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Langmuir 1996,11,4818-4822
Characterization of Mercaptoethylamine-ModifiedGold Electrode Surface and Analyses of Direct Electron Transfer to Putidaredoxin Ling Sang Wongt and Vincent L. Vilker**t Department of Chemical Engineering, University of California, Los Angeles, California 90024-0159
William T. Yap and Vytas Reipa Biotechnology Division, National Institute of Standards and Technology, Gaithersburg, Maryland 20899-0001 Received March 6, 1995. I n Final Form: August 18, 1995@ Direct electron transfer to putidaredoxin (Pdx) in solution from bare gold and mercaptoethylaminemodified gold electrodes was studied by cyclic voltammetry. The current response of Pdx at a bare gold electrode was irreversible and highly unstable. The modified electrode offered improved, but still impersistent, quasi-reversible current response. Surface-enhanced Raman spectroscopy revealed that mercaptoethylamine adsorbed on gold was in mixed trans and gauche conformations at 0.0 V (Ag/AgCl) initially. As the potential was changed anodically or cathodically over the range of +0.4 to -0.8 V, the adsorbate population became largely gauche in conformation. A link between the conformation change and deteriorationof Pdx current response at the electrodewas suggested. Simulationsof the electrochemical system yielded an estimated Pdx diffusion coefficientD = 1.4x lo-' cm2/sand the apparent heterogeneous electron transfer rate constant k" = 1.0 x 10-4 c d s .
Introduction Studies of electron transfer (ET) in biological systems encompass fundamental problems such as molecular recognition between redox partners, conformation and dynamics of inter- and intramolecular ET, and determination of thermodynamic and kinetic parameters of the ET process. Voltammetric study of redox proteins at solid electrodes has received wide attention in the last two decades due to improved functional electrodes for efficient coupling to solution p r ~ t e i n s . l - This ~ method has the advantage of permitting the study of a redox protein in the absence ofits physiologicalpartner. Effects of solution temperature, ionic strength, and pH on the formal potential and heterogeneous ET rate constant can also be determined by voltammetry. The success has also benefitted biosensor development and commer~ialization.~ We are interested in direct ET to putidaredoxin (Pdx) from a solid electrode because Pdx is critical for electron transport and catalytic turnover for cytochrome P-45OCam monooxygenase (EC 1.14.15.1, CPY101).6 P-45OC,, has been arguably the most important model system for the ubiquitous, general class of P-450 enzymes.' The potential +
Current address: Biotechnology Division, National Institute
of Standards and Technology, Gaithersburg, MD 20899. Abstract published in Advance A C S Abstracts, November 1, 1995. (1)Armstrong, F. A.; Butt, J. N.; Sucheta, A. Methods in Enzymology; Academic Press: San Diego, 1993;Vol. 227,pp 479-500. (2)Kinnear, K. T.; Monbouquette, H. G. Langmuir 1993,9,2255. (3)King, B.C.; Hawkridge, F. M.; Taniguchi, I. InRedorMechanisms and Interfacial Properties ofMolecules ofBiologica1Importance; Schultz, F. A,, Taniguchi, I., Eds.; The Electrochemical Society: Pennington, 1993;pp 56-62. (4) Guo, L. H.; Hill, H. A. 0. Advances in Inorganic Chemistry; Academic Press: New York, 1991;Vol. 36,pp 341-375. (5)Cass, A.E. G., Ed. Biosensors: a practical approach; IRL Press: London. 1990. (6)Lipscomb, J. D.;Sligar, S. G.; Namtvedt, M. J.; Gunsalus, I. C. J.Biol. Chem. 1976,251,1116. (7)Omura, T., Sato, R., Fujii-Kuriyama, Y., Eds. Cytochrome P-450; Kodansha: Tokyo, 1993. @
technological importance of P-450 enzymes has been recognized for a long time, but it has not materialized on a wider basis partly due to their requirements of expensive biological reducing sources and multiple redox partnem8 A mercaptoethylamine-modified gold electrode is found to be effective for direct electron transfer to putidaredoxin in aqueous s o l ~ t i o n This . ~ implies that perhaps expensive biological electron sources for P-450cam can be replaced by direct current. The modified surface is not yet optimized for rapid ET to Pdx,however. To improve the performance of the modified electrode, a better understanding of the properties of the modifier film is a prerequisite. While long chain alkanethiols and their derivatives adsorbed on gold have been studied extensive1y,l0-l2shorter alkyl chain compounds like mercaptoethylamine have not. The aim of this paper is to provide some fundamental understanding about the properties ofthe modified surface that effects ET to Pdx. Cyclic voltammograms of Pdx on bare and mercaptoethylamine-modified gold electrodes were compared. Kinetic parameters (heterogeneous ET rate constant and diffusion coefficientof Pdx) were extracted from data by simulation. Effects of changing potentials on the modifier film were investigated by surface-enhanced Raman spectroscopy (SERS). The relationship between the electrochemicalresponse of Pdx and the film properties obtained from SERS will be discussed.
