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Electrochemistry of Cytochrome c in Aqueous and Mixed Solvent Solutions: Thermodynamics, Kinetics, and the Effect of Solvent Dielectric Constant Niall J. O’Reilly and Edmond Magner* Department of Chemical and Environmental Sciences and Materials and Surface Science Institute, University of Limerick, Castletroy, Limerick, Ireland Received May 14, 2004. In Final Form: August 25, 2004 The solvent dielectric constant is considered an important factor in determining the redox potential of the heme-containing protein cytochrome c in solution. In this study, we investigate the electrochemical response of cytochrome c in aqueous/organic solvent mixtures (100% aqueous buffer, 30% acetonitrile, 40% dimethyl sulfoxide, and 50% methanol), reporting the redox potential (E°′), enthalpy, and entropy of reduction. The temperature dependence of the solvent dielectric constant () was also measured. The results show that alone cannot regulate the E°′ of cytochrome c in mixed solvent systems. The implications of the temperature dependence of on the validity of the thermodynamic data are also discussed. The effect of solvent and temperature on the electron-transfer rate constant, ks, was determined in each solvent mixture. A substantial increase in the activation energy for electron transfer was observed in 40% DMSO.
Introduction The selectivity of enzymes is of considerable interest in biocatalysis. In addition, the ability of enzymes to discriminate between different analyte molecules represents a powerful analytical tool in the field of biosensors. Many of these applications, however, require that the enzyme functions in a nonaqueous environment. Organic synthesis, for example, is carried out almost exclusively in organic solvents, whereas many organic analyte molecules are insoluble in water. Consequently, much research has been carried out in recent years to examine the behavior of protein molecules in organic and aqueous-organic media. Biocatalytic1-3 and biosensor4-9 applications in organic solvents have been well established. Studies of enzyme catalysis and selectivity,10-15 protein structure,16-21 * Author to whom correspondence should be addressed. Phone: (353) 61202629; fax: (353) 61213529; e-mail:
[email protected]. (1) Klibanov, A. M. Chemtech 1986, 354-359. (2) Klibanov, A. M. Acc. Chem. Res. 1990, 23, 114-120. (3) Klibanov, A. M. Nature 2001, 409, 241-246. (4) Beissenhirtz, M.; Scheller, F.; Lisdat, F. Electroanalysis 2003, 15, 1425-1435. (5) Diaz-Garcia, M. E.; Valencia-Gonzalez, M. J. Talanta 1995, 42, 1763-1773. (6) Saini, S.; Hall, G. F.; Downs, M. E. A.; Turner, A. P. F. Anal. Chim. Acta 1991, 249, 1-15. (7) Wang, J.; Naser, N.; Lopez, D. Biosens. Bioelectron. 1994, 9, 225230. (8) Campanella, L.; Giancola, D.; Gregori, E.; Tomassetti, M. Sens. Actuators, B: Chem. 2003, 95, 321-327. (9) Campanella, L.; Gatti, E.; Sammartino, M. P.; Sulpizio, A.; Tomassetti, M. Anal. Chim. 2003, 93, 35-43. (10) Das, P. K.; Caaveiro, J. M. M.; Luque, S.; Klibanov, A. M. J. Am. Chem. Soc. 2002, 124, 782-787. (11) Ke, T.; Wescott, C. R.; Klibanov, A. M. J. Am. Chem. Soc. 1996, 118, 3366-3374. (12) Ke, T.; Klibanov, A. M. J. Am. Chem. Soc. 1999, 121, 33343340. (13) Magner, E.; Klibanov, A. M. Biotechnol. Bioeng. 1995, 46, 175179. (14) Wescott, C. R.; Klibanov, A. M. J. Am. Chem. Soc. 1993, 115, 10362-10363. (15) Xie, Y.; Das, P. K.; Caaveiro, J. M. M.; Klibanov, A. M. Biotechnol. Bioeng. 2002, 79, 105-111. (16) Sivakolundu, S. G.; Mabrouk, P. A. J. Am. Chem. Soc. 2000, 122, 1513-1521. (17) Desai, U. R.; Klibanov, A. M. J. Am. Chem. Soc. 1995, 117, 39403945.
