Effect of Mono-CDNP Substitution of Lysine ... - ACS Publications

Figure 1 Cyclic voltammograms of native (a) and four mono-CDNP-substituted (b−e) cytochromes c adsorbed on HOOC−(CH2)6−SH modified Au electrodes...
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Effect of Mono-CDNP Substitution of Lysine Residues on the Redox Reaction of Cytochrome c Electrostatically Adsorbed on a Mercaptoheptanoic Acid Modified Au(111) Surface Shin-ichiro Imabayashi,*,† Takahiro Mita,† and Takashi Kakiuchi*,‡ Department of Chemistry and Biotechnology, Faculty of Engineering, Yokohama National University, Yokohama 240-8501, Japan, and Department of Energy and Hydrocarbon Chemistry, Graduate School of Engineering, Kyoto University, Kyoto 615-8510, Japan Received October 16, 2004. In Final Form: December 14, 2004 The effect of charge-inverting modification of single surface lysine residue on the electron transfer (ET) reaction of horse heart cytochrome c (cyt c) is examined for 12 different types of mono-4-chloro-2,5dinitrobenzoic acid substituted cyt c (mCDNPc) adsorbed on a Au(111) electrode modified with a selfassembled monolayer (SAM) of 7-mercapto-heptanoic acid (MHA). A negative shift in the redox potential by 10-35 mV as compared to that of native cyt c and a monolayer coverage in the range of 13-17 pmol cm-2 are observed for electroactive mCDNPc’s. The magnitude of the decrease in the ET rate constant (ket) of mCDNPc’s compared with that of native cyt c depends on the position of the CDNP substitution. For mCDNPc’s in which the modified lysine residue is outside of the interaction domain of cyt c with the SAM, the ratio of the ket of mCDNPc to that of native cyt c is correlated to the change in the dipole moment vector of cyt c due to the CDNP modification. This correlation suggests that the dipole moment of cyt c determines its orientation of adsorption on the SAM of MHA and significantly affects the rate of the ET. The CDNP modification of lysine residues at the interaction domain significantly decreases the rate, demonstrating the importance of the local charge environment in determining the rate of ET.

Introduction The electron transfer (ET) properties of proteins immobilized on the electrode strongly depend on their orientation on electrode surface.1-9 Understanding the factors that determine the orientation and conformation of proteins adsorbed on the electrode surfaces is, hence, important to optimize the ET properties of adsorbed proteins for biotechnological applications such as biosensors, biochips, bioreactors, and affinity chromatography. Self-assembled monolayers (SAMs) of thiol compounds are useful platforms for studying the above factors, because the interaction between proteins and SAM surfaces that have a large impact on the orientation and conformation of adsorbed proteins can be controlled by changing the headgroups of a SAM. In fact, recent studies with the aid * To whom correspondence should be addressed. E-mail: [email protected] (S.-i.I.); [email protected] (T.K.). † Yokohama National University. ‡ Kyoto University. (1) Tarlov, M. J.; Bowden, E. F. J. Am. Chem. Soc. 1991, 113, 18471849. (2) Cullison, J. K.; Hawkridge, F. M.; Nakashima, N.; Yoshikawa, S. Langmuir 1994, 10, 877-882. (3) Dick, L. A.; Haes, A. J.; Van Duyne, R. P. J. Phys. Chem. B 2000, 104, 11752-11762. (4) Chi, Q.; Zhang, J.; Nielsen, J. U.; Friis, E. P.; Chorkendorf, I.; Canters, G. W.; Andersen, J. E. T.; Ulstrup, J. J. Am. Chem. Soc. 2000, 122, 4047-4055. (5) Chi, Q.; Zhang, J.; Andersen, J. E. T.; Ulstrup, J. J. Phys. Chem. B 2001, 105, 4669-4679. (6) Haas, A. S.; Pilloud, D. L.; Reddy, K. S.; Babcock, G. T.; Moser, C. C.; Blasie, J. K.; Dutton, P. L. J. Phys. Chem. B 2001, 105, 1135111362. (7) Hansen, A. G.; Biosen, A.; Nielsen, J. U.; Wackerbarth, H.; Chorkendorf, I.; Andersen, J. E. T.; Zhang, J.; Ulstrup, J. Langmuir 2003, 19, 3419-3427. (8) Heering, H. A.; Wiertz, F. G.; Dekker, C.; de Vries, S. J. Am. Chem. Soc. 2004, 126, 11103-11112. (9) Imabayashi, S.; Mita, T.; Kakiuchi, T. Langmuir 2005, 21, 14701474.

