Langmuir 1994,10, 877-882
A Study of Cytochrome c Oxidase in Lipid Bilayer Membranes on Electrode Surfaces John K. Cullisont and Fred M. Hawkridge' Department of Chemistry, Virginia Commonwealth University, Box 842006, Richmond, Virginia 23284
Naotoshi Nakashima Department of Applied Chemistry, Nagasaki University, Nagasaki 852, Japan
Shimichi Yoshikawa Faculty of Science, Himeji Institute of Technology, Japan Received September 14,1993. I n Final Form: December 2,199P A membraneassociated enzyme, cytochrome c oxidase, has been put in lipid bilayer membranes attached to gold electrode surfaces. Goldhhiol self-assembly chemistry and deoxycholate dialysis procedures have been used to insert cytochrome c oxidase into a lipid bilayer on gold surfaces with a controlled orientation. Voltammetric and spectroelectrochemical results indicate that direct electron-transfer communication between the gold electrode surface and cytochrome c oxidase has been achieved. Moreover, immobilized cytochrome c oxidase can both reduce and oxidize solution-residentcytochrome c by appropriately controlling the applied electrode potential.
Introduction Chemical modification of surfaces has been an active area of research for a number of decades.lb In 1982 Taniguchi et al. used sulfur-containing compounds to modify gold electrodes! and in 1983 Nuzzo and Allara7 and Netzer and SagivS published papers describing the spontaneous self-assembly of ordered amphiphiles on electrode substrates. Since the publication of these papers, interest, in the spontaneous assembly of monolayers on electrodes has escalated.9 The incorporation of proteins into membranes on planar supports has been described previously by McConnell et al.l0and Nakanishi,ll and the use of cholate dialysis to incorporate proteins into membranes on glass slides has been described by Huang.12 The work described here combines spontaneous membrane assembly methods with enzyme reconstitution procedures to immobilize a membrane-resident mitochondrial redox enzyme onto an electrode surface. The method enables the study of the heterogeneous electron-transfer reaction between the immobilized cytochrome c oxidase and its
* To whom correspondence should be addressed. + Present address: Department of Chemistry, University of California at Riverside, Riverside, CA 92621. Abatractpublishedin Advance ACSAbstmcts, February 1,1994. (1) Adamson, A. W. In Phyical Chemistry of Surfaces,3rd ed.;Wiley: New York, 1976; pp 473-503. (2) Rabolt, J. F.; Sank, R.; Swalen, J. D. Appl. Spectrosc. 1980, 34, 517-521. (3) Richard, M. A.; Deutach, J.; Whitesides, G. M. J. Am. Chem. SOC. 1979, 200,66134626. (4) Polymeropouloe, E. E.; Sagiv, J. J. Chem. Phys. 1978,69, 18361847. (5) Techarner, V. V.; McConnell, H. M. Biophys. J. 1981,36,421-426. (6) T-chi, I.; Yoyosawa, K.; Yamaguchi, H.; Yaaukouchi, K. J. Electroanal. Chem. 1982,140,187-193. (7) Nuezo,R.G.;AUara,D.L.J.Am.Chem.Soc. 1983,105,4481-4483. (8) Netzer, L.; Sagiv, J. J. Am. Chem. SOC.1983,105,674-676. (9) Swalen,J. D.; Allara, D. L.;Andrade, E. A.; Chandross,E. A,;Garoff,
S.;Isreelachvili, J.; McCarthy, T. J.; Murray, R.; Pease, R. F.; Rabolt, J. F.; Wynne, K. J.; Yu, H. Langmuir, 1987,3, 932-950. (10) McConnell,H. M.; Watta,T. H.;Weis,R.M.; Brian,A. A.Biochim. Biophys. Acta 1986,864,95-106. (11) Nakanishi, M. FEBS Lett. 1984,176, 385-388. (12) Huang, L. Biochemistry 1986,24, 29-34.
native redox partner cytochrome c. The work described here may provide a means of constructing a membrane environmentin which to study how these complex enzymes orchestrate their redox chemistry in vivo. Initial experiments using Langmuir-Blodgett technology proved unproductive. In these experiments monolayers were prepared on the surface of tin oxide optically transparent electrodes. Subsequent experiments using thiol self-assembly methods showed that alkyvthiol monolayers on gold afford a more stable membrane assembly than the Langmuir-Blodgett technique. This stability is believed to be a result of the "covalent" linkage between the gold and the thiolate of octadecyl mercaptan. Thiols react strongly with gold to form a bond that is believed to be predominantly convalent with some ionic character.lgl8 The octadecyl mercaptan chains of wellordered monolayers have a lowest energy orientation when the hydrocarbon chain of the mercaptan is tilted between 20° and 40' from the surface The orientation of the chains at the surface is predominantly controlled by intermolecular van der Waals forces between the hydrocarbon chains. The rate of assembly of these monolayers on gold is dependent on the octadecyl mercaptan concentration, solvent, temperature, and chain length of the adsorbate molecule, as well as the cleanliness and structure of the (13) Evans, S. D.; Ulman, A. Chem. Phys. Lett. 1990,170,462-466. (14) Chidsey, C. E. D.; Liu, G.-Y.;bwntree, P.; Scoh, G. J. J. Chem. Phys. 1989,91, 4421-4423. (15) Bain, C . D.; Biebuyck, H. A.; Whitesides, G. M. Lungmuir 1989, 5,723-727. (16) Strong, L.; Whitesides, G. M. Langmuir 1988, 4, 546-558. (17) Widrig, C. A.; Chung, C.; Porter, M. D. J. Electroanal. Chem. 1991,310,335-359. (18) Bryant, M. A.; Pemberton, J. E. J. Am. Chem. SOC.1991, 113, 8284-8293. - -- . - -_ -.