denaturation behavior,18,19,22,23 and redox electrochemistry4,24-31 have all been performed in these solvent systems. The structure of proteins remains largely intact in anhydrous solvents. Using the amide I and amide III FTIR bands, Griebenow et al.19 found that the R-helical content of lyophilised hen-egg lysozyme was unchanged on suspension in anhydrous acetonitrile. This may be explained by the reduced mobility of the protein in anhydrous solvents, a factor which inhibits its folding into a more thermodynamically favored, denatured conformation. Proteins in mixed solvent solutions have this conformational freedom and are consequently more prone to denaturation. Thus, hen-egg lysozyme lost half of its R-helical content when placed in 60% acetonitrile.19 In contrast to this result, circular dichroism, resonance Raman,16 and 1H NMR24 studies have shown that the structure of the heme-protein cytochrome c is largely unchanged in mixed solvent media. Heme-containing proteins such as peroxidases and cytochromes have many potential uses and applications in the field of biocatalysis32 and biosensors.4,33 Cytochrome c is a well-characterized redox protein which has been (18) Wangikar, P. P.; Michels, P. C.; Clark, D. S.; Dordick, J. S. J. Am. Chem. Soc. 1997, 119, 70-76. (19) Griebenow, K.; Klibanov, A. M. J. Am. Chem. Soc. 1996, 118, 11695-11700. (20) Toba, S.; Merz, K. M. J. Am. Chem. Soc. 1997, 119, 9939-9948. (21) Wu, J.; Gorenstein, D. G. J. Am. Chem. Soc. 1993, 115, 68436850. (22) Klibanov, A. M. Tibtech 1997, 15, 97-101. (23) Schmitke, J. L.; Wescott, C. R.; Klibanov, A. M. J. Am. Chem. Soc. 1996, 118, 3360-365. (24) Battistuzzi, G.; Borsari, M.; Rossi, G.; Sola, M. Inorg. Chim. Acta 1998, 272, 168-175. (25) Borsari, M.; Bellei, M.; Tavagnacco, C.; Peressini, S.; Millo, D.; Costa, G. Inorg. Chim. Acta 2003, 349, 182-188. (26) Ci Li, Q.; Mabrouk, P. A. J. Electroanal. Chem. 1998, 455, 4548. (27) Grealis, C.; Magner, E. Chem. Commun. 2002, 816-817. (28) Grealis, C.; Magner, E. Langmuir 2003, 19, 1282-1286. (29) Mabrouk, P. A. J. Am. Chem. Soc. 1995, 117, 2141-2146. (30) Mabrouk, P. A. Anal. Chem. 1996, 68, 189-191. (31) O’Donoghue, D.; Magner, E. Chem. Commun. 2003, 438-439. (32) van Deurzen, M. P. J.; van Rantwijk, F.; Sheldon, R. A. Tetrahedron 1997, 53, 13183-13220. (33) Gorton, L.; Lindgren, A.; Larsson, T.; Munteanu, F. D.; Ruzgas, T.; Gazaryan, I. Anal. Chim. Acta 1999, 400, 91-108.
