J . Phys. Chem. 1990, 94, 8241-8246
8241
Adsorption and Redox Thermodynamics of Strongly Adsorbed Cytochrome c on Tin Oxide Electrodes James L. Willit+ and Edmond F. Bowden* Department of Chemistry, North Carolina State University, Raleigh, North Carolina 27695 (Received: December 27, 1989)
An examination of the redox and adsorption thermodynamics for adsorbed horse heart cytochrome c on fluorine-doped tin oxide electrodes has been performed using cyclic voltammetry with a nonisothermal electrochemical cell. Cytochrome c was found to adsorb strongly in neutral to slightly alkaline low ionic strength solutions with near-monolayer coverage resulting under optimum conditions. Variation in electroactive surface coverage with solution pH and ionic strength indicated that the adsorption is primarily controlled by electrostatic interactions. The surface formal potential of adsorbed cytochrome c was found t o be ca. +240 mV, which is 20-25 mV lower than the solution value. A reaction entropy, ASo,,, of ca. -75 to -80 J/(Kmol) was determined from the temperature coefficient of the surface formal potential. This value is about 25 J/(Kmol) more negative than the literature value for solution cytochrome c. These results are accounted for by a model in which ferricytochrome c adsorbs more strongly on tin oxide than ferrocytochrome c due to a greater degree of adsorption-induced structural alteration in the former species. The implications of this study for understanding similar formal potential shifts that result when cytochrome c binds to mitochondrial membranes and other biocomponents are discussed.
Introduction Over the past 15 years considerable attention has been directed toward understanding the interfacial electrochemistry of redox proteins.'-3 These efforts continue to be motivated by the potential for developing unique electrochemical strategies for the characterization of redox proteins, as well as by the potential for analytical and electrosynthetic applications. Several groups, including our own, have become interested in the properties and reactivity of irreversibly adsorbed redox proteins on solid electrodes. Adsorbed monolayers of cytochrome c and other proteins have been subjected to interfacial spectroelectrochemical characterization using techniques such as surface-enhanced resonance Raman spectroscopp and optical reflectance These studies have already contributed a great deal of insight into the structure and behavior of proteins on reflective metal electrodes. The electron-transfer mechanisms of irreversibly adsorbed redox proteins on solid electrodes have also received increased scrutiny.I2-I7 We have focused considerable effort in this regard on the cytochrome c/tin oxide system. It has been shown that cytochrome c will, with retention of its native redox potential, adsorb strongly on tin oxide electrodes in neutral pH, low ionic strength solutions.'~17The adsorption appears to be primarily electrostatic in origin, thus providing a useful electrochemical a n a l o g ~ eof '~ electrostatic protein-protein complexes.18-20 Cytochrome c has been shown to transfer electrons with tin oxide electrodes at quasi-reversible rates, with typical rate constants of 1-10 s-1.14-17 To gain a more fundamental understanding of the adsorbed cytochrome c electron-transfer reaction, as well as the adsorption reaction itself, a temperature-dependent electrochemical study has been conducted. In these experiments, the electron-transfer rate constant and the surface formal potential of adsorbed horse heart cytochrome c were measured between 8 and 35 OC for various solution conditions. The kinetic results, which include determinations of activation parameters and an estimate of electron-transfer distance, will be presented in a subsequent paper.21 In the present paper, measurements of the surface formal potential and its temperature dependence are used to determine the reaction entropy and enthalpy for the redox reaction of adsorbed cytochrome c. A comparison of these findings with results previously reported for the redox reaction of solution cytochrome 82-27 enables insight to be made into some aspects of the thermodynamics of cytochrome c adsorption. In particular, we address the question of why the oxidized form of cytochrome c adsorbs more To whom correspondence should be addressed. Chemical Technology Division, Argonne National Laboratory, Argonne, IL 60439.