of molecular dynamics simulation10,11 and surfaceenhanced infrared difference absorption spectroscopy12 revealed that the difference in the headgroup of the SAM gives rise to the change in the orientation of cytochrome c (cyt c) adsorbed on SAMs. Due to the large molecular dipole moment (325 D for ferric horse heart cyt c)13 and the clustering of cationic lysine residues on the heme edge, cyt c binds electrostatically to negatively charged SAM-modified electrode surfaces9,14-23 in a uniform orientation.3,24 Such a highly oriented protein monolayer is useful for studying the dependence of the ET of redox-active proteins on their orientation. The properties of ET of cyt c electrostatically adsorbed on the SAMs are found to be significantly influenced by the negative charge on the SAM surface, (10) Tobias, D. J.; Mar, W.; Blasie, J. K.; Klein, M. L. Biophys. J. 1996, 71, 2933-2941. (11) Nordgren, C. E.; Tobias, D. J.; Klein, M. L.; Blasie, J. K. Biophys. J. 2002, 83, 2906-2917. (12) Ataka, K.; Heberle, J. J. Am. Chem. Soc. 2004, 126, 9445-9457. (13) Koppenol, W. H.; Margoliash, E. J. Biol. Chem. 1982, 257, 44264437. (14) Collinson, M.; Bowden, E. F.; Tarlov, M. J. Langmuir 1992, 8, 1247-1250. (15) Song, S.; Clark, R. A.; Bowden, E. F.; Tarlov, M. J. J. Phys. Chem. 1993, 97, 6564-6572. (16) Nahir, T. M.; Bowden, E. F. J. Electroanal. Chem. 1996, 410, 9-13. (17) Clark, R. A.; Bowden, E. F. Langmuir 1997, 13, 559-565. (18) Bowden, E. F. Electrochem. Soc. Interface 1997, 6, 40-44. (19) Feng, Z. Q.; Imabayashi, S.; Kakiuchi, T.; Niki, K. J. Chem. Soc., Faraday Trans. 1997, 93, 1367-1370. (20) Imabayashi, S.; Mita, T.; Iida, M.; Feng, Z. Q.; Niki, K.; Kakiuchi, T. Denki Kagaku 1997, 65, 467-470. (21) Arnold, S.; Feng, Z. Q.; Kakiuchi, T.; Knoll, W.; Niki, K. J. Electroanal. Chem. 1997, 438, 91-97. (22) Knoll, W.; Pirwitz, G.; Tamada, K.; Offenha¨usser, A.; Hara, M. J. Electroanal. Chem. 1997, 438, 199-205. (23) Kasmi, A. E.; Wallace, J. M.; Bowden, E. F.; Binet, S. M.; Linderman, R. J. J. Am. Chem. Soc. 1998, 120, 225-226. (24) Du, Y.-Z.; Saavedra, S. S. Langmuir 2003, 19, 6443-6448.

10.1021/la047447w CCC: $30.25 © 2005 American Chemical Society Published on Web 02/15/2005