10.1021/la048796t CCC: $30.25 © 2005 American Chemical Society Published on Web 01/07/2005
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widely used as a model system to study electron transfer in proteins.34 The positively charged lysine residues surrounding the heme edge allow the protein to dock with negatively charged groups of its redox partners such as cytochrome c oxidase and cytochrome c peroxidase.35 This feature has been exploited to probe the redox electrochemistry of cytochrome c. Modified electrodes which contain groups capable of hydrogen-bonding with the lysine residues orient the heme toward the electrode surface, thus facilitating direct electron transfer.34,36 Cytochrome c has been shown to display quasi-reversible electrochemical behavior in a range of aqueous-organic solvent systems in both solution16,24,25 and immobilized form.4,37 The factors regulating the redox potential, E°′, of cytochrome c and other redox proteins are complex. Ligand binding effects,38 heme exposure to the solvent,39 the nature of the solvent medium,24,31 and heme-protein interactions40 all play a role. Measurement of E°′ as a function of temperature and the application of eq 1 allows the extraction of ∆Hrc°′ and ∆Src°′:
E°′ )
-∆Hrc°′ T∆Src°′ + nF nF
(1)
Ligand-heme interactions are thought to dominate the enthalpic term. For cytochrome c, the acceptor power of the methionine ligand stabilizes the ferroheme.38,41 Solvent reorganization effects42,43 and the more compact structure of the ferroheme form34 contribute to the entropic term. Among the factors proposed to regulate E°′ of cytochrome c is the dielectric constant of the solvent medium, ,4,16,24,25 the importance of which can be understood by considering the effects of electrostatic interactions on E°′. The E°′ of cytochrome c is lower in mixed solvent solutions than in water thus suggesting that the lower dielectric constant of the mixed solvent solution is responsible for the decrease in E°′. The of a solvent can also be lowered by increasing the temperature. For example, the of water decreases from 85.76 at 5 °C to 73.15 at 40 °C.44 A decrease of ∼16 mV in E°′ has been reported for cytochrome c over the same temperature range.25 According to eq 1, the change in E°′ with temperature is related to the entropy change, ∆Src°′. A fundamental principle of the thermodynamic approach is that ∆Src°′, ∆Hrc°′, and the system under study remain constant over the temperature range investigated. This is not the case in terms of solvent properties and it is possible that the decrease in E°′ with increasing temperature is simply reflecting the change in of the solvent. Under such conditions, it is difficult to know how much meaningful thermodynamic data can be obtained from E°′ vs T plots without first investigating the (34) Cytochrome c: A Multidisciplinary Approach; Scott, R. A., Mauk, A. G., Eds.; University Science Books: Sausalito, CA, 1996. (35) Millett, F. In Cytochrome c: A Multidisciplinary Approach; Scott, R. A., Mauk, A. G., Eds.; University Science Books: Sausalito, CA, 1996. (36) Eddowes, M. J.; Hill, H. A. O. J. Am. Chem. Soc. 1979, 101, 7113-7114. (37) Grealis, C. Characterisation of the Redox Properties of Cytochrome c in Non-Aqueous Solvents. Ph.D. Thesis, University of Limerick, 2003. (38) Moore, G. R.; Williams, R. P. J. FEBS Lett. 1977, 79, 229-232. (39) Stellwagen, E. Nature 1978, 275, 73-74. (40) Moore, G., R.; Pettigrew, G. W.; Rogers, N. K. Proc. Natl. Acad. Sci. U.S.A. 1986, 83, 4998-4999. (41) Tezcan, F. A.; Winkler, J. R.; Gray, H. B. J. Am. Chem. Soc. 1998, 120, 13383-13388. (42) Bertrand, P.; Mbarki, O.; Asso, M.; Blanchard, L.; Guerlesquin, F.; Tegoni, M. Biochemistry 1995, 34, 11071-11079. (43) Battistuzzi, G.; Borsari, M.; Ranieri, A.; Sola, M. J. Am. Chem. Soc. 2002, 124, 26-27. (44) Lange’s Handbook of Chemistry, 14th ed.; Dean, J. A., Ed.; McGraw-Hill: New York, 1992.