'Present address:
0022-3654/90/2094-8241$02.50/0
strongly on tin oxide than does the reduced form, as evidenced by a small negative shift in formal potential. The relevance of
( I ) Biological Electrochemistry; Dryhurst, G.; Kadish, K. M.; Scheller, F.; Renneberg, R.; Academic Press: New York; 1982; Vol. I. (2) Bowden, E. F.; Hawkridge, F. M., Blount. H. N . In Comprehensiue Treatise of Electrochemistry; Srinivasan, S.; Chizmadzhev, Y. A,, Bockris,
J. O'M., Conway, B. E., Yeager, E., Eds.; Plenum: New York, 1985; pp 297-346. (3) Armstrong, F. A.; Hill, H. A. 0.: Walton, N. J. Q.Rev. Biophys. 1986, 18. 263-322. (4) Cotton, T. M.; Schultz, S. G.; Van Duyne, R. P. J . Am. Chem. SOC. 1980, 102, 7960-7962. (5) Cotton, T. M. In Spectroscopy of Surfaces; Clark, R. J. H., Hester, R. E., Eds.; Wiley: New York, 1988; pp 91-153. (6) Niki, K.; Kawasaki, Y . ;Kimura, Y.; Higuchi, Y.; Yasuoka, N. Langmuir 1987, 3, 982-986. (7) Hildebrandt, P.; Stockburger, J. J. Phys. Chem. 1986,90,6017-6024. (8) Smulevich, G.; Spiro, T. G . J . Phys. Chem. 1985, 89, 5168-5173. (9) Taniguchi, 1.; Iseki, M.; Yamaguchi, H.; Yasukouchi, K. J . Electroanal. Chem. 1984, 175, 341-348. (IO) Hinnen, C.; Parsons, R.; Niki, K. J . Electroanal. Chem. 1983, 147, 329-337. ( 1 I ) Niwa, K.; Furukawa, M.: Niki, K. J. Electroanal. Chem. 1988, 245, 275-285. (12) Bowden, E. F.; Hawkridge, F. M.; Blount, H. N. J . Electroanal. Chem. 1984, 161, 355-376. (13) Niki, K., personal communication, 1989. (14) Yokota, T.; Itoh, K.; Fujishima, A. J. Electroanal. Chem. 1987, 216, 289-292. (15) Willit, J. L.; Bowden, E. F. J . Electroanal. Chem. 1987, 221, 265-274. (16) Willit, J. L.; Bowden, E. F. In Redox Chemistry and Interfacial Behavior of Biological Molecules; Dryhurst, G., Niki, K., Eds.; Plenum: New York, 1989; pp 69-79. (17) Collinson, M.; Willit, J. L.; Bowden, E. F. In Charge and Field Eflects in Biosystems II; Allen, M. J., Cleary, S . F., Hawkridge, F. M., Eds.; Plenum: New York, 1989; pp 63-76. (18) Salemme, F. R. Annu. Rev. Biochem. 1977, 46, 299-329. (19) Poulos, T. T.; Kraut, J. J . Biol. Chem. 1980, 255, 10322-10330. (20) Wendoloski, J. J.; Matthew, J . B.; Weber, P. C.; Salemme, F. R. Science 1987, 238, 794-797. (21) Willit, J. L.; Bowden, E. F., manuscript submitted for publication. (22) Taniguchi, V. T.; Sailasuta-Scott, N.; Anson, F. C.; Gray, H. B. Pure Appl. Chem. 1980, 52, 2275-2281. (23) Koller, K. B.; Hawkridge, F. M. J . Am. Chem. SOC.1985, 107, 74 12-74 17. (24) Koller, K. B.; Hawkridge, F. M. J . Electroanal. Chem. 1988, 239, 29 1-306. (25) Taniguchi, 1.; Iseki, M.; Eto, T.; Toyosawa, K.; Yamaguchi, H.; Yasukouchi, K. Bioelecrrochem. Bioenerg. 1984, 13, 373-383. (26) Taniguchi, 1.; Funatsu, T.; Iseki, M.; Yamaguchi, H.; Yasukouchi, K. J . Electroanal. Chem. 1985, 193, 295-302. (27) Watt, G.D.; Sturtevant, J. M. Biochemistry 1969, 8, 4567-4571.
0 1990 American Chemical Society
8242 The Journal of Physical Chemistry, Vol 94, No. 21, 1990
Willit and Bowden
"\
\
C
e
I
I
-0.41
-.-
h
0.60
0:20
0:40
0.00
E vs. NHE "*-r
I
I
I
k
j Figure 1. Diagram of nonisothermal electrochemical cell: (a) reference electrode capillary filled with 1 M KCI with a ceramic frit liquid junction, (b) Ace-thread connector, (c) Pt auxiliary electrode, (d) water jacket, (e) Ag/AgCl reference wire, (f) WE solution, (g) silicone rubber gaskets, (h) Lucite retainer plates, (i) Viton O-ring, 6) tin oxide electrode, (k) brass contact. The diameter of the cell cavity is 6.5 mm.
this question to physiological situations seems clear since similar shifts in formal potential have been reported for cytochrome c adsorbed or complexed to various biocomponents28 including mitochondrial membra ne^,^^^^^ phospholipid v e s i ~ l e s isolated ,~~~~~ respiratory enzymes,32and p h o ~ v i t i n . ~ ~ ~ ~ ~
Theory Discussions of the theoretical and practical aspects of these ~ ~ . ~recently ~ by experiments can be found in the l i t e r a t ~ r e , most Weaver.37 In this section a brief summary is given of the pertinent thermodynamics. In an electrochemical cell where the reaction of interest, Ox + ne Red, occurs at a potential Eo' vs NHE, the entropy and enthalpy of the reaction is given by eqs 1 and 2.22*36*37 The AS" = S0Rd - soox+ so"+- ( 1 /2)s0H2 (1)
-
AHo = HoRed - Hoax
+ H'H+ - ( 1 / 2 ) H o , ,
-0.4 0.60
I
I
1
0.20
0.40
I
0.00
Evs. NHE Figure 2. Cyclic voltammograms of cytochrome c adsorbed on a tin oxide electrode before and after background subtraction. Experimental conditions: pH 8, 15 mM ionic strength phosphate buffer, temperature = 24 "C. Panel A (top): adsorbed cytochrome c CVs (heavy line) with bare tin oxide background CVs (thin lines) superimposed. Panel B (bottom): same data after background subtraction. Potential sweep rates: (a) 100 mV/s, (b) 50 mV/s. "*-
I
(2)
absolute differences in partial molal entropy and partial molal enthalpy between Red and Ox reflect the entropic and enthalpic changes associated with the redox couple as a result of electron transfer. These quantities, defined in eqs 3 and 4, are the reaction entropy, ASorc,and reaction enthalpy, AHorc.37938
ASo, = S o R e d - soox AH',, = HoRed - HoOx
(3) (4)
