Langmuir 1993,9, 2255-2257
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Direct Electron Transfer to Escherichia coli Fumarate Reductase in Self-Assembled Alkanethiol Monolayers on Gold Electrodes Kathleen T. Kinnear and Harold G. Monbouquette' Chemical Engineering Department, University of California, Los Angeles, Los Angeles, California 90024-1592 Received July 27, 1 9 9 9 A strong cathodic current response to the presence of fumarate provides evidence of direct electron transfer to membrane-bound E. coli fumarate reductase in membrane-mimetic alkanethiol monolayers on gold electrodes. This electroenzymaticresponse is attenuated reversibly by the competitive enzyme inhibitor, oxaloacetate. The hydrophobic enzyme is coadsorbed with alkanethiols on gold in a novel detergent dialysis process. This approach could lead to novel sensors and biocatalytic devices and may provide an ideal means to study the electron transfer characteristicsof membrane-bound redox proteins in a readily manipulable membrane-mimetic medium.
Direct electron transfer between electrodes and redox gold have proven useful for the fundamental study of enzymesis of interest for the development of amperometric protein adsorption: and water-solubleredox enzymeshave biosensors and bioelectrocatalytic systems and for funbeen immobilized on such surfaces through both electrodamental study of redox centers in proteins.lb Such static and covalent atta~hment.~ Also, octadecyl merelectron exchange requires intimate contact between the captan adsorbed on a gold electrode has been used to enzyme and the electrode. Pyrolytic graphite electrodes anchor a phospholipid bilayer containing electroactive have proven particularly well suited for this purpose, cytochrome oxidase.1° In our case, a membrane-bound presumably due to the various functional groups available enzyme is embedded in an adsorbed alkyl mercaptan layer for enzyme interaction on the electrode ~ u r f a c e . lA~ ~ ~ ~ apparently in much the same way that these enzymes similar effect is obtained by chemically modifying the organize in lipid bilayers. surface of gold electrode^.^^^^^^^ Enzymes may adsorb We chose as our model system membrane-boundE. coli directly onto gold and other metal electrode surfaces, but fumarate reductase (FRD).ll FRD is a flavoproteinwhich the protein often has proved prone to denaturation, except catalyzes the terminal step in anaerobic electron transport at low tem~erature.~ when fumarate is utilized as a respiratory chain oxidant. Hydrophobic, membrane-bound enzymes such as DSimilar to E. coli succinate dehydrogenase, E. coli FRD fructose dehydrogenase (EC 1.1.99.8), D-gluconate dehyis composed of four protein subunits: FrdA (66 kDa) and drogenase (EC 1.1.99.4), and alcoholdehydrogenase(from FrdB (27 kDa) make up the hydrophilic, catalytic portion Gluconobacter suboxydans) have been observed to adsorb of the enzyme, and FrdC (15 kDa) and FrdD (13 kDa) on carbon paste, graphite, and metal electrodes and to comprisethe hydrophobicmembrane-boundanchor of the exhibit direct electron transfer.1ap2*3c However,the enzyme microenvironment in this configuration is far from that of enzyme. FrdA containsa covalentlybound FAD prosthetic ita native lipid bilayer. An alternative method based on group, while FrdB harbors three iron-sulfur centers. In interaction of proteins with lipid bilayers depositeddirectly addition to anchoring the complex in the membrane, the on electrodes has been developed.6 Well-characterized hydrophobicsubunits protect the iron-sulfur clusters from alkanethiol monolayerson gold' also present an attractive inactivation in the presence of oxygen and