Redox Reaction of Adsorbed Cytochrome c

which can be altered by the solution pH9,21,25 and the composition of mixed SAM systems.9,23,26 The dependence of the ET rate of cyt c on the negative surface charge density is in line with a recent molecular simulation study,27 which predicts that the orientation and bioactivity of cyt c electrostatically adsorbed on carboxyl-terminated SAMs are affected by the degree of dissociation of the carboxyl groups. Despite these foregoing studies, however, little is known about the detailed mechanism of how the orientation and ET properties of cyt c adsorbed on the SAMs are affected by the charged state of cyt c. This is in contrast with the fact that the effect of the substitution of the charged amino acid residues with neutral or oppositely charged residues has been studied in detail for the ET reactions between cyt c and its redox partners by using site-directed mutagenesis28-32 and chemical modification.13,33-35 The single lysine substitution with 4-chloro-2,5-dinitrobenzoic acid (CDNP) little affected the rate of the heterogeneous ET of cyt c dissolved in the solution at 4,4′-bipyridyl disulfide modified gold electrodes.36-38 Niki et al. recently investigated the ET kinetics of several mutants of rat cyt c immobilized on COOH-terminated alkanethiol SAMs focusing on the binding site of cyt c to the carboxylate terminus.39,40 They proposed that lysine 13 facilitates the most efficient ET pathway to the SAM carboxylate terminal. However, the effect of the change in the surface charge distribution on the orientation and ET properties of cyt c electrostatically bound to COOH-terminated SAMs on electrodes is still far from being fully understood. In the present work, we examined the effect of the change in the electrostatic properties on the ET reactions for mono-CDNP-substituted cyt c (mCDNPc) electrostatically immobilized on a SAM of 7-mercaptoheptanoic acid (MHA) by cyclic voltammetry (CV) and potential modulated UV-vis reflectance spectroscopy (also called electroreflectance spectroscopy, ERS). We herein report that the ET rate depends strongly on the position of the CDNPsubstituted lysine residue and can be correlated with the change in the dipole moment due to the substitution. (25) Avila, A.; Gregory, B. W.; Niki, K.; Cotton, T. M. J. Phys. Chem. B 2000, 104, 2759-2766. (26) Chen, X.; Ferrigno, R.; Young, J.; Whitesides, G. M. Langmuir 2002, 18, 7009-7015. (27) Zhou, J.; Zheng, J.; Jiang, S. J. Phys. Chem. B 2004, 108, 1741817424. (28) Qian, W.; Sun, Y. L.; Wang, Y. H.; Zhuang, J. H.; Xie, Y.; Huang, Z. X. Biochemistry 1998, 37, 14137-14150. (29) Burrows, A. L.; Guo, L. H.; Hill, H. A. O.; McLendon, G.; Sherman, F. Eur. J. Biochem. 1991, 202, 543-549. (30) Cutler, R. L.; Davies, A. M.; Creighton, S.; Waeshel, A.; Moore, G. R.; Smith, M.; Mauk, A. G. Biochemistry 1989, 28, 3188-3197. (31) Northrup, S. H.; Thomasson, K. A.; Miller, C. M.; Barker, P. D.; Eltis, L. D.; Guillemette, J. G.; Inglis, S. C.; Mauk, A. G. Biochemistry 1993, 32, 6613-6623. (32) Do¨pner, H. S.; Hildebrandt, P.; Rosell, F. I.; Mauk, A. G.; von Walter, M.; Buse, G.; Soulimane, T. Eur. J. Biochem. 1999, 261, 379391. (33) Rush, J. D.; Koppenol, W. H. Biochim. Biophys. Acta 1988, 936, 187-198. (34) Osheroff, N.; Borden, D.; Koppenol, W. H.; Margoliash, E. J. Biol. Chem. 1980, 255, 1689-1697. (35) Speck, S. H.; Koppenol, W. H.; Dethmers, J. K.; Osheroff, N.; Margoliash, E.; Rajagopalan, K. V. J. Biol. Chem. 1981, 256, 73947400. (36) Hill, H. A. O.; Page, D. J.; Walton, N. J.; Whitford, D. J. Electroanal. Chem. 1985, 187, 315-324. (37) Hill, H. A. O.; Whitford, D. J. Electroanal. Chem. 1987, 235, 153-167. (38) Tominaga, M.; Hayashi, K.; Taniguchi, I. Anal. Sci. 1992, 8, 829-836. (39) Niki, K.; Pressler, K. R.; Sprinkle, J. R.; Li, H.; Margoliash, E. Russ. J. Electrochem. 2002, 38, 63-67. (40) Niki, K.; Hardy, W. R.; Hill, M. G.; Li, H.; Sprinkle, J. R.; Margoliash, E.; Fujita, K.; Tamimura, R.; Nakamura, N.; Ohno, H.; Richards, J. H.; Gray, H. B. J. Phys. Chem. B 2003, 107, 9947-9949.