Experimental Section Materials. Putidaredoxin (Pdx) was purified from an Escherichia coli DH5a clone which was kindly supplied by Professor J. A. Peter~0n.l~ Growth and purification protocols are detailed (8) Schubert, F.; Scheller, F.; Mohr, P. Pharmazie 1985,40, 233.
(9)Wong, L. S.; Vilker, V. L. J.Ebctroanal. Chem., in press. (10)Rowe, G. K.; Creager, S. E. Langmuir 1994,10,1186. (11)Garrell, R.L.; Chadwick, J. E. Colloids Surf., A 1994,93,59. (12)Prime, K. L.; Whitesides, G. M. Science 1991,252,1164. (13)Peterson, J.A.;Lorence, M. C.;Amameh, B. J.Biol. Chem. 1990, 265,6066.
This article not,subject to U S . Copyright. Published 1995 by the American Chemical Society
Analyses of Direct E T to Putidaredoxin
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elsewhere.14J5 Briefly, the clone was grown for 12 h in 2 x YT media and another 12 h in TB media to a final cell density of 10 g/L. The culture was then harvested and lysed (one freezethaw cycle lysozyme) and the lysate centrifuged. Pdx was recovered from the supernatant by gradient ion-exchange (DEAE, Pharmacia) and size-exclusion (S-200 HR, Pharmacia) chromatography.16 The purified protein gives an absorbance ratio && A2751 L 0.4. Before each experiment, Pdx was passed through a size-exclusion column (G-10, Pharmacia) to remove the stabilizer added during the purification and to exchange the buffer. All other chemicals were of the highest commercial grade (Aldrich, Fisher) and were used as received. Electrochemical Measurements. A single-compartment, three-electrode cell was employed. The cell consisted of a 1mm gold disk working electrode (Cypress), a miniature Ag/AgCl reference electrode (Cypress), and a platinum wire auxiliary electrode. The gold electrode was cleaned with chromic-sulfuric acid and polished with 1and 0.05 p m alumina (Buehler). The electrode was sonicated thoroughly and rinsed with copious amount of Milli-Q water before use. For modification, the electrode was immersed in a 10 mM solution of mercaptoethylamine solution for 1-2 days. The electrochemical cell with Pdx was purged with Ar for 15 min and was Ar-blanketed during the experiment. Voltammetry was done with a n Omni-90 analog potentiostat (Cypress) with data output to a n Ly18300 X-Y recorder (Linseis). SERS Measurements. For SERS experiments, gold electrodes were electrochemically roughened before modification. A gold electrode was first polished with 0.05pm alumina to a glossy finish and sonicated in deionized water for 10 min. Then the gold electrode was made SERS active by cycling the potential between +1.2 and -0.3 V i n 1M KC1 solution six times. Between each scan, the anodic potential was held for 10 s and the cathodic potential for 30 s. The electrode was rinsed with deionized water, sonicated for 5 min, and immersed in a 10 mM mercaptoethylamine solution for 1-2 days. The activated surface was stable for several hours if it was kept under water. SERS spectra were recorded with a n air-tight, three-electrode setup: a stationary gold disk (2 m m 4, ALFA) working electrode, a miniature Ag/AgCl reference electrode (BAS), and a circular, platinum auxiliary electrode. The cell was purged with Ar for 15 min before and also throughout the experiment. Raman excitation was provided by a model LSR3OP helium-neon laser (Aerotech) at 632.821 nm. The power at the samplewas typically 20 mW, corresponding to 0.64 W/cm2. Raman scattering was collected at 90" with respect to the exciting beam by a n fll.2 camera !ens (Nikon) and focused into the entrance slit of the monochromator (Spex 14018) set at 200 pm. Elastic scattering was eliminated with a SuperNotchPlus holographic laser line filter (Kaiser). A Spectrum One liquid nitrogen cooled CCD detector (Spex) was used for detection.