Using a nonisothermal electrochemical cell, we can measure (28) Nicholls, P. Biochim. Biophys. Acta 1974, 346, 26 1-3 10. (29) Dutton, P. L.; Wilson, D. F.; Lee, C.-P. Biochemistry 1970, 9, 5077-5082. (30) Vanderkooi, J.; Erecinska, M.; Chance, B. Arch. Biochem. Biophys. 1973, 154, 531-540. (31) Kimelberg, H. K.; Lee, C . P. J. Membr. Biol. 1970, 2, 252-262. (32) Vanderkooi, J.; Erecinska, M. Arch. Biochem. Biophys. 1974, 162, 383-391. (33) Peterson, L. C.; Cox, R. P. Biochem. J . 1980, 192, 687-693. (34) Yoshimura, T.; Matsushima, A.; Aki, K. Biochim. Biophys. Acta 1980,625, 100-108. (35) deBethune, A. J.; Licht, T. S.; Swendeman, N. J. Electrochem. SOC. 1959, 106, 6 16-625. (36) Lin, J.; Breck, W. G. Can. J. Chem. 1965, 43, 766-771. (37) Yee, E. L.; Cave, R. J.; Guyer, K. L.; Tyma, P. D.; Weaver, M. J. J . Am. Chem. SOC.1979, 101, 1131-1 137. (38) Hupp, J. T.;Weaver, M. J. J . Electroanal. Chem. 1983, 145,43-51.
-0.2 0.60
0.40
0.20
I
0.00
E vs. NHE Figure 3. Background-subtracted 50 mV/s cyclic voltammograms as a function of temperature. Solution conditions: pH 8, 15 mM ionic strength phosphate buffer. Temperatures: (a) 28 "C, (b) 20 OC, (c) 8
"C.
ASo, directly. In this cell the temperature of the thermally isolated reference electrode (RE) half-cell is held constant while varying the temperature of the working electrode (WE) half-cell. From the temperature coefficient of the nonisothermal formal can be calculated by using eq 5.37 AHo, potential Eo'ni, ASo,, can then be determined from eq 6 , ASo, = nF(dEO',i/dT)
(5)
Adsorption of Cytochrome c on Tin Oxide
+ TASO,,
AH",, = -nFEo'ni,25
where Eofnj.25is the measured formal potential vs N H E at 25 "C.
Experimental Section Reagents and Materials. All solutions were prepared by using water purified by a Milli-Q system with an Organex-Q final stage (Millipore). Monobasic and dibasic potassium phosphate were reagent grade (Fisher). Tris, i.e., tris(hydroxymethy1)aminomethane (Trizma Base, reagent grade), and cacodylic acid (hydroxydimethylarsine oxide, 98% pure) were obtained from Sigma. Tris was used as received, whereas the cacodylic acid was recrystallized twice from 50/50 hexane/2-propanol. K3Fe(CN)6 was reagent grade (Fisher). Horse heart cytochrome c (Type VI, Sigma) was purified c h r o m a t ~ g r a p h i c a l l yon ~ ~ (carboxymethy1)cellulose (CM-52, Whatman), concentrated by using an Amicon ultrafiltration cell with a YM-5 membrane (Amicon Corp.), and passed through a desalting column (Bio-Gel P6-DG, Bio-Rad) equilibrated with Milli-Q water. The purified cytochrome c was stored at 4 "C as a ca. 35 pM solution in I O mM ionic strength, pH 7 phosphate buffer and used within four weeks of purification. During the 4-week period, no degradation of the electrochemical response was observed. Furthermore, the absorbance at 695 nm, indicative of an intact methionine-iron bond,40 remained constant over this period. Fluorine-doped tin oxide was donated by PPG Industries as an -0.4 pm thick film of 20 Q/sq resistivity on a 5-mm glass substrate. Apparatus. The nonisothermal electrochemical cell diagrammed in Figure 1 is similar to the design of Koller and Hawkridge23*24 and features a horizontally mounted working electrode (WE) with a geometric area of 0.32 cm2. The temperature of the WE half-cell was controlled to f0.2 "C with a circulating water bath. A Teflon-shrounded type K (copper/constantan) thermocouple was used to monitor the solution temperature. The platinum auxiliary electrode was coiled around the capillary tubing of a Ag/AgCI (1 .O M KCI) reference electrode. Thermal isolation of the reference electrode was achieved with a 45 cm length of 3 mm 0.d. capillary tubing. As shown in Figure I , this bent tubing was filled to a level slightly below the level of the solution in the cell to prevent 1 M KCI from flowing into the cell. The entire cell was enclosed in a grounded, wire mesh Faraday cage. All experiments were performed using an EG&G Princeton Applied Research Model 362 scanning potentiostat. Analog potential and current outputs from the potentiostat were digitized and stored by a Nicolet 3091 digital oscilloscope. The digitization rate was adjusted to -5 points per millivolt. Using Nicolet PC/31 software, the stored waveforms were transferred to the hard disk of an IBM PC via the serial communications cable. A QuickBASIC program, written in-house, performed all the data analysis, smoothing, and background subtractions discussed below in the Data Analysis section. Procedures. The fluorine-doped tin oxide electrodes were cleaned by sequential 20-min sonications in Alconox, isopropanol, and twice in Milli-Q water. The electrodes were then equilibrated in I O mM ionic strength, pH 7 phosphate buffer solution for 16-20 h at room temperature. After equilibration, an electrode was installed in the nonisothermal cell, and the cell was filled with 2.0 mL of a selected buffer. Background cyclic voltammograms (CVs) were then acquired and stored, using scan rates of 1000, 500, 200, 100, and 50 mV/s at 12 and 24 "C. To obtain a more reproducible background, the potential was scanned continuously until a stable current response was obtained, typically within five potential sweeps. Only data obtained at the 50 and 100 mV/s sweep rates were used to determine the formal potentials reported in this paper.41 A sub(39) Brautigan, D. L.; Ferguson-Miller, s.; Margoliash, E. Methods Enzymol. 1978, 530, 128-164. (40) Schejter, A.; George, P. Biochemistry 1964, 3, 1045-1049.