relationship between E°′ and . A knowledge of in mixed solvent media and how it varies with temperature is therefore required. While much progress has been made in the development of models to predict the of mixed solvent media,45 the experimental measurement of is still required, particularly for mixtures which display nonideal behavior. For biosensor applications, rapid electron transfer is often desirable to ensure a reasonable response time and linearity.4 Recently, it has been shown that the activation energy of electron transfer for cytochrome c immobilized onto TiO2 modified SnO2 electrodes was significantly higher in 95% glycerol than in aqueous buffer.28 It is therefore of interest to determine if this trend is also followed in the solution-phase electrochemistry of cytochrome c in the presence of organic solvents. In this paper, we report the experimental measurement of the dielectric constant as a function of both solvent composition and temperature and attempt to relate these data to temperature-dependent behavior of E°′ of cytochrome c. We also report the effect of temperature on the electron-transfer kinetics in each solvent medium. Materials and Methods Reagents. Horse heart cytochrome c, potassium ferricyanide (99%), sodium chloride (99.5%), alumina ( 0.94). A break is clearly visible in the E°′ versus T plots for the three mixed solvent solutions representing a transition from the low- to high-temperature native conformers. Such biphasic behavior has been observed previously for cytochrome c in similar solvent mixtures and in aqueous solution at alkaline pH.24,25,56 The onset of the high-temperature conformer was characterized by a progressive deterioration of the response, with ∆Ep increasing with temperature. The transition from the lowto high-temperature conformer has been attributed to a conformational change in the oxidized form, possibly involving the deprotonation of lysine residues on the heme crevice.57 This would explain the trend toward more irreversible behavior after the break temperature as the lysine residues are considered important in orienting the heme toward the electrode surface.36 It has been suggested24,25 that a decrease in the solvent polarity would stabilize the high-temperature conformer. This is supported by the dielectric constant data reported here (discussed below), where the break temperature decreased with decreasing , although no break temperature was observed in aqueous buffer up to 40 °C. Table 1 records the E°′ 25°C, ∆Hrc°′, ∆Src°′ (for the lowtemperature conformer) and the break temperature in each solvent. This thermodynamic data is in reasonable agreement with that reported previously24,25,58 with pH differences a likely explanation for some discrepancies. The enthalpy and entropy changes are compensative with the enthalpic term being the dominant factor in all solvents (Table 2). The presence of organic solvent in the solution increases both ∆Hrc°′ and ∆Src°′ with respect to the aqueous buffer with the overall effect of decreasing E°′. A decrease in solvent ordering would be expected on reduction, resulting in an entropy increase. The heme iron, however, is shielded somewhat by its axial ligands, thus minimizing such an effect. In addition, a neutral, reduced heme would allow increased interaction between solvent dipoles and the heme propionates.42 This, coupled with the more compact nature of the ferroheme,34 may dominate the entropy term resulting in the negative ∆Src°′ values observed in all solvents. Reduction of cytochrome c in mixed solvent systems instead of water is favored on entropic grounds. Solvent-containing mixtures are less polar than aqueous buffer solution and might consequently interact with the heme propionates to a lesser extent. The tendency of the reduced protein to change to a more compact form may also be minimized in the presence of an organic solvent. These factors may explain the higher values of ∆Src°′ in the solvent mixtures. In contrast, the reduction of cytochrome c in mixed solvent media is disfavored on enthalpic grounds with respect to aqueous buffer. This behavior is puzzling as the ferroheme might be expected to be more stable in a less polar solvent. Indeed, this is the case for immobilized cytochrome c in 95% glycerol.28 Axial ligation is the main contributor to the enthalpic term. Moore and Williams (56) Battistuzzi, G.; Borsari, M.; Sola, M.; Francia, F. Biochemistry 1997, 36, 16247-16258. (57) Christen, R. P.; Nomikos, S. I.; Smith, E. T. J. Biol. Inorg. Chem. 1996, 1, 515-522. (58) Koller, K. B.; Hawkridge, F. M. J. Am. Chem. Soc. 1985, 107, 7412-7417.
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Figure 1. Cyclic voltammograms of (A) 0.7 mM cyt c in aqueous buffer, (B) 0.55 mM cyt c in 30% acetonitrile, (C) 0.6 mM cyt c in 40% DMSO, and (D) 0.4 mM cyt c in 50% methanol. Conditions: ν ) 0.02 V s-1, T ) 298 K (50% methanol, T ) 293 K).