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Experimental Section Reagents. MHA was synthesized from 7-bromoheptanenitrile.41,42 Horse heart cyt c (Sigma type VI, horse heart) was purified using cation exchange column (Whatman CM-52) chromatography following the method proposed by Margoliash43 and stored at 4 °C. 4-Chloro-2,5-dinitrobenzoic acid (CDNB) was purchased from Aldrich, recrystallized from benzene, and dried under reduced pressure. Water was distilled and purified with a Milli-Q system (Millipore Co.). All other chemicals were of reagent grade and used without further purification. Gold substrates were prepared by vapor deposition of gold (99.99% purity) onto freshly cleaved mica.44 Preparation of Mono-CDNP Cytochromes c (mCDNPc’s) by Chemical Modification. The method for preparing mCDNPc’s is well established.37,43,45,46 Briefly, a cyt c solution was prepared by dissolving the purified cyt c in 0.2 mol dm-3 sodium carbonate buffer at pH 9. A CDNB solution was prepared by dissolving the recrystallized CDNB into about 10% of the final reaction volume of 0.2 mol dm-3 sodium carbonate buffer and titrating back to pH 9.0 with 1 mol dm-3 NaOH. The solutions of CDNB and cyt c were mixed and diluted with 0.2 mol dm-3 sodium carbonate buffer so that the final concentrations of cyt c and CDNB were 1 and 5 mmol dm-3, respectively. The reaction of CDNB with lysine residues on the cyt c surface was allowed to proceed at room temperature for 11 h under a nitrogen atmosphere. After potassium ferricyanide was added to the solution to make cyt c fully oxidized, cyt c was promptly separated from excess CDNB and its hydrolysis byproduct by column chromatography on Bio-Gel P-4 (BioRad Co.). The 0.2 mol dm-3 carbonate buffer for CDNP-substituted cyt c was replaced with 25 mmol dm-3 sodium phosphate buffer at pH 7.8 using ultrafiltration. The modified protein mixture oxidized with ferricyanide was chromatographed on a column of CM-cellulose (Whatman CM-52) using a linear gradient between 1 L each of 25 and 150 mmol dm-3 sodium phosphate (pH 7.8) and was separated into multi-, di-, and mono-CDNP-substituted and unreacted native cyt c species depending on the number of substituted lysine. The number of substituted CDNP per cyt c molecule determined spectroscopically according to the method of Margoliash et al.45 ranges from 1.0 to 1.3 for the four major fractions A, B, C, and D where the mCDNP substitution was expected. This indicates that these fractions mainly contain mCDNPc’s, and these four fractions were further separated into individual mCDNPc’s using Margoliash’s method.46 Comparing the elution profile and the order of elution for the components in each fraction A, B, C, or D with those in the previous study,46 we identified the mono-CDNP-lysine 13, 72, 86, 87, 8, 27, 73, 7, 25, 39, 60, and 99 cytochromes c. The detailed procedure for the separation of mCDNPc’s from the four major fractions is given in the Supporting Information. Sample Preparation and Electrochemical Measurements. The single-component SAM of MHA was formed by placing a gold substrate into a 1 mmol dm-3 ethanolic solution of MHA for 20 h. The substrate was rinsed with ethanol and then dried in air. A small amount of 10 mmol dm-3 sodium phosphate buffer (pH 7) containing 200 µmol dm-3 mCDNPc or native cyt c was placed on the surface of the thiol-modified gold substrate. After storing the substrate for 1 h at 4 °C, loosely adsorbed cyt c was removed from the substrate by rinsing the substrate repeatedly with the phosphate buffer. The cell configurations for evaporated gold substrates used in the electrochemical measurements have been described previously.19,44 An ET rate constant for cyt c, ket, adsorbed on the SAM was evaluated by the frequency (41) Troughton, E. B.; Bain, C. D.; Whitesides, G. M.; Nuzzo, R. G.; Allara, D. L.; Porter, M. D. Langmuir 1988, 4, 365-385. (42) Organic Syntheses Collective Vol. I, 2nd ed.; Gilman, H., Ed.; John Wiley & Sons: New York, 1967; pp 131-132. (43) Brautigan, D. L.; Ferguson-Miller, S.; Margoliash, E. Methods Enzymol. 1978, 53D, 128-165. (44) Imabayashi, S.; Iida, M.; Hobara, D.; Feng, Z. Q.; Niki, K.; Kakiuchi, T. J. Electroanal. Chem. 1997, 428, 33-38. (45) Brautigan, D. L.; Ferguson-Miller, S.; Margoliash, E. J. Biol. Chem. 1978, 253, 130-139. (46) Osheroff, N.; Brautigan, D. L.; Margoliash, E. J. Biol. Chem. 1980, 255, 8245-8251.