The Journal of Physical Chemistry, Vol. 94, No. 21, I990 8243 sequent paper2' will focus on the temperature-dependent kinetic results evaluated at all five scan rates. After the cell was rinsed three times with Milli-Q water, cytochrome c was adsorbed by refilling the cell with 100 p L of ca. 35 p M cytochrome c in IO mM ionic strength, pH 7 phosphate buffer. This adsorption step was performed with the cell at room temperature and the cytochrome c solution at 4 "C at the time of addition. After I O min, the cytochrome c solution was removed. Then the cell was rinsed again three times with Milli-Q water and refilled with 2.0 mL of the same protein-free buffer solution used to acquire the background CVs. CVs were then taken of the adsorbed cytochrome c using the same procedure described above for obtaining reproducible background currents. Potential sweep rates were randomly applied at each temperature. The first set of CVs was taken at 8 "C. The temperature was increased in 4 "C intervals up to 28 "C, and then increased to 35 "C. This procedure was examined for systematic error by decreasing the temperature to one of the prior settings during the temperature ramp. For example, a typical temperature series was 8, 12, 16, 20, 24, 16, 28, 35 "C. Data at the replicate temperatures within each series were found to be in excellent agreement. Three complete sets of data were acquired for each of the five solution conditions investigated in this work. A new electrode was used to obtain each data set. Liquid Junction Potentials and Uncompensated Resistance. Liquid junction potentials (E,,) were calculated42 by using tabulated ionic mobilities.43 For the five solution conditions studied, values of E,, ranged only from 2.6 to 3.7 mV. Because the estimated liquid junction potential is small and insensitive to solution composition, the reported potentials were not corrected for this factor. All peak potentials were corrected for uncompensated iR. However, this was primarily a concern in the determination of the electron-transfer kinetics21and had negligible impact on the formal potential measurements reported here. For example, the largest uncompensated resistance was 7.7 kR at pH 8, 1.6 mM ionic strength. At a 100 mV/s sweep rate, the resultant i R corrections to the anodic and cathodic peak potentials, +4 and -4 mV, respectively, had no net effect on the determination of Eofsur[
Data Analysis. The monolayer to submonolayer coverages of adsorbed cytochrome c resulted in voltammetric peak currents that were small and superimposed on sloping backgrounds (see Figure 2A). Therefore, digital background subtraction was used to improve the precision and accuracy of peak potential determinations. A computer program identified the beginning and end of a single potential sweep cycle and performed a point-by-point subtraction of the appropriate background current4 from the total current in the presence of adsorbed cytochrome c. A typical background-subtracted CV is shown in Figure 2B. Peak currents, potentials, areas, and full width at half-maximum (fwhm) values were determined by using the background-subtracted CVs. Formal potentials were taken to be (Ep,a Ep,,)/2 at 50 and 100 mV/s, which were the slowest sweep rates empl~yed.~' A linear regression fit of Eo'surfvs temperature data for each replicate was used to calculate a surface formal potential at 25 "C, Eo'surf,25. At high current sensitivities the background and faradaic current data contained considerable noise (primarily 60 Hz). To
+
(41) EoJsurf values showed little dependence on scan rate up to 200 mV/s, although deviations were noted at 500 and 1000 mV/s. The use of 50 and 100 mV/s data for determinations of Eo'rurl values was therefore deemed to be an acceptable procedure. These results also indicate that the transfer coefficient is near 0.5. (42) Bard, A. J.; Faulkner, L. R. Electrochemical Methods; Wiley: New York, 1980; Chapter 2. (43) Millazo, G. Electrochemistry: Theoretical Principles and Practical Applications; Elsevier: New York, 1963; pp 60-61. (44) Background voltammograms were compared over the temperature range 8-28 OC and only subtle changes with temperature were found. To correct for these subtle changes, background CVs were acquired at 12 and at 24 "C. By use of these two sets of background CVs, the greatest difference in temperature between the adsorbed CVs and the background CVs used for background subtraction was 4 O C .