+
Theory Section The Faradaic current for the reaction Ox is given by17
+ e- =.
Red
( 1 -a)F(E -E")/RT iF = FAk"[Co(O,t)e-fl(E-Eo)'RT - C,(O,t)e 1
(1) where F is the Faraday constant, A is the electrode area (cm2),k" is the heterogeneous rate constant (cmls), CO and CRare the concentrations of Ox and Red, respectively, a is the transfer coefficient, E the working electrode potential corrected for the uncompensated resistance R,, R is the gas constant (Jlmol K), and T i s the temperature (14)Koe, G . S.; Vilker, V. L. Biotechnol. Prog. 1993,9, 608. (15) Roome, P. W.; Peterson, J. A. Arch. Biochem. Biophys. 1988, 266, 32. (16)Certain commercial equipment, instruments, and materials are identified in this paper to specify adequately the experimental procedure. In no case does such identification imply recommendation or endorsement by the National Institute of Standards and Technology, nor does it imply that the material or equipment is necessarily the best available
for the purpose. (17) Bard, A. J.;Faulkner, L. R. Electrochemical Methods; John Wiley & Sons: New York, 1980.
-0.8
-0.6
-0.4
-0.2
0.0
potential, V
Figure 1. Cyclic voltammograms of putidaredoxin (Pdx), with scan rate = 0.02 V/s at a (a) bare gold electrode with 500 p M Pdxin 20 mM sodium phosphate and 50 mM sodium perchlorate buffer at p H 7.4 and argon purged; (b) mercaptoethylaminemodified gold electrode with 100pM Pdx i n 50 mM HEPES and 100 mM KC1 buffer at p H 7.4 and argon purged.
(K).Large peak separations in the cyclic voltammograms and its scan rate dependence suggest a quasi-reversible electron transfer a t the electrode.l* The formulation of the boundary value problem has been well-documented.17J9,20 The solution of this boundary value problem by Laplace transform gives the followingintegral equation which relates the dimensionless current, defined as x i/FAC*(nDvFfRT)112, to the potential (E):19,20
I (F1RT)vt with v being the scan rate ( V I S ) , q = k"/(nDovFfRT)112,and D = DO = DR.
where y
Current vs potential curves were obtained by solving the integral equation numerically21 for various experimental parameters. In particular, the peak separations for different values of scan rate, R,, and q were calculated and compared to the experimental values.
Results and Discussion Voltammetry. Shown in Figure l a is the cyclic voltammogram of 500 pM Pdx on a freshly polished, clean bare gold electrode. In the presence of such high protein concentration, a cathodic peak was detected in the first scan. But the current response deteriorated rapidly with time, and no peak attributable to a redox reaction was distinguishable after three cycles. C u r r e n t response from Pdx was not recovered after resting the system a t open (18)Greef, R.; Peat, R.; Peter, L. M.; Pletcher, D.; Robinson, J. Instrumental Methods in Electrochemistry; Ellis Horwood Limited: Chichester, England, 1985; Chapter 6. (19) Adrieux, C. P.; Garreau, D.; Hapiot, P.; Pinson, J.; Saveant, J. M. J. Electroanal. Chem. Interfacial Electrochem. 1988,243, 321. (20) Nicholson, R. S. Anal. Chem. 1966,37, 1351. (21) Nicholson, R. S.; Olmstead, M. L. In Electrochemistry; Mattson, J. S., Mark, H. B., MacDonald, H. C., Jr., Eds.; Marcel Dekker: New York, 1972.