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Figure 2. Midpoint potentials and the amounts of electroactive protein of native and 12 mono-CDNP-substituted cytochromes c adsorbed on HOOC-(CH2)6-SH modified Au electrodes determined by CV. The order on the horizontal axis is based on the order of elution from the column of CM-cellulose for mCDNP-cyt c.

Figure 1. Cyclic voltammograms of native (a) and four monoCDNP-substituted (b-e) cytochromes c adsorbed on HOOC(CH2)6-SH modified Au electrodes in 0.01 mol dm-3 sodium phosphate buffer at pH 7: initial potential, +0.3 V; sweep rate, 20, 50, 100, and 200 mV s-1; electrode area, 0.5 cm2. The lysine residue at position 72 (b), 87 (c), 7 (d), or 60 (e) was substituted by CDNP. dependence of the ER response according to the procedure described previously.47,48 The ER response was measured in the frequency range between 4 and 300 Hz with a potential modulation of 50 mV at the potential and wavelength where the maximum ER response was obtained (typically at the midpoint potential of CV and 419 nm, respectively). All electrochemical measurements of cyt c were performed at 25 ( 2 °C in deaerated 10 mmol dm-3 sodium phosphate buffer. The ionic strength was adjusted to 30 mmol dm-3 by adding an appropriate amount of NaCl. The potential, E, was referred to a Ag|AgCl|saturated KCl electrode.

Results and Discussion Redox Properties of mCNDPc’s. Figure 1 shows cyclic voltammograms of native and four mono-CDNPlysine 72, 87, 7, and 60 cytochromes c adsorbed on the SAM of MHA. A pair of CV peaks appeared around 0 V and the peak current was proportional to the sweep rate in all cyclic voltammograms as expected for the CV response of adsorbed species.49 The R, β, and Soret bands appeared at 550, 520, and 420 nm, respectively, in the ER (47) Sagara, T.; Igarashi, S.; Sato, H.; Niki, K. Langmuir 1991, 7, 1005-1012. (48) Feng, Z. Q.; Sagara, T.; Niki, K. Anal. Chem. 1995, 67, 35643570. (49) Laviron, E. J. Electroanal. Chem. 1974, 52, 395-402.