The Journal of Physical Chemistry, Vol. 94, No. 21, 1990
8244
TABLE I: Thermodynamic and Surface Coverage Parameters for Adsorbed and Solution Cytochrome c
Willit and Bowden
w
r z
Adsorbed Cytochrome c"
vj 5
. >
7.0 7.0 8.0
243 (4) 239 (5) 240 (5) 234 (5) 240 (2)
1.1 11
1.6
8.0
15
8.0
132
-91 (24) -78 ( 5 ) -65 (20) -79 (9) -67 (14)
-51 (7) -46 ( I ) -42 (5) -46 (3) -43 (4)
15 15 13 14
g g g g
7
g
0
Lu 077
,! 0
I 10
Pqb E 0'roln.25~'
7.0 7.0 7.0 7.0 7.0 7.0 7.9
50 110
200 200 200 200 200 7.9 200 8.0 200 8.0 200
mV
AS Ore, AH Orc'C J / ( K m o l ) kJ/mol
270 259 259 268 264 (0.8) 256 ( I )
-62.3 -48.9 -49.3 -51.9
-44.6 -40.9 -39.7
-56.1 -53.1
258
-43.1
266 264 ( I ) 256 ( I )
-51.5 -53.1
-42.2 -40.5 -37.7 -4 1 -41.4
-59.4
-42.4
-41.3
30
20
30
T i "C
Solution Cvtochrome C " pH mM
20
buffer
phosphate phosphate phosphate/NaCIO, phosphate/NaCI Tris/cacodylate phosphate phosphate/NaCIO, phosphate/NaCI Tris/cacodylate phosphate
ref 22 27 25 25 23 23 25
26 24 24
values reported for cytochrome c adsorbed on tin oxide in the presence of phosphate buffer. b p signifies ionic strength. Potentials (25 "C) are v5 VHE. dValues shown represent the averages obtained from linear regression fits of six E "'surf vs T data sets. Le., three replicates each a t 50 and 100 m V / s (see Experimental Section). 95% confidence intervals are given in parentheses. eAHo,c values were calculated from Eo'ni,25 and ASorCvalues by using eq 6. 95% confidence intervals a r c given in parentheses for adsorbed cytochrome c results. rEstimated error limits are f 2 pmol/cm2. gThis work. "Values were obtained either by calorimetry2' M by nonisothermal electrochemistry, namely, cyclic voltammetry at indium o ~ i d eand ~ ~bis(4-pyridyl) . ~ ~ disulfide modified gold25*26electrodes, and mediated thin-layer spectropotentiostatic measurements at gold minigrid electrodes.22'Standard deviations in parentheses are from original references.
W
I
z
ui >
.
> O
w 022-1 0
10
"All
improve the S / N ratio, all background-subtracted current data were subjected to an 1 1-point moving average smooth, followed by a 101-point Savitzky-Golay quadratic smooth.45 With a data density of 5 points/mV, this 101-point smoothing interval had a width of 20 mV. Because the ratio of the smoothing interval width to the fwhm was 50.2, negligible distortion ( < I % ) of peak potentials and areas resulted from this smoothing procedure.45 Results Ecaluation of Nonisothermal Cell Performance. In order to test that the cell was operating nonisothermally, AS",, of K3Fe(CN), was determined from cyclic voltammetric measurements of E O ' , , as a function of temperature. For pH 7, 0.20 M ionic strength, Tris/cacodylate buffer, a reaction entropy of -141 J / (Kmol) was determined by using eq 5. The corresponding literature value, estimated from Lin and B r e c k ' ~data ~ ~ at 0.20 M ionic strength. is -146 J/(K-mol). This level of agreement is generally taken to indicate satisfactory operation of a nonisot her ma 1 cel I Cyclic Volrammetry of Adsorbed Cytochrome c. Figure 2A shows typical cyclic voltammetric responses for adsorbed cytochrome c a t near-monolayer coverage. These responses are superimposed on the background CVs for the bare electrode. Figure 2B shows the same data after background subtraction. Two criteria were used in selecting solution conditions for these experiments. First, solutions were used in which cytochrome c was in its native conformation. Because ferricytochrome c undergoes a pH-dependent conformational change with a pK, of ca. 9.3,46solutions more alkaline than pH 8 were not used. Second, with each experiment lasting several hours, only solutions com.z2.23336,37
(45) Enke, C. G.; Nieman. T. A. Anal. Chem. 1976, 48, 705A-712A.
(46) Dickerson, R. E.; Timkovich, R. In The Enzymes; Boyer, P. D., Ed.: Academic: S e w York, 1975; Vol. XI-A, pp 397-547.
T i "C Figure 4. Plots of surface formal potentials of adsorbed cytochrome c as a function of temperature. Solution conditions were the same as Figures 2 and 3. Top panel shows 50 mV/s data. Bottom panel shows 100 mV/s data. The panels show three replicate data sets, each acquired at a separate electrode. The lines shown are individual linear regression fits to each data set.