4820 Langmuir, Vol. 11, No. 12, 1995
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T
A
spectrum recorded at 0.0 V (Ag/AgCl) showed strong features at 650 and 730 cm-’ which were assigned to C-S stretches on the basis of data for similar compounds.25 The doublet arises because the C-S vibration is sensitive to the conformation of the adjacent C-C bond which may be gauche or trans. The trans conformation is indicated by the higher wavenumber band of the doublet. The ratio of ITJIGis related, but not proportional, to the ratio of trans- and gauche-configured adsorbate. A ratio of IdIG 1in the initial spectrum recorded a t 0.0 V (Figure 2) indicated that the adsorbate was in mixed conformation before potential stepping. When the potential was stepped to -0.4 V (Ag/AgCl),IG (650 cm-l) increased slightly while IT (730 cm-l) decreased significantly. Similar spectra were recorded at +0.2 and -0.8 V, irrespective of the sequence of potential changes. This implies that the initial mixed conformation is not stable with respect to potential change and that the adsorbate shifts to predominantly the gauche configuration. However, a t +0.4 V, IT increased and IC decreased compared to those of the spectrum a t +0.2 V, suggesting a transformation of some adsorbate from the gauche to the trans conformation. The phenomena may be explained partly by electrostatic attraction between the negativeljl charged gold electrode below +0.2 V and the NH3+group. Although the apparent pKa for the amine group in a similar compound (ethylamine) is 10.63,26it may shift several pH units due to the interaction with the metal surface and/or the interfacial electric field. We have observed (not shown) stronger Raman bands due to the buffer anions when the mercaptoethylamine was adsorbed, suggestive of the increase in excess surface positive charge due to the protonated amine end group. The adsorbate gauche conformation will be favored if the amine group becomes attracted to gold, thus the ethyl chain ofthe adsorbate inclines in order to be parallel to and closer to the surface. When the potential is a t $0.4 V, the electrode becomes positively charged and repels the NH3+ group, favoring the trans conformation which has the amine group farther from the surface. This would also provide more surface enhancement for the C -S stretch by making it closer to the surface normal. An analogous observation is made for ethanethiol, the structure of which in the solid state i s g a u ~ h ewhile ,~~ trans is favored in the case of propanethiol. With alkyl chain length longer than four, surface-adsorbed alkanethiols are shown to be mostly in the trans conformation which corresponds to crystalline-like packing seen in the spectra of solid compounds.25Our adsorbate with only a n ethyl chain and a charged terminal group falls outside of this range. The dominance of thegauche conformation in our system may be attributable to two factors: (1)the short alkyl chain which provides insufficient (hydrophobic) driving force for crystalline-like packing and (2) the electrostatic interaction which provides attraction between the surface and the amine group. However, between 0.0 and -0.8 V where Pdx electrochemical experiments were performed, the diminished IT/ IG suggests that mercaptoethylamine was largely in the gauche conformation. It is possible that the change from a mixed trans and gauche to a mainly gauche population of chemisorbed mercaptoethylamine upon successive potential cycling is the cause of the deteriorating current response from Pdx. It could happen if the trans-conformed adsorbate is more effective in preventing Pdx from unfolding or changing to a conformation unfavorable to
-
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1
I
I
I
I
1
1
i
500
550
600
650
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Raman shift, c m l
Figure 2. Surface-enhanced Raman spectra of mercaptoethylamine-modified gold electrode in 0.1 M Na~S04.Arrows indicate bands associated with the backbone conformations (gauche (G)and trans (T))as represented schematically above the graph (see text).