spectra (data not shown) of mCDNPc’s adsorbed on the SAMs of MHA, which agree well with the difference absorption spectrum between the reduced and oxidized states of cyt c.50 This means that the structure around the heme of electroactive cyt c was preserved after the CDNP modification and the adsorption on the SAM. The midpoint potential (E1/2) and the amount of electroactive cyt c (Γ) obtained from cyclic voltammograms are shown for 12 mCDNPc’s in Figure 2a,b, respectively. The E1/2 values of mCDNPc’s varied from -15 to 10 mV, which are more negative than that of native cyt c (20 mV), but there is no clear dependence of the magnitude of the potential shift on the position of the modified lysine residue. The change in the net charge of -2 caused by the monosubstitution of lysine with CDNP should stabilize the ferric state.51-53 This seems to explain, qualitatively, the negative shift of E1/2, although it is reported that the significant negative shift of redox potential was not observed for the single lysine substitution of cyt c in the previous studies.37,38,54-56 The Γ values of mCDNPc’s and (50) Hinnen, C.; Nguyen van Huong, C.; Rousseau, A.; Dalbera, J. P. J. Electroanal. Chem. 1979, 95, 131-146. (51) Moore, G. R.; Pettigrew, G. W. In Cytochromes c: Evolutionary, Structural, and Physicochemical Aspects; Springer-Verlag: Berlin, 1990; p 324. (52) Aviram, I.; Myer, Y. P.; Schejter, A. J. Biol. Chem. 1981, 256, 5540-5544. (53) Smith, H. T.; Staudenmeyer, N.; Millett, F. Biochemistry 1977, 16, 4971-4974. (54) Staudenmeyer, N.; Smith, M. B.; Smith, H. T.; Spies, F. K.; Millett, F. Biochemistry 1976, 15, 3198-3205. (55) Staudenmeyer, N.; Ng, S.; Smith, M. B.; Millett, F. Biochemistry 1977, 16, 600-604. (56) Brautigan, D. L.; Ferguson-Miller, S.; Margoliash, E. J. Biol. Chem. 1978, 253, 140-148.

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Figure 3. ET rate constants of native and 12 mono-CDNPsubstituted cytochromes c adsorbed on HOOC-(CH2)6-SH modified Au electrodes estimated by ERS. The order on the horizontal axis is based on the order of elution from the column of CM-cellulose for mCDNP-cyt c.

native cyt c are in the range between 1.3 × 10-11 and 1.7 × 10-11 mol cm-2. These values correspond to the calculated theoretical maximum coverage of cyt c, 1.5 × 10-11 mol cm-2,57 suggesting that the adsorbability of cyt c was little affected by the CDNP modification. ET Rates between mCNDPc and Electrodes. The rate constants for the ET between cyt c and the electrode (ket) estimated by ERS48 are represented in Figure 3 for native cyt c and all mCDNPc’s obtained. The ket for native cyt c adsorbed on the SAM of MHA is 1170 ( 240 s-1, which is comparable to the value, 1000 s-1, reported for a native cyt c/SAM system with a similar monolayer thickness.19,25 The ket values for mCDNPc’s in which the modified lysine was on the nonbonding domain of cyt c (lysine 39, 60, or 99) were close to that of native cyt c. In contrast, the ket values of other CDNP derivatives in which one of lysine residues surrounding the heme crevice was modified were less than half of the value for native cyt c. It is interesting that the modification of the lysine residue which is located in the closest proximity to the heme edge (lysine 13), which is the area where the ET is most likely to occur, did not result in a lower ET rate compared with the modification of the lysine residue on the periphery of the heme (lysine 8, 27, 73, 86, or 87). This suggests that the change in the ET rate is not simply attributable to the local electrostatic effect. In fact, Margoliash et al. reported that the differences in the electrostatic potential in front of the heme edge were small among the mCDNPc’s and were not correlated with the relative reactivity of the mCDNPc’s for the protein reaction partners.13 The dipole moments of the mCDNPc’s differ from that of native cyt c by as much as 140 D in magnitude and 45° in direction13 due to the CDNP modification at a different point on the cyt c surface. For the direction of the dipole, the intersection of the dipole moment vector with the protein surface for mCDNPc’s is shifted from that for native cyt c (the intersection is shown by “N”) as shown in Figure 4.33 Zhou et al. demonstrated by a combined Monte Carlo and molecular dynamics simulation approach that the direction of the dipole of cyt c determines the final orientation of adsorbed cyt c on the carboxylterminated SAMs, whose degree of dissociation is 50%, in such a way that the dipole vector points toward the SAM surface.27 This means that the mCDNPc having a highly displaced dipole moment should have an orientation considerably different from that of native cyt c on a (57) Fedurco, M. Coord. Chem. Rev. 2000, 209, 263-331.