patible with substantial and robust coverage were used. This second requirement prohibited the use of ionic strengths > 100 mM at pH 7 and of all pH 6 solutions. For these conditions the cytochrome c coverage was unacceptably small and nonpersistent, presumably due to decreased electrostatic attraction (vide infra). For four of the five solutions studied, electroactive surface coverage was on the order of 1 monolayer. Assuming a surface roughness factor of 2 and a single molecule adsorption area of ca. 1500 A2, the density of a theoretical, close-packed monolayer of cytochrome c is ca. 20 pmol/cm2. Experimental coverage, I', was found to be 13-1 5 pmol/cm2 except in pH 8, 131 mM ionic strength solutions, where the cytochrome c coverage was 7 pmol/cmz (see Table I). At the solution conditions studied, the electroactive coverage remained essentially constant for several hours as temperature was varied between 8 and 28 "C. However, upon increasing the temperature to 35 "C, an irreversible decrease in both the rate of electron transferz1and the electroactive surface coverage was observed. Determination of Thermodynamic Parameters. Figure 3 shows 50 mV/s CVs for adsorbed cytochrome c acquired at a single electrode at three temperatures. A negative shift in formal potential with increasing temperature is evident. The effect of temperature on electron-transfer kinetics2' accounts for the larger negative shifts observed for the anodic peak in Figure 3. That is, due to the quasi-reversible electron-transfer kinetics, the decrease in peak separation with increasing temperature causes negative and positive shifts in the anodic peak and the cathodic peak, respectively. When this effect is superimposed on a negative E"'surf shift, results such as those shown in Figure 3 are expected. Although these CVs exhibit some quasi-reversibility, the accurate values was not precluded since the elecdetermination of EDfsurf trochemical transfer coefficient (a)is near 0.515*4' Figure 4 is a plot of E"',, as a function of temperature for three replicate sets of data at pH 8, 15 mM ionic strength. The surface formal potentials at 25 "C determined from these data, and from similar data acquired at other solution conditions, are presented in Table I. These values for Eo'surf.2S are shifted ca. 20-25 mV negative of the solution formal potential, E0'soln,2S, of cytochrome c in phosphate buffers at 25 0C.z2-27
The Journal of Physical Chemistry, Vol. 94, No. 21, 1990 8245
Adsorption of Cytochrome c on Tin Oxide AGO = -25kJ/mol
AH,;
= -41
kJ/mol
AS;, = -55J/ K mol
e-+
cyt c
e-+
cyt c
I
J
'
,
( I ~ I ) ~ ~ ~ ~ (3)
'
CYt C(Wsoln
J
(1)
7 CYt C(")ads AGO = -23 kJ/mol
AH;,
= -46 kJ/mol
AS;,
= -80 J/
K mol
Figure 5. Thermodynamic cycle depicting redox and adsorption thermodynamics of strongly adsorbed cytochrome c. Thermodynamic parameters refer to the reduction of solution and adsorbed cytochrome c, reactions 3 and 1, respectively. A G O is calculated with respect to NHE. Approximate values have been given with confidence intervals omitted for clarity. Refer to text for further detail.
Table I also presents values of AS",, obtained for adsorbed cytochrome c on tin oxide. The ASo,,values at lower ionic strengths exhibit a larger variance as reflected in the 95% confidence intervals given in parentheses. This increased variance may result from electrode-to-electrode surface variability that becomes more evident at low ionic strengths. A comparison with literature values for solution cytochrome c, given in the bottom half of Table I, indicates that a negative shift in AS",, of approximately 25 J/(Kmol) occurs upon adsorption. Due to the different ionic strength requirements of adsorption and solution experiments, some caution is required in making this comparison. It should be noted that there is a slight temperature-dependent change in pH with phosphate buffers that could conceivably impact these results. For example, a 10 mM ionic strength phosphate buffer adjusted to pH 7.8 at 25 OC has a pH of 7.6 at 8 OC, and a pH of 8.0 at 35 "C. However, our results, as well as studies with solution cytochrome c,23-27indicate that E"' and AS",, are relatively insensitive to pH in the range of 7-8. With due consideration of these factors, we conclude that the results in Table 1 demonstrate a significant decrease in AS",, upon adsorption.
Discussion Electrostatic Adsorption. Evidence for the governing role of electrostatics in the adsorption of cytochrome c on tin oxide has been presented e l ~ e w h e r e . ' ~The ' ~ results obtained in the present study provide further confirmation of this role. For example, maximal, near-monolayer, coverage was observed in pH 7-8, low ionic strength, solutions as shown by the first four entries of Table I. Strong Coulombic attraction is understandable for these conditions in light of the basicity of cytochrome c (IEP = and the acidity of the tin oxide surface (IEP 5.5).48 However, upon increasing the ionic strength and/or decreasing the pH toward the tin oxide IEP, a weakened Coulombic interaction is expected and observed. Thus, only small and nonpersistent cytochrome c coverage resulted in pH 7 solution at higher (100 mM) ionic strength and in all pH 6 solutions, ranging in ionic strength from 1 to 100 mM (data not shown). Diminished coverage was also observed at pH 8 as a result of increasing the ionic strength from 15 to 132 mM. However, the Coulombic attraction for this condition was strong enough to yield substantial, albeit nonmaximal, coverage (see fifth entry in Table I). In summary, the observed variation in surface coverage as a function of pH and ionic strength is due primarily to electrostatic interactions between cytochrome c and tin oxide. Redox and Adsorption Thermodynamics. The cycle shown in Figure 5 relating the redox thermodynamics to the adsorption thermodynamics is similar to those previously proposed for the adsorption of cytochrome c on membranes,49as well as for the
-
(47) Barlow, G. H.;Margoliash, E. J . Biol. Chem. 1966,241, 1473-1477. (48) Ahmed, S.M. In Oxides and Oxide Films, Diggle, J . W., Ed.; Marcel Dekker: New York, 1972; Vol. 1, pp 319-517.