are required environment for gentle, stabilizing immobilization of for the enzymeto exchangeelectronswith quinones.ll FRD hydrophobicproteins. The alkyl chainsof the chemisorbed normally reduces fumarate with menaquinol serving as species effectively mimic the ion barrier properties of the the electron donor. The FAD group and the three ironorganized hydrocarbon chains in lipid bilayers. SAMs on sulfur centers of FRD: 1[2Fe-2Sl, 2[4Fe-4Sl, and 3[3Fe4S1, all exhibit standard redox potentials more negative, Abstract published in Advance ACS Abstracts, September 1, @
1993. (1)(a) Ikeda, T.; Fushimi, F.; Miki, K.; Senda, M. Agric. Bioi. Chem. 1988,52, 2655-2658. (b) Guo, L. H.; Hill, H. A. 0.; Lawrance, G.A.; Sanghera, G.S.; Hopper, D. J. J.Electroanal. Chem. 1989,266,379-396. (c) Guo, L.-H.; Hill, H. A. 0.; Hopper, D. J.; Lawrance, G. A.; Sanghera, G. S. J. Bid. Chem. 1990,265,1958-1963. (2) (a) Ikeda, T.; Mataushita, F.; Senda, M. Biosens. Bioelectron. 1991, 6, 299-304. (b) Ikeda, T.; Miyaoka, S.; Miki, K. J.Electroanal. Chem. 1993,352,267-278. (3)(a) Burrows, A. L.; Hill, H. A. 0.; Leese, T. A.; McIntire, W. S.; Nakayama, H.; Sanghera, G. S. Eur. J. Biochem. 1991,199,73-78.(b) Gorton, L.; Jijnseon-Petterseon,G.; Cs6regi,E.; Johaneson, K.; Dominguez, E.;MarkeVarga,G.Analyst 1992,117,1235-1241.(c) Ikeda,T.;Miyaoka, S.;Matsushita, F.; Kobayashi, D.; Senda, M. Chem. Lett. 1992,847-850. (4)Armstrong, F. A.; Hill, H. A. 0.; Walton, N. J. Acc. Chem. Res. 1988,21,407-413. (5)Armstrong, F. A. Struct. Bond. 1990,72, 137-221. (6)(a) Tien, H.T.; Salamon, Z.Bioeleetrochem. Bioenerg. 1989,22, 211-218. (b) Schuhmann, W.; Heyn, S.-P.; Gaub, H. E. Adu. Mater. 1991,3,388-391. (c) Salamon, Z.;Tollin, G. Bioelectrochem. Bioenerg. 1991,25,447-454. (d) Tien, H.T.; Salamon, Z.; Ottova, A. Crit. Reu. Biomed. Eng. 1991, 18,323-340. (e) Salamon, Z.;Tollin, G. Bioelectrochem. Bioenerg. 1992,27,381-391.
(7)(a) Finklea, H. 0.; Avery, S.; Lynch, M.; Furtach, T. Langmuir 1987,3,409-413.(b) Porter, M. D.; Bright, T. B.;Allara, D. L.; Chideey, C. E. D. J. Am. Chem. Soc. 1987, 109, 3559-3568. (c) Bain, C. D.; Troughton, E. B.;Tao, Y.-T.; Evall, J.; Whitesides, G. M.; Nuzzo, R. G. J.Am. Chem. SOC.1989,111,321-335.(d) Whitesides, G.M.; Laibinie, P. E. Langmuir 1990,6,87-96.(e) Alves, C.A.; Smith, E. L.; Porter, M. D. J. Am. Chem. SOC.1992,114,1222-1227. (8)Prime, K. L.; Whitesides, G.M. Science 1991,262,1164-1167. (9)(a) Tarlov, M. J.; Bowden, E. F. J. Am. Chem. SOC.1991, 113, 1847-1849. (b) Collinson, M.; Bowden, E. F.; Tarlov, M. J. Langmuir 1992,8,1247-1250.(c) Willner, I.; Katz, E.; Riklin, A.; Kasher, R. J.Am. Chem. SOC. 1992,114,10965-10966.(d) Amador, S.M.; Pachence, J. M.; Fischetti, R.; McCauley, J. P.; Smith, A. B.;Blasie, J. K. Langmuir 1993, 9,812-817. (10)Cullison, J. K.; Hawkridge, F. M.; Nakashima, N.; Hartzell, C. R. In Charge and Field Effects in Biosystems--3; Allen, M. J., Cleary, S. F., Sowers, A. E., Shillady, D. D., Eds.; Birkhluser: Boston, MA, 1992; PP 29-40. (11)Ackrell, B. A. C.; Johnson, M. K.; Gunsalus, R. P.; Cecchini, G. In Chemistry and Biochemistry of Flauoenzymes; Mtiller, F., Ed.; CRC Press: Boca Raton, FL, 1992; Vol. 111, pp 229-297.
0743-7463/93/2409-2255$04.00/00 1993 American Chemical Society
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2256 Langmuir, Vol. 9,No. 9,1993
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Figure 1. Steady-state CVs of FRD in SAMs on gold in the presence of 0 mM (short dashes), 0.375 mM (long dashes), and 2.0 mM fumarate (solid curve). All CVs were conducted at room temperature in 50 mM HEPES (pH 7.55) and 100 mM NaCl under a blanket of argon at a scan rate of 50 mV/s.
