy)2PVPCl, with a molar ratio of 1:5 of osmium to poly(4-vinylpyridine) (PVP) (17) Hartzell, C. R.; Beinert, H.; van Gelder, B. F.; King, T. S. Methods Enzymol. 1978, 53, 54. (18) Mimms, L. T.; Zampighi, G.; Nozaki, Y.; Tanford, C.; Reynolds, J. A. Biochemistry 1981, 20, 833. (19) Goss, L. A.; Charych, D. H.; Majda, M. Anal. Chem. 1991, 63, 85.
Lindholm-Sethson et al.
Figure 2. Typical cyclic voltammogram for a gold electrode modified with a polyelectrolyte film containing Os(bipy)2 in deaerated 2 mM HEPES buffer, pH 7.0, and 50 mM K2SO4. The arrow indicates the scan direction. The total amount of osmium complex was estimated from integration of the anodic and cathodic peaks. Eo′ ) 420 mV vs SHE, ΓOs ) 5 × 10-10 mol cm-2, v ) 20 mV/s, and A ) 0.16 cm2. monomer.20 It was self-assembled from a 2.4 mM solution (PVP monomeric concentration) in 20 mM H2SO4. All polyelectrolyte adsorption steps took place for 20 min at room temperature. This polyelectrolyte-modified gold electrode was mounted in a three-electrode thin layer flow cell as a part of a flow injection analysis (FIA) system. The effective electrode area was 0.16 cm2. Deaerated 2 mM HEPES buffer, pH 7.0, and 50 mM K2SO4 kept under argon atmosphere were pumped through the system. Cyclic voltammetry was performed until stable voltammograms were obtained. Liposome fusion was accomplished at the exposed electrode surface by the recirculation of 1 mM liposome or proteoliposome dispersion in the system for 2 h, followed by buffer solution for 15 min. Cyclic voltammetry was performed also during fusion. A stock solution of reduced cytochrome c was prepared by reduction with sodium dithionate of a 1-2 mM solution of cytochrome c in deaerated HEPES buffer and subsequent gel chromatography on a Sephadex PD 10 column. It was thereafter kept under argon in a test tube sealed with a septum. The electrode was polarized to +640 mV vs SHE, and 0.05 mL aliquots of different concentrations of reduced cyt c were injected at one of the valves. Dilutions were prepared immediately prior to each new injection. The concentration range was 1-35 µM, and the exact concentration was determined photometrically from the difference in absorbance at 550 and 541 nm (a550 - a541) with ) 21.0 mM-1 cm-1. Ellipsometry was used to determine the apparent mean thickness of the biological film, after electrochemistry, washing, and gently drying in an argon stream. The refractive index of lipid films is reported to be in the range 1.4-1.6, and we use 1.45, which is the same as that for protein films.11 Measurements were performed at five different spots, and a mean value was calculated.
Results and Discussion The gold electrode modified with a three-layer polyelectrolyte film was characterized with cyclic voltammetry after it was mounted in the flow cell of the FIA system. Consecutive cyclic voltammograms were collected until stable voltammograms were obtained (Figure 2). Cyclic voltammetry was also performed during fusion with the proteoliposomes, which resulted in significantly higher anodic currents, due to oxidation of cyt oxidase in the liposome suspension. When fusion and subsequent wash(20) Larsson, H. J. Electroanal. Chem. 1994, 365, 229.
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Figure 3. Oxidation peaks upon repeated injections of 0.05 mL of 0.027 mM reduced cyt c in the FIA system with cytochrome oxidase. The arrow indicates injection of 0.8 mL of 0.010 M sodium azide, which inhibits the enzyme, evident as a smaller amplitude of the oxidation peak at simultaneous cyt c injection. The reversibility of azide inhibition is also evident. The flow rate was 0.24 mL min-1, and the eluent was oxygen-free 2 mM HEPES buffer, pH 7.0, and 50 mM K2SO4 in all solutions.
ing were completed, slightly elevated anodic currents could still be observed at positive potentials as compared to those of the original voltammogram. This indicates the presence of small amounts of oxidase residing on the surface of the modified electrode. The oxidation of reduced cytochrome c was investigated with the planar gold electrode polarized to +640 mV vs SHE. The substrate was transported to the electrode surface by injection at a six-port valve into the flow stream in the tubing upstream. We varied the concentration of substrate and registered the current for different experimental conditions. As azide reversibly inhibits the enzyme and is easily washed away, we found it most appropriate and simple to use the inhibited enzyme preparation for determination of unspecific electron transport (background) and the same preparation in the absence of azide to determine total (specific and unspecific) electron transport.11 The amplitudes obtained for the azideinhibited preparation were negligible at very low cyt c concentrations (