(19) Porter, M. D.; Bright,T. B.; Allara, D. L.; Chidsey, C. E. D. J. Am. Chem. SOC.1987,109,3559-3568. (20) Ulman,A.; Eilers, J. G.; T h a n , N. Langmuir 1989, 5, 114711 52.
(21) Nuzzo, R. G.; Dubois, L. H.; Allara, D. L. J. Am. Chem. SOC.1990, 112,558-569.
0743-7463/94/2410-0877$04.50/00 1994 American Chemical Society
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gold substrate prior to modification.22 At room temperthem on to the second pair of prosthetic groups, heme a3 ature octadecyl mercaptan monolayers on gold are stable and The second pair forms a binuclear redox for months in air, aqueous solutions, and ethanol.22 center that is believed to be the active site for the reduction of dioxygen to water. The approximate formal potentials The dialysis procedures used in conjunction with the of these four redox sites are as follows: heme a, 215 mV; self-assembly methods are based on those found in the heme as, 350 mV;47cub, 350mV; CUa, 190mV.48 However, literature for reconstituting membrane-resident enzymes these redox sites can also be shown to be more complex into vesicles in s0lution,2~and are similar to those used by and dependent upon cooperativecoupling of these values.'B Huang.12 For reconstituting enzymes of the oxidative The general experimental rationale described in this phosphorylation chain, detergents such as cholate or paper is as follows. A stable submonolayer of octadecyl deoxycholate are used to disperse the membrane phosmercaptan is initially formed on a gold substrate. These pholipids and the enzyme. The removal of the detergent electrodes are then placed into dialysis tubing along with through dialysis%then promotes the formation of vesicles deoxycholate, biological amphiphiles, buffer, and the that contain the reconstituted enzyme. enzyme. These electrodes are then dialyzed against buffer Cholate and deoxycholate are both bile-salt detergents. to remove the deoxycholate which drives the formation of One side of the molecule is hydrophobic and the other is bilayers onto the gold substrates. Voltammetric and hydrophilic. When added to a vesicle solution,they insert spectroelectrochemical experiments conducted on these into the membrane bilayer and disperse the components modified electrodesprovide data that show direct electronof the vesicle. The structure of the cholate molecule transfer communication between the gold substrate and enables it to stabilizethe hydrophobic edges of the bilayer cytochrome c oxidase. The data indicate that by approby minimizing their contact with aqueous solution.26 priately controlling the potential the immobilized cytoCytochrome c oxidase has been reconstituted into chrome c oxidase can both reduce and oxidize solutionvesicles with considerable success. Using cholate dialysis resident cytochrome c. Th goal of this enzyme immoprocedures, between 75% and 100% of the reconstituted bilization scheme is to provide a membrane structural cytochrome c oxidase molecules are unidirectionally environment for studying the mechanisms of electron oriented with their cytochrome c binding sites facing ~ ~ in cytochrome c oxidase. outward, mimicking the orientation found in u ~ u o . ~ ~transfer Cytochrome c oxidase is the terminal enzyme of the Experimental Section oxidative phosphorylation chain, and it catalyzes the fourelectron reduction of dioxygen to water.% The electrons Instrumentation. The cyclic voltammetry and differential pulse voltammetry experiments were performed using a PAR used to reduce oxygen are received from ferrocytochrome 174 polarographic analyzer. For the potential step chronoabc at the outer face of the inner membrane of the sorptometryexperimentam an IBM PC AT microcomputersystem mito~hondrion.