binding of anions to cytochrome c.28 Reactions 1 and 3 in this figure are the one-electron-transfer reactions of adsorbed and solution cytochrome c, respectively. The thermodynamic parameters indicated for these reactions apply to the reduction offerricytochrome c. Reactions 2 and 4 in this figure are the adsorption/desorption reactions for ferricytochrome c and ferrocytochrome c, respectively. The approximate values given in Figure 5 have been taken from Table I, with confidence limits omitted for clarity. The formal negative shift potential of cytochrome c undergoes a 20-25" upon adsorption on tin oxide electrodes. As indicated in Figure 5 , this corresponds to a 2 kJ/mol difference in AGOada,the free energy of adsorption, between the oxidized and reduced forms of the protein. With P = exp(-AGad,/R7'), a surface form of the Nernst equation can be written ass0 Eo'surf,2S
=
Eo'soln.25
- ( R T / n F ) In
(@ox/@rd)
(7)
where Pled and Po, are the adsorption coefficients of ferrocytochrome c and ferricytochrome c, respectively. The observed and E0'soln,25corresponds to a 20-25 mV difference in Eo'surf,25 Pox/@red ratio of 2, indicating that ferricytochrome c adsorbs to tin oxide more strongly than does ferrocytochrome c. Similar behavior has been observed previously for cytochrome c binding to various membra ne^,^^-^' to respiratory enzymes,32and to the phosphoprotein p h ~ s v i t i n . ~In~ ,all ~ ~of these studies, negative formal potential shifts ranging in magnitude from 20 to 50 mV were taken to indicate that the oxidized form of cytochrome c binds more strongly than the reduced form. A similar conclusion has been reached in 31PNMR studies of cytochrome c association with lipid m~ltilayers.~'Further discussion of tin oxide results in the context of these other studies is presented below. By comparison of the redox thermodynamics of solution and adsorbed cytochrome c, differences in the adsorption thermodynamics of ferricytochrome c and ferrocytochrome c on tin oxide can be discerned. A value for AS", of approximately -75 to -80 J/(Kmol) was measured for the reduction of adsorbed cytochrome c. Compared to the AS",, values of -49 to -62 J/(Kmol) for the reduction of solution cytochrome c,22-27this represents a negative shift of approximately 25 J/(Kmol) (see Table I). Thus, it can be concluded that the entropy of adsorption, hSoads, of ferricytochrome c is ca. 25 J/(K.mol) greater than that for ferrocytochrome c. Measurements of the formal potential and reaction entropy can be used to calculate the reaction enthalpy, AH",,, for both solution and adsorbed cytochrome c (see Table I and Figure 5 ) . A comparison of these values indicates that AH", is 4-6 kJ/mol more exothermic for adsorbed cytochrome c than for solution cytochrome c. This difference in AH",,indicates that of ferricytochrome c is 4-6 the enthalpy of adsorption, AHoads, kJ/mol less exothermic than for ferrocytochrome c. These small, yet significant, differences in and AHoads provide a basis for explaining why ferricytochrome c adsorbs more strongly than ferrocytochrome c on tin oxide. It is well-known that the oxidized form of cytochrome c is less stable and more susceptible to structural alteration than is the reduced form. Ferricytochrome c is, for example, more susceptible to thermal denaturation, pH-induced denaturation, and proteolytic digestion, than is the ferrous form.46,s2-56These differences in physicochemical behavior have been attributed to a redox-de(49) Vanderkooi, J.; Erecinska, M.; Chance, B. Arch. Biochem. Biophys. 1973, 157, 531-540.
(50) Laviron, E. J . Elecrroanal. Chem. 1979, 100, 263-270. (51) Waltham, M. C.; Cornell, B. A.; Smith, R. Biochim. Biophys. Acta 1986,862, 451-456. (52) Margoliash, E.; Schejter, A. Ado. Protein Chem. 1966, 21, 113-286. (53) Moore. G. R.: Huann. Z.; Elev. C. G . S.; Barker. H. A.; Williams. R. J. P.Faraday Discuss. d e m . Sac.- 1982, 74, 31 1-329. (54) Osheroff, N.; Borden, D. Koppenol, W. H.; Margoliash, E. J . Biol. Chem. 1980, 255, 1689-1697. (55) Salemme, F. R. In Frontiers of Biological Energetics; Dutton, P. L., Leigh, J. S., Scarpa, A,, Eds.; Academic Press: New York, 1978; Vol. 1, pp 83-90. (56) Bosshard, H. R.; Zurrer, M. J . Biol. Chem. 1980, 255, 6694-6699.