at -55 mV and -20 mV, -330 mV, and -70 mV, respectively,I2 than the succinate/fumarate couple (30 mV) consistent with ita role as a reductase. Recently, direct electron transfer between the water-soluble catalytic subunita of both E. coli succinate dehydrogenase and fumarate reductase and a graphite electrode surface have been dem0n~trated.l~ However, loss of enzyme activity was reported with continued voltammetric ~yc1ing.l~ We use the complete FRD complex, including the two protective hydrophobicsubunita, to anchor the enzyme in a self-assembled alkanethiol layer on gold. The hydrophobic enzyme is codeposited with alkanethiols on gold in a novel one-step process. The enzyme (10-20pM) and alkanethiol (0.05 mM) are solubilized in HEPES buffer (50 mM, pH 7.55) and 100 mM NaCl with 75 mM n-octyl 8-D-glucopyranoside. The preparation is dialyzed against 4-5 X 500 mL deoxygenated 50 mM HEPES buffer (pH 7.55) for 18h in the presence of a clean,141.6mm diameter gold electrode using a 6000-8000 M W cutoff dialysis bag. Best results were obtained for a heterogeneous alkanethiol layer formed from a 6040 mix of octadecyl mercaptan (OM) and dodecyl mercaptan (DM). The shorter chain of DM may allow closer approach of the enzyme to the electrode surface. Cyclic voltammograms (CVs) conducted in 50 mM HEPES and 100 mM NaCl under a blanket of argon do not show oxidation and reduction current peaks in the absence of substrate, presumably due to low enzyme surface coverage and the resolution limita of our instrumentation.15 When fumarate is added to the system, however, catalytic waves are observed whose intensity increases with fumarate concentration (Figure 1). A cathodic response has been detected at fumarate concentrations as low as 75 pM. The onset of catalytic activity occurs close to the formal reduction potential for the (12) (a) Cammack, R.; Patil, D. S.;Condon, C.; Owen, P.; Cole, S. T.; Weiner, J. H. In Fhuim and Flauoproteim; Bray, R. C., Engel, P. C., Mayhew, S. G.,E&.; Walter de Gruyter: New York, 19W, pp 551-554. (b) Simpkin,D.;Ingledew, W. J. Biochem. SOC.Tram. 1984,22,500-501. (c) Cammack, R.;Patil, D. S.;Weiner, J.H. Biochim. Biophys. Acta 1986, 870,546551. (13) (a) Sucheta, A.; Ackrell, B. A. C.; Cochran, B.; Armstrong, F. A. Nature 1992,356,361-362. (b) Sucheta, A.; Cammack, R.; Weiner, J.; Armstrong, F. A. Biochemistry 1993,32, 5455-5465. (14) Cyclic voltammograms were conducted with a Bioanalyticnl Lafayetta, IN) CV-1B interfaced to a Macintosh IIcx with Systems (W. a National Instruments (Austin, TX) Lab-NB board and software (LabVIEW 11). An Ag/AgCl reference electrode and a platinum wire counter electrode were used for all studies. (15) Gold disk working electrodes (diameter = 1.6 mm;A,, = 0.02 cm2)were polished with 5-, 1-, and 0.05-jim alumina (with sonication between each size alumina powder), treated briefly with aqua regia, repolished with 0.05-pm alumina briefly, and subjected to repeated scans from 1.4 to -0.3 V in 0.5 M HSO. before each use.
0.6 ' I m
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Figure 2. Inhibition of FRD electroenzymologyby oxaloacetate. A typical catalytic CV is obtained for the uninhibited FRD electrode in 1 mM fumarate (solid curve). However, after conducting CVs in 0.75 mM oxaloacetate in the absence of fumarate (no electroenzymatic activity observed), an enzyme electrode exhibita no catalytic response upon transfer back to a 1 mM fumarate solution (dashed c w e ) . FRD activity is recovered fully after incubation of the electrode in the 1 mM fumarate solution overnight. All CVs were conducted at room temperature in 50 mM HEPES (pH 7.55) and 100 mM NaCl under a blanket of argon at a scan rate of 50 mV/s.