~sThe molecular weight of the bovine with a 12-bit Data Translation DT2801A analog-to-digital and complex has been estimated to be between 166 OOO and digial-to-analogboard was used to acquire data and generate 210 OOO Da,29931and cytochrome c oxidase is believed to potential step. A commercial Data Translation software packcontain up to 13 subunits.3- Approximately half of the age, DT/Notebook, was used in conjunction with the data enzyme is embedded within this mitochondrialmembrane acquisition board. The potentialwaveformsused were generated while the other half protrudes into the digitally. The instrumentationused in the apectroelectrochemical experiments has been described pre~iously.5~ Cytochrome c oxidase contains four redox sites, two Electrochemical Cells. The cell used in all experiments, a-type hemes (hemes a and as) and two copper atoms (Cu, except the potentialstep chronoabsorptometry experiments, was and cub). The four prosthetic groups can be divided into adapted by Kine2 from a design originally developed by two pairs. One pair, heme a and CUa,ae-ll1are redox centers B0wden.M In brief, the cell was machined from Lucite and that accumulate electronsfrom ferrocytochromec and pass designed to accommodate a planar electrode. The electrode is clamped onto the cell, and an O-ringsealsthe contactand defies (22) Bain, C. D.; Troughton, E. B.; Tao, Y. T.;Evall, J.; Whitesides, the electrode area. Electrical contact to the working electrode G. M.; NUZZO, R. G. J. Am. Chem. SOC.1989,111, 321-335. is made using a brass shim. The reference electrode used in all (23) Eytan, G. D. Biochim. Biophys. Acta 1982,694,186202, experiments was a Ag/AgC1(1.0M KCl) reference electrode. A (24) Racker, E. J. Membr. Biol. 1972,10, 221-235. coiled platinum wire was used as the auxiliary electrode. (25) Small, D. M.; Penkett, S. A.; Chapman, D. Biochim. Biophys. Acta 1969,176, 178-189. For the potential step chronoabsorptometry experiments a (26) Casey, R. P.; Ariano, B. H.; Ami, A. Eur. J. Biochem. 1982,122, thin-layer cell designed by Bowden et al. was u8ed.M In brief, 313-318.
(27) Nicholls, P.;Hildebrandt, V.;Wriggleaworth,J. M. Arch. Biochem. Biophys. 1980,204, 533-543. (28) Palmer, G. Pure Appl. Chem. 1987,59,749-758. (29) Capaldi, R. A.; Malateeta, F.; Darley-Uehmar, V. M. Biochim. Biophys. Acta 1983, 726,135-148. (30) h i , A. Biochim. Biophys. Acta 1980,594, 231-252. (31) Capaldi, R. A. Biochim. Biophys. Acta 1982,694,291-306. (32) Kadenbach, B.; Jauraech, J.; Hartman, R.; Merle, P. Anal. Biochem. 1983,129,517-529. (33) Kadenbach, B.; Merle, P. FEBS Lett. 1981,135,l-11. (34) Verheul, F. E. A. M.; Boonman, J. C. P.; Draijer, J. W.; Muijers, A. 0.;Borden, D.; Tan, D. E.; Margoliaeh, E. Biochim. Biophys. Acta 1979,548,397-416. (35) Vanderkooi,G.;Senior,H.E.; Capaldi, R. A.;Hayashi, H. Biochim. Biophys. Acta, 1972,274,38-48. (36) Henderson, R.; Capaldi, R. A.; Leigh, J. S. J . Mol. Biol. 1977,112, 631-648. (37) Fuller, S. D.; Capaldi, R. A.; Henderson, R. J.Mol.Biol.l979,134, 305-327. (38) Fuller, S. D.; Capaldi, R. A.; Henderson, R. Biochemistry 1982, 21,2525-2529. (39) DePiere, E.; Emter, L. Annu. Rev. Biochem. 1977,&,201-262. (40) Beinert, H.; Hansen, R. E.; Hartzell, C. R. Biochim. Biophys. Acta 1978,423,339-365. (41)Yong, F. C.; King, T. E. J. Biol. Chem. 1972, 247, 6384-6388.
(42) Eglington, G. G.; Johneon, M. K.; Thompson, A. J.; G d i , P. E.: Greenwood. C. Biochem. J. 1980.191., 319-331. -~ - - ~ '(43) Wharton, D. C. In Metal Io& in Biological Systems; Sigel, H., Ed.;Marcel Dekker: New York, 1974; Vol. 3, p 157. (44)Lindsey, J. S.;Owen, C. S.;Wilson, D. F. Arch.Biockm. Biophys. _ 1971,169,492-505. (45) Erecinska, M.; Wileon, D. F. Arch. Biochem. Biophys. _ _ 1978,188, 1-14. (46)Powers, L.; Chance, B.; Ching, Y.; Angiollo, P. Biophys. J. 1981, 34,465-498. (47) Schroedl,N. A.;Hartzell, C. R. Biochemistry 1977,16,4961-4971. (48) Anderson, J. L.: Kuwana, T.: Hartzell, C. R. Biochemistn, 1976. 15,3847-3855. (49) Hendler, R. W.; Westerhoff, H. V. Biophys. J. 1992, 63, 1586-
..