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The Journal of Physical Chemistry, Vol. 94, No. 21, 1990
pendent difference in dynamic properties. Thus, although cytochrome c is considered to be a quite rigid protein in both redox forms,s7the oxidized form does exhibit a greater degree of dynamic freedom and structural f l e ~ i b i l i t y . ~It~ -is~reasonable ~ to expect that the oxidized form of cytochrome c should also be more susceptible to structural alteration as a result of interfacial forces at solid/aqueous interfaces. Our findings that soa& for ferrifor fercytochrome c is larger by ca. 25 J/(K.mol) than 4Soads rocytochrome c clearly supports this proposal. This conclusion is also consistent with general views regarding protein adsorption at solid/aqueous interfaces. That is, proteins that exhibit greater structural flexibility are known to undergo more significant structural alterations upon adsorption resulting in stronger binding than is observed with more rigid proteins.58 Such adsorption-induced structural alterations increase the degree of disorder in the protein and should give rise to positive contributions to both d S o a d s and AHoads.58The results described above are consistent with this model in that more positive values for the entropy and enthalpy of adsorption were in fact observed for the more strongly adsorbing ferric form of cytochrome c . Although the exact nature of the structural alterations in adsorbed ferricytochrome c on tin oxide are not known, we can speculate that a slight loosening or disruption of the heme crevice is a likely possibility. The conformational flexibility of this region of the cytochrome c structure, in particular the lower left side,s7has been implicated in denaturation phenomena that ultimately lead to a rupture of the Fe( 111)-sulfur bond.53954,56*57.s9 Although rupture of this bond is not a consideration here, a lesser structural alteration of the same region of the molecule does provide a reasonable explanation for the observed thermodynamic behavior of ferricytochrome c on tin oxide. The conformational stability of the heme crevice has also been invoked previously by Yoshimura et al.34in a somewhat different manner to explain the altered reactivity of cytochrome c when it is complexed to phosvitin. As suggested above, the tin oxide electrode bears resemblance to certain biological media with respect to its ability to bind cytochrome c. It has been found in the present work that the solution formal potential of horse cytochrome c shifts negative by 20-25 mV upon adsorption on tin oxide. Similar negative shifts have been reported for horse cytochrome c bound to phosvitin (20-30 mV),33-34cardiolipin-lecithin vesicles (45-50 mV),30*3' mitochondrial membranes (50-60 mV),29,30and isolated cytochrome oxidase and reductase (35-40 mV).j2 Shifts of these ranging magnitudes translate into binding constant ratios, @ox/&,. from 2 for a -20-mV shift, to 7 for a -50-mV shift. In all of these cases, it has been shown that there is clearly a substantial, if not dominant, electrostatic contribution to the binding. The membranous lipid systems, however, are seen to give rise to a signif(57) Williams, R . J . P. Z . Phys. Chem. 1988, 269, 387-402. (58) Lyklema, J . Colloids Surf. 1984, 10, 33-42. (59) Myer, Y . P.; MacDonald, L. H.; Verma. B. C.; Pande. A . Biochemistry 1980, 19, 199-207
Willit and Bowden icantly larger differential binding between the two redox forms than does tin oxide, phosvitin, and isolated respiratory enzymes. This probably reflects additional nonelectrostatic binding components in the case of lipid membranes, Le., hydrophobic interactions and/or the effect of diffusional mobility of lipids in the membrane.m3 Clearly, additional work is desirable in order to discern the origins of these binding affinity differences and to assess their physiological significance in the function of cytochrome c. It seems evident from the present study that the determination of formal potentials and reaction entropies through the use of a nonisothermal cell arrangement can provide fundamental information that will be essential for realizing this objective. Conclusions
Cytochrome c adsorbs strongly on tin oxide at near-monolayer coverage in neutral to slightly alkaline low ionic strength solutions, primarily as a result of electrostatic attraction. Under these solution conditions, the adsorbed cytochrome c is electroactive and exhibits near-native redox properties. From variable-temperature voltammetric measurements of the nonisothermal formal potential, a reaction entropy of ca. -75 to -80 J/(Kmol) was determined for adsorbed cytochrome c. This value is some 25 J/(K-mol) more negative than the corresponding solution value. It thus appears that greater structural changes accompany the electron transfer when cytochrome c is adsorbed. Upon adsorption, a 20-25" decrease in formal potential was also observed, which indicates that the oxidized form of cytochrome c adsorbs more strongly by ca. 2 kJ/mol than the reduced form. Differences in the enthalpies and entropies of adsorption between the two redox forms support a model in which ferricytochrome c undergoes more structural alteration upon adsorption than ferrocytochrome c. The conclusions drawn from this study of adsorbed cytochrome c on tin oxide may prove useful in understanding similar formal potential shifts that have been observed for more physiologically relevant ads or bent^.^*-^^ Furthermore, the electrochemical approach described here for characterizing cytochrome c adsorption and redox thermodynamics should be applicable in similar characterizations of other redox proteins. Acknowledgment. This work has benefited from helpful discussions with Professors J. H. Reeves and J . T. Hupp. Thanks to Dr. John Sopko and PPG for providing the tin oxide material. We gratefully acknowledge support for this work by the National Science Foundation (CHE-8820832). Registry No. F,, 7782-41-4; cytochrome c, 9007-43-6; tin oxide, 1332-29-2. (60) Brown, L. R.; Wuthrich, K . Biochim. Biophys. Acta 1977, 468, 389-410. (61) Papahadjopoulous, D.; Mascarello, M.; Eylar, E. H.; Isac, T. Biochim. Biophys. Acta 1975, 401, 317-335. (62) Letellier, L.; Schechter, E. Eur. J . Biochem. 1973, 40, 507-512. (63) Vincent, J. S.; Levin, I . W. Biochemisrry 1988, 27, 3438-3446.