Figure 4. Schematic representation of the front surface of ferrycytochrome c: the shade represents the porphyrin plane, the numbers within the dashed circle are the R-carbon position of lysine residues, and the bare numbers show the intersections of the dipole moment vectors with the protein surface for CDNP derivatives where a lysine of the indicated number is substituted. This figure is based on Figure 1 in ref 33.

carboxyl-terminated SAM. The alteration of the dipole moment vector due to the lysine modification should result in the change in the adsorbed orientation, leading to the change in the ET rate between the adsorbed cyt c and the electrode. In the case of the modification of the back surface lysine residues (39, 60, and 99) where the displacement of the dipole vector is slight,13,33 the reduction in the ET rate tended to be small as shown in Figure 3. Relationship between the Dipole Orientation and the ET Rate of mCDNPc. In the dynamic docking58,59 and gating60 models that successfully explained the interprotein ET reactions, it is considered that the orientation of the protein-protein complex that is optimal for the binding is not always optimal for the ET. If this is the case for the ET of cyt c electrostatically adsorbed on carboxyl-terminated SAMs, the reorientation from the adsorbed orientation to the position optimal to ET requires the rotation of the dipole of cyt c in an external electric field generated by the negative charge on the SAM surface. This reorientation obviously needs the activation for ET. To examine the relationship between the dipole moment and the ET rate for mCDNPc, we used the model13 which successfully explained the rate of ET between cyt c reductase, cyt c oxidase, or cyt c peroxidase and the CDNPderivatized cyt c in which one of the lysines outside of the interaction domain (except lysines 13, 27, 72, 86, and 87) was substituted. The work (U) needed to rotate the dipole of the mCDNPc through an angle θ in an external electric field (E) to achieve the orientation at which the ET can occur is given by13

U ) µE(1 - cosθ)

(1)

where µ is the dipole moment of the mCDNPc and θ is the angle of the dipole moment of mCDNPc between the adsorbed orientation and the orientation at which the (58) Liang, Z.-X.; Nocek, J. M.; Huang, K.; Hayes, R. T.; Kurnikov, I. V.; Beratan, D. N.; Hoffman, B. M. J. Am. Chem. Soc. 2002, 124, 6849-6859. (59) Ren, Y.; Wang, W.-H.; Wang, Y.-H.; Case, M.; Qian, W.; McLendon, G.; Huang, Z.-X. Biochemistry 2004, 43, 3527-3536. (60) Hoffman, B. M.; Ratner, M. A. J. Am. Chem. Soc. 1987, 109, 6237-6243.

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Figure 5. Relative ET rate constants of 12 mono-CDNPsubstituted cytochromes c adsorbed on HOOC-(CH2)6-SH modified Au electrodes plotted as a function of the value of µ(1 - cos θ).

ET occurs. An increase in the activation energy (Ea) by an amount U would decrease the ET rate. If the preexponential factor A is the same, the relative activity, r, which is defined as the ratio of ET rate constants ket/ket (at the optimum orientation), is given by

r ) Ae-(Ea+U)/kT/Ae-Ea/kT ) e-U/kT

(2)

where k is the Boltzmann constant. Figure 5 shows the ln r vs µ(1 - cos θ) for 12 mCDNPc’s adsorbed on MHA-modified gold electrodes. The dipole moment of mCDNP- or native-horse heart ferro cyt c and the angle between the dipole vectors of mCDNP- and native-ferro cyt c13 were used as the µ and θ values, respectively. The large deviation from the linear dependence is observed for the mCDNPc’s modified at lysine 13, 27, 72, 73, or 86 that are located around the exposed heme edge. A recent study using surface-enhanced IR difference absorption spectroscopy revealed that lysines 13, 27, 72, and 86 are involved in the binding site of native cyt c to the carboxyl-terminated SAM.12 The CDNP modification at the interaction domain of cyt c with the SAM would also have short-range effects such as local charge environment and steric hindrance, leading to an ET rate that is too low to be explained only by the change in dipole moment. For the mCDNPc’s where one of lysine residues outside the binding site is substituted, the linear relationship between ln r and µ(1 - cos θ) was obtained as indicated by the dotted line in Figure 5, supporting the applicability of the above model to the present cyt c|MHA SAM|Au systems. The electric field strength, E, which cyt c molecules experience on the surface of the MHA SAM was estimated to be 1.4 × 107 V m-1 from the slopes of the dotted line using eq 1. This value is comparable to the magnitude of the apparent electric fields of the enzyme reaction partners of cyt c such as cyt c oxidase, cyt c reductase, cyt c peroxidase, and sulfite oxidase (0.9-3.9 × 107 V m-1).13 Effect of External Electric Field Generated by the Electrode. The electric field experienced by the adsorbed cyt c consists of two components: (1) a local electric field at the binding domain built up by the array of carboxylate groups at the SAM surface and (2) an external electric field generated by the metal electrode. Kuznetsov et al. suggested the effectiveness of the latter to orient cyt c molecules at glassy carbon electrodes.61 In agreement with the direction of the dipole axis of cyt c, a negative electrode potential with respect to the potential of zero charge (pzc) orients cyt c molecules such that the ET between cyt c and (61) Kuznetsov, B. A.; Byzova, N. A.; Shumakovich, G. P. J. Electroanal. Chem. 1994, 371, 85-92.