1 find -""
(60) Bancroft, E. E.; Sidwell, J. S.; Blount, H. N. Anal. Chem. 1981,
53,1390-1394. (51) Re%d, D. E.; Hawkridge, F. M. Anal. Chem. 1987,59,2334-2339. (52) King,B. C. Ph.D. Dhrtation,VirginiaCommonwealthUniversity, 1988. (53) Bowden, E. F. Ph.D. Dissertation, Virginia Cmmonwealth University, 1982. (54) Bowden, E. F.; Cohen, D. J.; Hawkridge,F. M. Anal. Chem. 1982, 54, 1005-1007.
Cytochrome c Oxidase in Lipid Bilayer Membranes the cell formed a sandwich with the enzyme-modified gold electrode on one side and a quartz window on the other, with a thin layer of solution (Le,, 150 pm) between. A bifurcated light pipe was positioned flush against the quartz window. The visible light at 550 nm was focused on one arm of the light pipe, directed to the surface of the electrode, and then reflected light was collected by the light fibers forming the other arm. The light exiting this arm of the light guide was then focused onto the photomultiplier tube. The difference in the molar absorptivity between reduced and oxidized cytochrome c at 550 nm is approximately 21 OOO M-l cm-lSM In all of the modified electrode experimenta the cells were assembled under 0.1 M phosphate buffer, pH 7.4. This was a precautionary measure taken to avoid exposureof the membranemodified electrode surfaces to air to prevent the forces present at air-water interfaces from disrupting the membrane assemblies. Reagents. The water used in all of these experimenta was first deionized and then further treatedwith a Milli RO-4/Milli-Q system (Millipore Corp.) to exhibit a resistivity of 18 M cm-l upon delivery. The phosphate used to prepare the 0.1 M phosphate, pH 7.4, buffers was ACS reagent grade. The gold foil (Aldrich,99.99 % ) used for electrodeswas polished with alumina polish (Beuhler, Alpha Micropolish I1 deagglomerated alumina) and a polishing cloth (Fisher, Gamal polishing cloth). The vapor-depositedelectrodes (Evaporated Metal Films, Inc.) had a thin film of CrOz or TiOl with approximately 10002000 A of gold covering the oxide layer. The vapor-deposited electrodes were cleaned in an air plasma (Harrick Scientific, Inc.) and were hydrophilic upon removal from the plasma cleaning chamber. Hydrophilicity is a good qualitative measure for assessing the cleanliness of gold surface5.B The electrodes were removed from the plasma cleaning chamber and immediately immersed in water until used to reduce contamination of the gold surface from air-born organics. Octadecylmercaptan (Aldrich,98 % ) was usedwithout further purification. Ethanol (US1 Chemical Co., 100%)was used as a solvent for the octadecyl mercaptan. The electrodes were taken out of the water bath, rinsed with ethanol, and then placed into the octadecyl mercaptan/ethanol mixture. Following the modification, the electrodes were rinsed generously with ethanol and water, and then once again placed in water until further use. Bovine cytochrome c oxidase from two different preparations was used. The two isolation procedures used were the Hartzell and Beinert preparations7 and the Yoshikawa and Caughey preparation.% Bovine cytochrome c oxidase samples were received directly from Yoshikawa's laboratory or purified in our laboratory from fresh beef hearts." One modification in the Yoshikawa and Caughey preparation was required. The cytochrome c oxidase did not always appear in the expected ammonium sulfate precipitation fraction. After each precipitation, adropfromeachfraction wasaddedtoacuvette containing reduced cytochrome c and the decrease of the 550-nm band of cytochrome c was monitored. The fraction that gave the most rapid decrease in the 550-nm band contained the more active cytochrome c oxidase and was the fraction that was saved for subsequent use. Cytochrome c (Sigma Chemical Co., type VI) was further purified using a (carboxymethy1)cellulose (Whatman, CM-52) cation-exchangecolumn followinga published procedure.S@The purified cytochrome c was collected from the ion-exchangecolumn and concentrated to 5 mL using an Amicon ultrafiltration system (AmiconCorp. Model 8050 ultrafiltration cell). The cytochrome c was reduced using sodium dithionite and passed through a desalting column (Bio-Gel, P-6DG). In order to avoid contamination of the cytochrome c with dithionite or with reaction products, only the first half of the elution band was collected. The concentration was determined using a Hewlett-Packard (55) Van Gelder, B. F.; Slater, E. C. Biochim. Biophya. Acta 1962,58, 593-595. (56) Smith, T. J. Colloid Interface Sci. 1980, 75, 51-65. (57) Hartzell. C. R.: Beinert, H. Biochim. Biophys. - . Acta 1974, 368, 318'-338. (58)Yoehikawa, S.; Choc, M. G.; O'Toole, M. C.; Caughey, W. S. J. Biol. Chem. 1977,252,5498-5508. (59) Brautigan, D. L.; Fergueon-Miller, S.; Margoliaah, E. Methoda Enzymol. 1978,630, 131-132.