Figure 2. E°′ vs T plots of cytochrome c in aqueous buffer (]), 30% acetonitrile (0), 40% DMSO (/), and 50% methanol (O). Data points represent the mean E°′ at each temperature (1 standard deviation (n ) 3). ν ) 20 mV s-1.
have shown that the length of the Fe-S bond between the heme and the methionine ligand has a significant effect on the redox potential.38 It is possible that small perturbations in the protein structure occur in mixed solvent solutions which alter the Fe-S bond length. Indeed, UV spectra of cytochrome c in mixed solvents 30% acetonitrile and 40% DMSO show small changes in the molar absorptivity of the 695-nm charge-transfer band which is a signature of the methionine ligation.16 Figure 3 illustrates the effect of temperature on for each of the solvent mixtures (in the absence of protein, buffer, and electrolyte), with the values at 25 °C reported
in Table 1. The values obtained in water over this temperature range differ by no more than 1.3% from those reported previously.44 Most noteworthy is the high of the 40% DMSO solution which is very similar to that of water (74.0 and 77.8 at 25 °C, respectively) and in good agreement with that reported previously (∼75.6 interpolated from data in ref 59). Given that for pure DMSO at 20 °C is 48.9,44 this result is somewhat surprising and underscores the need for the experimental measurement of for solvent mixtures. DMSO/water mixtures display other nonideal characteristics including unusual freezing point, diffusion coefficients, and density.60 The of the 30% acetonitrile and 50% methanol mixtures are largely as expected on the basis of their component properties. Sivakolundu et al. reported that the redox potential of cytochrome c decreased linearly with increasing solvent content (r2 > 0.937) in a range of different solvents and attributed this result to the decreasing dielectric constant of the solvent medium.16 While a similar effect is observed here, it would appear that this trend is fortuitous and due to compensative enthalpic and entropic contributions. For example, 30% acetonitrile has the lowest solvent content of the three mixed solvent solutions investigated and yet has the largest change in ∆Hrc°′ and ∆Src°′ in comparison to cytochrome c in aqueous buffer. Similarly, a plot of E°′ versus at a given temperature shows no correlation (Figure 4) nor do similar plots of ∆Hrc°′, ∆Src°′, and ks versus (data not plotted). The change in with temperature is reasonably consistent across all solvent (59) Kaatze, U.; Pottel, R.; Scha¨fer, M. J. Phys. Chem. 1989, 93, 5623-5627. (60) Strader, M. L.; Feller, S. E. J. Phys. Chem. A 2002, 106, 10741080.
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Table 1. Redox Potential, ∆H °rc′, and ∆S °rc′ of Native Cytochrome c in Each Organic Solvent and Conformer Transition Temperature and Dielectric Constant (E, in the Absence of Buffer/Electrolyte)a
a
solvent
E°25°C ′ (V vs SHE)
-1 mol-1) ∆S°′ rc (J K
-1 ∆H°′ rc (kJ mol )
break temp °C
25°C
100% aq 50% CH3OH 40% DMSO 30% CH3CN
0.265 ( 0.001 0.239 ( 0.001b 0.239 ( 0.002 0.247 ( 0.002
-54.2 ( 1.5 -44.8 ( 2.9 -32.5 ( 2.6 -21.2 ( 1.0
-41.6 ( 0.4 -36.1 ( 0.8 -32.6 ( 0.8 -30.1 ( 0.4
23.9 ( 1.0 33.9 ( 0.2 28.6 ( 0.8
77.8 ( 1.1 58.9 ( 0.8 74.0 ( 1.0 68.6 ( 1.8
Values are based on the mean of three individual determinations (1 standard deviation. b 20 °C value. Table 2. Breakdown of E°′ into Its Enthalpic and Entropic Componentsa
solvent
E°25°C ′ (V vs SHE)
-∆H°′ rc/nF (V)
b T∆S°′ rc/nF (V)
100% aq 50% CH3OH 40% DMSO 30% AcN
0.265 0.239c 0.239 0.247
0.431 0.368 0.338 0.312
-0.167 -0.132 -0.100 -0.066
a The differences between the experimental and calculated values of E°′ arise from experimental error. b 25 °C. c 20 °C.