circuit potential. The transient response could be reproduced if the electrode was fastidiously cleaned and polished again. This result is not surprising as direct ET to cytochrome c from a bare gold surface is possible if the surface is pretreated by certain procedures.22 Surface fouling by hydrophobic contaminants present in the atmosphere and solution has been postulated to be a cause of response decay.22 Also, the possibility of irreversible adsorption of Pdx cannot be excluded in our case. When a mercaptoethylamine-modifiedelectrode is used, a pair of redox peaks can be observed at lower (100 pM) Pdx concentration (Figure lb). The features are sustainable for about 10 cycles but can be regained by resting the system at open circuit potential. Thus, the mechanism for the deterioration of current is probably different from that in the case of a bare electrode. Since the deterioration is not irreversible, we may argue that the film is not displaced by irreversible adsorption of Pdx. In the case of cytochrome c, the role of a modifier like bipyridyl disulfide might be to maintain the protein in conformations that are conducive to ET.23 Thus, the temporary loss of ET to Pdx in our system can be due to a change in film properties which corresponds to a loss in its conformationmaintaining function. To this end, the results of probing the adsorbate conformation by SERS are presented below. SERS. Figure 2 shows a series of SER spectra of mercaptoethylamine-modified gold electrodes held a t different potentials over the range of -0.8 0.4 V (vs Ag/AgCl) in a 0.1 M Na2S04 solution a t pH 7. The initial
+
(22) Bowden, E. F.; Hawkridge, F. M.; Blount, H. N. J . Electroanal. Chem. Interfacial Electrochem. 1984, 161, 355. (23)Sagara, T.; Murakami, H.; Igarashi, S.; Sato, H.; Niki, K. Langmuir 1991, 7, 3190. (24) Allinger, N. L.;Hickey, M. J. J . A m . Chem. SOC.1975,97,5167.
(25)Bryant, M. A.; Pemberton, J. E. J . A m . Chem. SOC.1991,113, 546. (26) Lange’s Handbook of Chemistry; Dean, J. A,, Ed.;McGraw Hill: New York, 1985; pp 5-37.
Analyses of Direct ET to Putidaredoxin -0.8
potential, V -0.4
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Langmuir, Vol. 11, No. 12, 1995 4821 0.0
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a 175
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Figure 4. Peak separations,AE,, as functions of for various R,: (a) 175 000, (b) 100 000, (c) 10 000 8 and filled circles (0) are experimental AE,'s as a function of -In v, top abscissa.
Figure 3. Cyclic voltammograms of Pdx at the mercaptoethylamine-modifiedelectrode for various scan rates: (a)0.005, (b) 0.01, (c) 0.02, (d)0.05, and (e)0.10Vfs. Conditions: 100pM Pdx in 50 mM HEPES and 100 mM KC1 buffer at pH 7.4 and
argon purged.
ET. SERS is a powerful tool for studying interactions of surface modifiers with protein^.^^!^^ Further studies of P& direct electrochemistry combiningelectrochemicaland spectroscopic methods are being carried out. Determination of Kinetic Parameters. Figure 3 shows cyclic voltammograms of Pdx at the mercaptoethylamine-modified Au electrode a t various scan rates. The large peak separations, which increase with scan rate, and the roughly equal cathodic and anodic peaks are characteristic of a quasi-reversible process. Figure 4 shows plots of calculated peak separation, AE,,versus In 1/, for various R,. By plotting the experimental AEp)s vs -In v1I2 and then superimposing the experimental curve upon the best fitting calculated AE,vs In 1/, curve, a value of ln(k"I(nDvFIRT)Y2)can be graphically estimated from the horizontal shift of the origin of the abscissa of the experimental plot from that of the calculated plots. A plot of experimental AE,vs -In vU2is also shown in Figure 4 as the curve with filled circles with abscissa axis on top. The experimental plot can be translated horizontally to coincide with the plot with curve c for which R, = lo4 R. After the translation, the abscissa origin was shifted -3.6 units which gives an estimated value of 0.30 for k"lDY2. Nonlinear regression analysis was also applied to the set of experimental AEp)s, employing eq 2, to obtain the parameters which give the least squares deviations of the experimental AE, from the calculated values for all five (27)Zhou, C.; Cotton, T. M.; Qu, X.; Lu, T.; Dong, S. In Redoz Mechanisms and Interfacial Properties of Molecules of Biological Importance; Schultz, F. A., Taniguchi, I., Eds.; The Electrochemical Society: Pennington, 1993; pp 63-74. (28)Taniguchi, I.; Iseki, M.; Yamaguchi, H.; Yasukouchi, K. J . Electroanal. Chem. Interfacial Electrochem. 1984,175, 341.