Langmuir, Vol. 10, No. 3, 1994 879 Model 8452A diode array UV/vis spectrophotometer and using a molar absorptivity for reduced cytochrome c of 29 500 M-1 cm-l at 650 nmaM The dialysis experiments were modeled after procedures used to reconstitute cytochrome c oxidase into vesicles in solution.Two different types of biological amphiphilee were used in these experimenta, (DOPE) L-phosphatidylethanolamine, dioleoyl (Sigma, 99%1, and (DOPC) L-phosphatidylcholine, dioleoyl (Sigma, 99 % ). These amphiphiles were stored in a chloroform/ methanol solvent. Approximately 25 mg of DOPE and 6 mg of DOPC were injected into a 10-mL round-bottom flask. The storage solvent was removed by evaporation (Buchi, RE111 Rotovap). The amphiphiles were then rinsed with anhydrous ether (J. T. Baker, >98%), and the ether was removed via evaporation. Ether was used once more to rinse the amphiphilee and was then removed by evaporation. This procedure was followedto ensure complete removal of the chloroform/methanol storage solvent. Once the storage solvent had been removed 50 mg of sodium deoxycholate (Sigma) was added to the round-bottom flask with 3 mL of 0.1 M phosphate buffer, pH 7.4. The flask was wrapped with aluminum foil to avoid any photodegradation of the amphiphilee, and the mixture was stirred at 4 "C until all of the amphiphile residue had dissolved. Dialysis tubing (molecular weight cutoff 3500, Spectrapor, Spectrum Medical Industries, Inc.) was soaked in water to remove glycerin and sulfides from the membrane prior to use. Between 2 and 5 mg of cytochrome c oxidase was added to the buffered amphiphde/deoxycholate mixture. When the cytochrome c oxidase from the Hartzell and Beinert oxidase preparation was used, the enzyme was added to the amphiphile/ deoxycholate mixture and then stirred at 4 "C for 30 min. The mixture was then sonicated for 5 min and finally stirred again for 30 min. The mixture was then passed through a 1-pm filter to remove the particulates from the mixture. The cytochrome c oxidase from the Yoshikawa/Caughey preparation was added to the amphiphile/deoxycholate mixture and stirred for 6 min, and it formed a solution. The results obtained in this work did not depend on which cytochrome c oxidase sample was used. Unless otherwise indicated, the Hartzell and Beinert oxidase preparation was the sample studied. The octadecyl mercaptan modified electrodes were put into the dialysis tubing with the oxidase mixture. The air inside the tubing was removed before tying the ends of the dialysis membrane. The electrodes were then dialyzed against 0.1 M phosphate buffer, pH 7.4, for 2-6 daya at 4 OC under stirred conditions. The phosphate buffer was changed twice daily. Following the dialysis, the dialyais bag was cut open with a clean razor blade in a container of 0.1 M phosphate buffer. Latex glovea and Teflon-coatedtweezerswere used at all times to handle the electrodes, being careful to only touch the edges of the electrodes. A 50-mL beaker was immersed in the buffer, and one of the electrodes was put into the beaker without allowing the electrode surface to contact air. A pipet was used to remove as much of the buffer as possible while still keeping the electrode submerged. The beaker was rinsed with fresh buffer, several times to remove any vesicles containing cytochromeoxidase. The electrode was next placed into a large container of buffer, and the electrochemical cell was amembled under the surface.
Results and Discussion Figure 1 shows an idealized model of a gold electrode following modification and immobilization of cytochrome oxidase onto the electrode surface. The gold surface actually is not atomically smooth, and contains on the atomic scale a high degree of surface roughness, as well as grain and edge boundaries. The amphiphiles are also not (60)Caaey, R. P. Biochim. Biophys. Acta 1984, 768,319-347. (61) Hinkle, P. C.; Kim, J. J.; Racker, E. J. Biol. Chem. 1972, 247, 1338-1339. (62) Zhang, Y.; Georgevich, G.; Capaldi, R. A. Biochemietry 1984,23, 5616-5621. (63) Zhang, Y.; Capaldi, R. A,; Cullis, P. R.; Madden, T. D. Biochim. Biophya. Acta 198S,808,206-211.
880 Langmuir, Vol. 10, No.3, 1994
Cullison et al.
G==i
s"r-0
ommmm0
Solution
Cytochrome c Oxidase
Au s-o ~ rdJced CYtC
0 s--0
O~ xidized
cyt c
Figure 1. A model of the enzyme-immobiliedelectrodesurface. The octadecyl mercaptan is the molecule containingthe sulfur, S, head group, and the biological amphiphilesare shown as the two tailed moieties. Cyt is an abbreviation for the word cytochrome.