Figure 5. Plot of E°′ vs in aqueous buffer ([), 30% acetonitrile (0), 40%, DMSO (/), and 50% methanol (O) as a function of temperature.
Figure 3. Effect of temperature on dielectric constant of water (]), 30% acetonitrile (0), 40% DMSO (/), and 50% methanol (O). Values represent the mean dielectric constant, , at each temperature (1 standard deviation (n ) 3).
Figure 4. Plot of E°′ vs at 293 K for each solvent mixture.
mixtures and, comparing Figures 2 and 3, it is apparent that the temperature dependence of the cytochrome c redox potential in each solvent does not mirror the temperature dependence of . This is borne out in Figure 5 where plots of E°′ versus determined over a range of temperatures are shown. While these plots are linear in each solvent (both E°′ and decrease linearly with temperature), the slopes of these lines vary between solvent mixtures. Thus, alone cannot regulate E°′, in agreement with the results reported by Beissenhirtz et al. for immobilized cytochrome c in mixed solvent solutions.4 These dielectric measurements were made in the absence of electrolyte. However, Wang and Anderko have predicted that at a given
temperature and ionic strength, the decrease in due to the presence of NaCl will be constant, regardless of the solvent composition.45 Moore et al. have already cautioned against the use of one particular factor, such as heme exposure to the solvent, to explain E°′ in different redox proteins.40 Similarly, it would appear that the factors affecting the E°′ of cytochrome c in different solvent media are complex and cannot be conveniently attributed to single properties such as solvent content or dielectric constant. It has been observed that the data obtained in 40% DMSO appears anomalous when compared to the other solvent solutions examined. Whether DMSO/water mixtures are a special case or simply symptomatic of a wider range of solvent mixtures is unclear on the basis of the data available. If 40% DMSO is indeed anomalous, then it is likely that dielectric constant does have some bearing on E°′ in different solvents. In fact Smith has shown, through dielectric modeling, that much of the change in E°′ of cytochrome c in aqueous media can be accounted for by changes in brought about by varying the temperature and pressure.61 Also, the dielectric constant of the protein itself changes with temperature which in turn has an effect on E°′.57 Therefore, the temperature/E°′ profiles of cytochrome c are likely to be due to three factors: redox thermodynamics, temperature dependence of the solvent dielectric constant, and temperature dependence of the protein dielectric constant. It is not possible to determine the extent of each factor on the temperature dependence of E°′. Thus, attributing this dependence completely to thermodynamic considerations will result in the underestimation of both ∆Src°′ and ∆Hrc°′ (i.e., values will be calculated as being more negative than they actually are). In this context, interpretation of the enthalpy and entropy data in Table 2 is difficult. The electron-transfer rate constant, ks, was determined by the Nicholson method.55 At low scan rates, the separation between the anodic and cathodic peaks (∆Ep) remains reasonably constant. Graphs of the kinetic (61) Smith, E. T. J. Am. Chem. Soc. 1995, 117, 6717-6719.
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Figure 6. Plot of Ψ vs (scan rate)-1/2 in aqueous buffer at 298 K ([) and 40% DMSO at 278 K (0). Table 3. Summary of Kinetic Properties of Cytochrome c in Each Solvent Mixture. Values Are Based on the Mean of Three Individual Determinations (1 Standard Deviation solvent
ks25°C × 102 (cm s-1)
DO25°C × 106 (cm2 s-1)
Ea (kJ mol-1)
100% aq 50% CH3OH 40% DMSO 30% AcN
2.44 ( 0.22 0.89 ( 0.24* 0.45 ( 0.09 2.39 ( 1.20
1.15 ( 0.18 0.13 ( 0.04a 0.20 ( 0.01 0.86 ( 0.07
14.0 ( 2.5 18.3 ( 7.3 57.0 ( 2.6
a Measured at 15 °C because of the onset of the high-temperature conformer.