0.15
0.25
0.35
0.4
k / f i , s1I2
Figure 5. Average deviation of the calculated from the experimentalpeak separationsas functionsofko/Dmfor various R,: (0)1000, (0) 10 000, (A) 50 000, and ( 0 )100 000 8.
scan rates. Figure 5 shows examples of plots of root mean square deviations of AE,,6 = tZn((AEp)n,exp - (AEp)n,ca~c)21 nl li2, as functions of k"lDli2for various values of R,. All curves exhibit sharp minima a t values of k"lD1/2equal to ca. 0.3. Analyzing the peak separations for various scan rates yielded a value of 0.31 f 0.03 s1I2 for k"lDY2and ca. lo4 R or less for R,. The k"lD1" estimated is not sensitive to R, as long as R, I lo4R which was our case. By comparing the magnitudes of the experimental peak currents with that of the corresponding simulated x, a value of 1.4 x cm21s is estimated for the diffusion coefficient (D) which is comparable to Taniguchi's value for a similarlysized f e r r e d ~ x i n The . ~ ~ apparent heterogeneous electron transfer rate constant, k", is determined to be 1.0 x cmls from the ratio kolD1/2. Another feature of the cyclic voltammograms for the Pdx reaction a t the mercaptoethylamine-modified Au electrode is the large background current relative to the Pdx redox current. The capacitive charging current was estimated from the vertical separation of the forward and reverse scan, giving a capacity of 29 pF/cm2 for the mercaptoethylamine-modified Au electrode. However, (29) Nishiyama, K.; Ishida, H.; Taniguchi, I. J . Electroanal. Chem. 1994,373,255.
4822 Langmuir, Vol. 11, No. 12, 1995
Wong et al.
Conclusions
0.01
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!2
z
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Figure 6. Background current at the mercaptoethylaminemodified electrode in 50 mM HEPES and 100 mM KC1 buffer at pH 7.4: (a)total background current, solid line; (b)capacitive charging current, long dash; (c) and (d)total charging currents ( A ) cathodic scan, (v)reverse scan]. Conditions c d = 0.23 pF, R, = lo4 S2 and scan rate = 0.02 VIS. after the capacitive charging current was subtracted from the background current, significant residual current could still be seen for both the forward and the reverse scans (Figure 6). The process contributing to this residual current is being investigated.
The electrochemical response of Pdx on a well-cleaned, bare gold electrode was irreversible and highly unstable. We have obtained a more stable and quasi-reversible response with lower Pdx concentration at a mercaptoethylamine-modified gold electrode. The improved performance a t a modified electrode is expected to be dependent of the orientation and integrity of the adsorbed film. From our electrochemical and SERS studies, the mercaptoethylamine layer on gold displayed mixed trans andgauche conformations as determined on the basis of the observed ratio (ZTIIG)before potential stepping. A change of applied potential quickly led to a sharp decrease in z T / z G , although the modifier remains adsorbed on the gold substrate. This conformational change is hypothesized to cause the film to lose its conformation-supportive role for Pdx and lead to current deterioration with potential cycling in our modified electrode experiments. Simulations provided an estimate of the diffusion coefficient of 1.4 x cm2/sfor Pdx, and despite the nonidealities in the voltammograms, the ratio k"lDu2can be determined to within 10%precision by either graphical or nonlinear regression analysis.
Acknowledgment. We gratefully acknowledge support for this research from the National Science Foundation (Grant CATS 9313009), the DuPont Company, and the National Institute of Standards and Technology. LA950173F