IV I
1
I
I
0.6
0.4
0.2
I
0.0
Potential (Volts vs. NHE)
Figure 2. Cyclic voltammograms of an "oxidase"-modified electrode. The gold substrateused was a gold foil electrode. The scan rates (mV/s)are (A) 10, (B),20, and (C)50. The electrode area is 0.87 cm2;the buffer is 60 mM phosphate buffer, p.H 7. This behavior was stable for over 24 h. The initial potential is +0.62 V.
oriented normal to the gold surface as discussed earlier. In highly organized octadecyl mercaptan monolayers the chains are tilted approximately 30' from the surface normal.1D This organization of the chains is an energy minimization process which is primarily controlled by the intermolecular interactions and distance between the chains. The tilt angles in the system shown in Figure 1 are not known. It is anticipated, however, that the average tilt of the chains in these experiments deviates from the expected 30°found in well-organized octadecyl mercaptan monolayer assemblies. When modifying the gold electrode, octadecyl mercaptan concentrations and modification times were chosen so that the octadecyl mercaptan did not cover the entire gold surfacezzto allow areas where the cytochrome c oxidase could partition into the bilayer during the dialysis procedure. On the basis of experimental data to be discussed later in this section, it is proposed that the cytochromec oxidase incorporates into the membrane in aunidirectional fashion with the cytochrome a3 and cub sites closest to the gold electrode substrate. With the enzyme in this orientation the hydrophilic domains on the surface of the enzyme can minimize their interactions with the hydrophobic interior of the membrane which significantly lowers the energy of the system.a Figure 2 shows cyclic voltammograms taken at a cytochrome oxidase modified electrode. From the total charge under the background-corrected cyclic voltam-
0
1 ,.-ia'
_._... -..-.-..--
0.0
0.2
I
0.4
< 0.6
Potential (Volts vs. NHE) Figure 3. Cyclic voltammograms of immobilized cytochrome oxidase. (A) Solid line, cyclic voltammogram of immobilized cytochrome oxidase; dashed line, cyclic voltammogram after the addition of a 50 NMsolution of reduced cytochrome c. The scan rate is 50 mVIs, the electrode area is 0.87 cm2,and the solution is 60 mM phosphate buffer, pH 7. (B)Solid line, cyclic voltammogram of dialysis-treatedgold with no oxidase present; dashed line, cyclic voltammogram following the addition of a 25 pMsolutionofreducedcytochromec. Thescanrate,temperature, electrode area, and buffer are the same as in (A).
metric peaks, the surface coverage of immobilized oxidase was estimated to be 50% assuming a molecular radius of 20 A65 and four redox sites. This estimate could deviate considerably from the actual coverage. Factors such as residual oxygen present within solution or within the membrane could affect the total charge obtained. The voltammograms in Figure 2 show both oxidation and reduction waves. These waves are bracketed around a potential of approximately 400 mV versus the normal hydrogen electrode. This is the approximate potential of the cytochrome a3 redox site.Bs These data suggest that the cytochrome c oxidase is inserted into the membrane in a unidirectional orientation with the cytochrome a3 redox site closest to the electrode. Figure 3 shows two cyclic voltammograms taken at the same electrode as used in Figure 2 but with a change in initial scan direction. The solid line shows a cyclic voltammogram of the cytochrome c oxidase modified electrode at a scan rate of 50 mV/s. The dashed line is the response following the addition of reduced cytochrome c to solution. Upon the addition of reduced cytochrome c to solution, the anodic current increased considerably. The enhanced anodic current response is well removed from the formal potential of cytochrome c, 260 mV versus the normal hydrogen electrode.67 The increase in the current upon the addition of the reduced cytochrome c is consistent with electron transfer from ferrocytochrome c in solution, through the cytochrome c oxidase, to the electrode. Since the cytochrome c oxidase appears to be reacting close to ita native redox potential, the cytochrome a3 redox site communicating with the electrode may be in a state similar to ita redox state found in vivo. Figure 3B shows the cyclic voltammetric response obtained in a control experiment. (64) Creighton, T. E. Proteins: Structures and MolecularProperties; W. H. Freeman and Co.: New York, 19% Chapter 4. (65) Fuller, 9. D.; Capaldi, R. A,; Henderson, R. J. Mol. Bid. 1979,134, 305-327. (66) Schroedel, N. A.; Hartzell, C. R. Biochemistry 1977, 16, 13271333. (67) Cusanovich, M.A. In Bioorganic Chemistry; van Tamelen, E. E., Ed.;Academic Press: New York, 1978; Vol. IV, p 117.
Cytochrome c Oxidase in Lipid Bilayer Membranes
Langmuir, Vol. 10, No. 3, 1994 881
1u ,
....
-'B
0.0
0.2
0.4
0.6
0.8
Potential (Voltsvs. NHE)
Figure 4. (A) Cyclic voltammogram of an oxidase-modified vapor-deposited gold electrode taken upon assembly of the cell. (B)Cyclic voltammogram on the same modified electrode as in scan 1, after the addition of a 180 pM solution of reduced cytochrome c, and the storage of the cell at 4 "C for 4 days. The electrode area is 0.87 cm*; the buffer used is 0.1 M phosphate buffer, pH 7.4. The scan rate is 50 mV/s.