parameter, Ψ, versus ν-1/2 were therefore plotted for scan rates with a ∆Ep value g70 mV after which point ∆Ep increased consistently with scan rate. Figure 6 shows typical plots for 100% aqueous buffer and 40% DMSO. Good linearity was observed in all solvents. The ks values and diffusion coefficients in each solvent are outlined in Table 3. While the diffusion coefficients in aqueous buffer, 30% acetonitrile and 40% DMSO, are in good agreement with those reported by Sivakolundu et al.,16 the ks values are approximately 7 times larger. The compensation for solution resistance in this work may explain this discrepancy. ks in aqueous phosphate buffer is in reasonable agreement with that reported by Koller et al. (0.8 × 10-2 cm s-1)58 and Eddowes and Hill (1.86 × 10-2 cm s-1).36 While the presence of 30% acetonitrile makes significant changes to the redox thermodynamics, it does not appear to adversely affect the rate of electron transfer of cytochrome c with values of ks very similar to that in aqueous buffer. There is an increased variability in the data in 30% acetonitrile, although similar variability has been reported previously in aqueous buffer.58 40% DMSO has the most dramatic effect on the redox kinetics, inducing a 5-fold drop in ks. Arrhenius plots for each solvent system are shown in Figure 7 with the corresponding activation energy for electron transfer, Ea, reported in Table 3. No discernible trend in ks was observed in 30% acetonitrile although the inherent variability of the method, compounded with a low activation energy, might mask such a trend. Ea in aqueous buffer of 14 kJ mol-1 is very similar to that reported by Koller and Hawkridge (∼16 kJ mol-1 estimated from the data in Figure 5 of ref 58). Ea in 50% methanol is comparable to that in aqueous buffer although there is considerable uncertainty in the value of Ea in this solvent mixture. The temperature-dependent behavior of ks is
Figure 7. Plot of ln(ks) vs T-1 in aqueous buffer ([), 30% acetonitrile (0), 40% DMSO (/), and 50% methanol (O). Data presented is that of an individual determination. Errors in ks at 25 °C in Table 3 are representative of all temperatures.
particularly pronounced in 40% DMSO with a 4-fold increase in Ea in this mixture with respect to aqueous buffer. The effect of 40% DMSO on the electron-transfer properties of cytochrome c is curious. The of this solvent mixture is very similar to that of water. In addition, 1H NMR, circular dichroism (CD),24 and resonance Raman spectroscopy16 have shown minimal differences in the protein structure of cytochrome c in 40% DMSO compared to aqueous solution. Despite this, the Ea required for electron transfer in 40% DMSO is 4 times larger than that in aqueous buffer. Similarly, a large increase in Ea was reported for immobilized cytochrome c on transfer from aqueous buffer to 95% glycerol, despite showing almost identical resonance Raman spectra.28 The reason for this is unclear although a small conformational change around the heme crevice which would partially block the path of electron transfer to the electrode may be responsible. Indeed, a CD study by Sivakolundu et al. concluded that changes occurred in heme-polypeptide interactions in the vicinity of the heme active site when DMSO concentrations exceeded 25%.16 The electron-transfer properties do not appear to be significantly affected in 30% acetonitrile or 50% methanol which is consistent with small conformational changes in these solvents. Conclusion Solvent dielectric constant alone cannot explain the redox behavior of cytochrome c in mixed solvent solutions although it undoubtedly plays a role. Thus, while thermodynamic evaluation of E°′ can be useful in elucidating general trends with solvent composition, care must be taken when interpreting such data which may be compromised by temperature-dependent solvent and protein dielectric effects. ks in 40% DMSO is significantly suppressed compared to aqueous buffer. Nonetheless, fast, reversible electrochemistry is possible for cytochrome c in the other mixed solvents examined. Acknowledgment. We would like to thank Prof. Khalil Arshak for the use of the impedance analyzer and Dr. Jakki Cooney and Dr. Todd Kagawa for the use of protein purification equipment. Funding from the Irish Research Council for Science, Engineering, and Technology is gratefully appreciated. LA048796T