succinate/fumarate couple (30 mV). The role of the enzyme in the observed phenomena is confiied by the reversible inhibition of the reduction current response by the strong competitive inhibitor, oxaloacetate (Figure 2).lS Oxaloacetate also has been shown to inhibit the electroenzymatic oxidation of succinate to fumarate by FRD in micelles." In additional control studies, no catalytic response with increasing fumarate concentration was observed for the bare gold electrode, for the case where enzyme adsorption was attempted in the absence of alkanethiol, or for an electrode with an adsorbed alkanethiol layer formed through detergent dialysis in the absenceof FRD. The immobilized enzymesystem is stable at room temperature for at least several days despite repeated rinsing and cyclic voltammetry Studies run to steady state. However while conducting cyclic voltammograms at 36 OC, the system shows complete loss of activity in a matter of hours. Partial activity is recovered upon incubation overnightin buffer at room temperature. These results have been reproduced several times wing both pure FRD18 and a preparation that includes some membranelipid.lg Efforts to further optimizethis system for improved enzyme loading and catalytic activity are currently in progress. Cyclic voltammograms, conducted with and without an alkanethiol layer formed by detergent dialysis, in 0.5 M HzSO4 suggest that the electrode surface is modified in a way similar to that observed for SAMsformed from organic solvents. In Figure 3 the characteristic reduction in the gold oxide removal peak is observed indicating impeded access of HzO to the electrode surface and a typically high fractional surface coverage of 0.98.20 Further studies are underway to more fully assess the quality of alkanethiol SAMs formed by detergent dialysis as opposed to those assembled from ethanol or chloroform. (16) Ache& B.A.C.;Cochran,B.;Cecchini,G.Arch.Biochem.Biophys. 1989,268,2644. (17) Kinnear, K. T.;Monbouquette, H. G.Biotechnol. Bioeng. 1993, 42,140-144. (18) Morningstar, J. E.; Johnson, M. K.; Cecchini, G.; Ackrell, B. A. C.; Kearney, E. B. J. Biol. Chem. 1985,260,13631-13638. (19) Johneon, M. K.; Kowal, A. T.; Morningstar, J. E.; Oliver, M. E.; Whittaker, K.; Gunealue, R. P.; Ackrell, B. A. C.; Cecchini, G.J. Biol. Chem. 1988,263,14732-14738. (20)Sabatani, E.;Rubinstein, I.; Maw, R.; Sa+, J. J. Electroanal. Chem. 1987,219,365-371.
Letters
Langmuir, Vol. 9, No. 9, 1993 2267 those envisioned for immobilized lipid bilayer systems,6d*22 where membrane proteins, including receptors and ion channels,are incorporated intoSAMs. Also, if the surface concentration of redox protein can be increased such that noncatalytic electron exchange is observed, this system may provide an ideal means to study the electron transfer characteristics of membrane-bound redox proteins in a readily manipulable membrane-mimeticmedium. -20 1.5
1
0.5
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-0.5
Potential (V vs Ag/AgCI) Figure 3. Passivation of a gold electrode surface by an OM/DM S A M formed through detergentdialysis. The characteristic gold oxide removal peak observed in a steady-stateCV of the untreated electrode (dashed curve) is reduced dramatically in a CV of the surface-coated electrode(solid curve). Both CVs were conducted at room temperature in 0.5 M HzSO, at a scan rate of 60 mV/s.
Our simple one-step process for self-assembling an alkanethioUenzyme layer on gold through detergent dialysis may provide a means to reconstruct other redoxactive, membrane-bound sensory and catalytic proteins in a stabilizing membrane-mimeticmicroenvironmenton gold electrodes. Others have demonstrated that selective ligands integrated into SAMs have potential in the Our approach could developmentof ion and pH lead to novel sensors and biocatalytic devices, analogous to
Acknowledgment. K. T. Kinnear acknowledges support from the UCLA Women and Minority Graduate Assistantship Program and the UCLA Training Program in Biotechnology (NIGMS, USHHS-1232-GM-08375).We are grateful to G. Cecchini (Veterans Administration Medical Center, University of California, San Francisco) for the gift of E. coli fumarate reductase and for several useful discussions. (21) (a) Rubinetein, I.; Steinberg, S.; Tor, Y.;Shanzer, A.; Sagiv, J. Nature 1988,332,426-429. (b) Nelson, A. J. Chem.SOC.,Faraday !Pram. 1991, 87, 1851-1856. (c) Hickman, J. J.; Ofer, D.; Ldbinia, P. E.; Whitaides, G. M.; Wrighton, M. S . Science 1991, 252, 688-691. (d) Steinberg,5.;Rubinetein,I. Langmuir 1992,8,1183-1187. (e) Stainberg, S.; Tor, Y.; Sabatani, E.; Rubmetein, I. J. Am. Chem. SOC.1991, 113, 5176-5182. (22) (a) Krull, U.J.; Thompson, M.Trende A d . Chem. 1986,4,9096. (b) Tien, H. T.J. Clin. Lab. Anal. 1988,2,256-264. (c) Stanger, D. A.; Fare, T.L.; Cribbs, D. H.; Ruin, K. M. Biosene. Bioelectron. 1992, 7, 11-20.