The dialysis was performed in the same manner as in the above experiment, except no cytochrome c oxidase was put into the dialysis membrane. It is anticipated that in the control experiments a bilayer, similar to the bilayer formed when the oxidase is present, forms on the electrode surface. In Figure 3b the solid l i e is the response observed at the bilayer electrode in buffer alone. The dashed line is the response observed after reduced cytochrome c was added to a concentration of 25 pM. These data show that in the control experiment the electrochemical communication between the cytochrome c and the electrode is blocked or occurring at a very low rate. The results of this control experiment reinforce the idea that the oxidase is reacting directly with the electrode and that cytochrome c is being oxidized by the immobilized oxidase. This model provides an example of an integral enzyme-modified electrode in which there is evidence that the orientation of the enzyme is understood. Moreover, this architecture is designed to replicate what is known about this enzyme's native environment. It is also one of the few examples of an enzyme reacting directly at an electrodeem The cyclic voltammetry of an oxidase-modified vapordeposited gold electrode is shown in Figure 4. This electrode was modified for 1min at a concentration of 10-4 M octadecyl mercaptan in ethanol. The dialysis in this experiment was performed for 4 days, changing the buffer solution three times. It was believed that a longer dialysis time would yield a more stable membrane system at the electrode surface by removing more of the deoxycholate. In this experiment cytochrome c oxidase isolated using the Yoshikawa and Caughey procedure was used.68 The cyclicvoltammetric response of an enzyme-modified vapordeposited gold electrode in buffer alone is shown in Figure 4A. If this voltammogram is enlarged, an anodic peak at approximately 450 mV versus the normal hydrogen electrode is evident. Again, the formal potential of the redox process is close to the formal potential of the cytochrome as site of cytochrome c oxidase. There is a dramatic time dependence of the heterogeneous reaction between the solution-resident reduced cytochrome c and the immobilized cytochrome c oxidase. Upon addition of cytochrome c after dialysis there is a small enhancement of the anodic current similar to that shown in Figure 3A. The response at the same electrode after adding the reduced cytochrome c to the cell and then (68)Paddock, R.M.;Bowden, E.F.J. Electroanol. Chem. 1989,260, 487-494.
0.2 0.4 Potential (Volts vs NHE)
Figure 5. Differential pulse voltammograms of an oxidasemodified electrode. (A) Differential pulse voltammetricresponse observed at the oxidase-modifiedelectrode in buffer alone. (B) The solid line is the response observed after the addition of reduced cytochrome c, and the dashed line is the response observed after doubling the cytochrome c concentration to 180 fiM. The electrode is 0.87 cm*, the modulation amplitude is 5 mV, the scan rate is 2 mV/s, and the buffer is 0.1 M phosphate, pH 7.4.
storing the cell at 4 OC for four days is shown in Figure 4B. The anodic current is enhanced by over an order of magnitude. Time dependenciesin catalytic currents were frequently observed. More than a bilayer may be present on the surface of the electrode initially. Following the dialysis procedure, the electrodes have a film that can be seen on the surface of the electrode. The visible film can be removed by gently flushing a stream of buffer over the surface. This suggests that multilayers are present at the surface after dialysis, and some may still be present even after all the visible f i i has been removed. Multilayers have been observed by Huang12using similar procedures. After equilibration at 4 OC for 4 days some of the multilayers on the surface may have desorbed from the surface of the electrode so that better communication between the ferrocytochrome c in solution and the immobilized cytochrome c oxidase on the electrode occurs. The reproducibility of these experiments was about 15%, and there were also concerns over the control experiments which sometimesgave anomaloussurface waves that were similar but not identical to the voltammetric waves observed in the oxidase experiments. It is not believed that oxidative desorption of the thiol from the gold is responsible for these waves. At the pH values used the oxidative desorption takes place at potentials more positive than the solvent oxidation for alkanethiols longer than n-butanethiol.17 Differential pulse voltammetry and spectroelectrochemical experiments were also conducted. The results from a differential pulse voltammetric experiment are shown in Figure 5. The top scan is the differential pulse voltammetric response observed at the oxidase-modified electrode in buffer alone. The solid line at the bottom is the response observed after the addition fo reduced cytochrome c to solution, and the dashed line at the bottom is the response after a second addition of reduced cytochrome c to solution. Upon the addition of reduced cytochrome c the redox peak of the species shown reacting in the top scan splits into two peaks. The peak at the more negative potential apparently is not dependent on the concentration of cytochrome c, while the peak at the more positive potential does depend on the addition of reduced cytochrome c. The cytochrome c could not be reacting directly with the gold electrode because the formal potential of the cytochromec is approximately 260 mV versus the normal
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Figure 6. Potential step chronoabsorptometry experiments. (dashed line)repeated potential stepsof 10-minduration starting at +0.73 V and stepping to -0.17 V versus NHE. (solid line) change in 550-nm absorbance versus time. The electrode is an oxidase-modified vapor-depositedgold electrode. The solution initially contained 130 pM reduced cytochrome c. hydrogen electrode. This is additional evidence that cytochrome c is being oxidized by the immobilized cytochrome c oxidase and that electrons are then being mediated to the gold substrate surface through the cytochrome c oxidase. Potential step chronoabsorptometry experiments were also conducted. In this experiment a thin-layer cell, described in the Experimental Section, was used in conjunction with a bifurcated light guide. The potentuial of the electrode was modulated between -0.17 and +0.73 V versus the normal hydrogen electrode. The potential is stepped 600 mV more negative and 300 mV more positive than the formal potential of the cytochrome c oxidase. From the data described earlier it is apparent that the reduction of the cytochrome c oxidase is more difficult than the oxidation. This choice of potentials drives the reduction to alarger extent for this reason. As the potential was being modulated the a-band of reduced cytochrome c a t 550 nm was monitored. The dashed line in Figure 6 shows the potential step sequence used in these experiments, and the solid line shows the absorbance changes monitored at 550 nm as a function of time. These results show that cytochrome c can be both oxidized and reduced through cytochrome c oxidase when the potential is stepped to the values shown. It is evident that the rate of reduction of cytochrome c is less than the rate of its oxidation from the magnitude of the absorbance changes shown. The response shows evidence of kinetic limitations in the reduction of th cytochrome c. It is proposed that by stepping to large enough negative overpotentials one can reduce the cytochrome a3 site which shifts the direction of the flow of the electrons and the equilibria toward the reduction of the other redox sites in the cytochrome c oxidase.60 In this mechanismelectrons will be transferred from the cytochrome a3 site in the oxidase to the outer cytochrome a and Cu, redox sites in the molecule. Ferricytochrome c would then be reduced by these redox sites. The spectroelectrochemical data are significant because they suggest that the barrier for the flow of electrons from the cytochrome a3 site to solutionresident cytochrome c is not very high. However, the equilibrium clearly lies in the direction of electron flow from the cytochrome c to the cytochrome a3 site. This method could prove in the future to be a unique method for studying the electron-transfer reaction between cytochrome c and cytochrome c oxidase.
Control experiments were conducted with membranes containing no cytochrome c oxidase, and there was no change in the absorbance at 550 nm as a function of potential. There was some decrease in absorbance over a couple of hours, showingthat ferrocytochromec oxidation was taking place; however, the rate of this oxidation was not dependent on the applied potential. Diffusion of oxidized cytochrome c formed a t the auxiliary electrode of the thin-layer cell into the path of the bifurcated light guide could explain this result given the length of the experiment. Both the differential pulse voltammetric and spectroelectrochemical experiments show clear evidence that electrons are being mediated to and from the cytochrome c via the immobilized cytochrome c oxidase. More work still needs to be performed to optimize dialysis times and sample volumes. By varying the dialysis times and sample volumes, conditions that favor bilayers over multilayers could be found. Experiments such as these should also help in improving reproducibility.
Conclusions The results presented show direct electrochemical communication both between cytochrome c oxidase and an electrode substrate and between cytochrome c oxidase and reduced cytochrome c in solution. The cyclic voltammetric peaks are bracketed around a potential of 400 mV vs NHE, suggesting that the cytochrome a3 site is communicatingwith the electrode. The time dependencies observed in some cyclic voltammetric experiments are proposed to be due to the formation of multilayers on the electrode surface. A series of single potential step chronoabsorptometry (SPSCA) experiments were performed in which only the concentration of reduced cytochrome c was monitored so that any contributions from background interferences were minimized. The data suggest that both the reduction and oxidation of the cytochrome c was being mediated by the cytochrome c oxidase in the membrane at the electrode surface. Ubiquinone can also be turned over within these and a next logical step would be to incorporate the quinone into a membrane with cytochrome c reductase. If the ubiquinone could then be turned over, perhaps it could serve as a mediator to communicate with the immobilized cytochrome c reductase. The isolation of cytochrome c reductase from beef hearts is now being pursued in our laboratorywith these experiments in mind. With additional research aimed at understanding and controlling the experimental variables in the procedures used, the procedures described in this paper should provide a new means for studying and improvingour understanding of membrane-bound redox enzymes. Acknowledgment. We appreciate the assistance of Professor James Terner in the cytochrome c oxidase preparations performed in our laboratory. We would also like to acknowledge the support of the National Science Foundation (Grant NSF CHE9111786) for funding this research. (SS! Xiaoling, Y.; C.ullieon,J. K.; Sun,S.; Hawkridge, F. M. In Charge and AeldEffectsrnBiosystem-II; Allen,M. J., Cleary, S. C.,Hawkridge, F. M., Eds.; Plenum: New York, 1990, pp 81-90.