the electrode favorably occurs, whereas a potential more positive than the pzc promoted an electrochemically inactive orientation.61 Pope and Buttry determined the change in the electric field magnitude at the Au|dodecanethiol SAM|electrolyte interface to be in the range of 106-107 V m-1 per volt in the potential range between -0.6 and +0.2 V vs saturated calomel electrode (SCE) from the Stark shifts of the fluorescence from a cationic probe immobilized in the SAM.62 This means that the electric field at a given potential depends on the potential difference between the potential and the pzc. The pzc of the alkanethiol-adsorbed gold electrode was reported to be in the range between -0.3 and -0.5 V vs Ag|AgCl|saturated KCl, whereas a OH- or COOH-terminated SAM on gold has a more positive pzc.63-67 As the potential difference between the pzc of the MHA-modified gold electrode and the redox potential of cyt c should be less than 0.5 V, the strength of the external electric field at the adsorbed site of cyt c on the SAM of MHA around the midpoint potential of cyt c should be in the range of 105-106 V m-1. This is about an order of magnitude smaller than the value obtained from Figure 5. It is considered that the local electric field originating from carboxylate groups at the SAM surface plays a more important role in determining the orientation and ET properties of cyt c adsorbed on 7-MHA-modified Au(111) electrodes under the present conditions. Comparison of the Effect of the CDNP Modification on the ET of Cyt c Adsorbed on Electrodes with That of Cyt c Dissolved in Buffer. Hill et al.36,37 and Tominaga et al.38 reported that when cyt c was dissolved in solution the single lysine substitution with CDNP little affected the rate of the heterogeneous ET at 4,4′-dithiodipyridine (DTDP)-modified gold electrodes. The observed ET rate constant of cyt c can be expressed as the product of the unimolecular ET rate and the association constant between cyt c and DTDP on the electrode surface. They attributed the small change in the heterogeneous ET rates to the absence of precise donor/acceptor compatibility (cyt c/DTDP compatibility), which is normally a major factor in regulating interprotein ET. In contrast, the previous39,40 and the present works revealed that the single lysine substitution by site-directed mutagenesis or chemical modification reflected in the ET rate of cyt c electrostatically adsorbed on carboxyl-terminated SAM-modified electrodes. This difference in the effect of the lysine substitution between adsorbed cyt c and cyt c dissolved in a buffer solution probably comes from the fact that the possible rate-limiting step, either the mass transfer or adsorption/ desorption processes, is excluded in the diffusionless voltammetry for adsorbed cyt c systems. Ataka and Heberle recently reported that the structure of adsorbed cyt c on a SAM of DTDP is different from that on the carboxyl-terminated SAM,12 indicating that the pyridine moiety interacts with cyt c in a completely different manner than lysine binding sites. This is another possible reason for the different effect of the lysine substitution on the ET. (62) Pope, J. M.; Buttry, D. A. J. Electroanal. Chem. 2001, 498, 7586. (63) Sondag-Huethorst, J. A. M.; Fokkink, L. G. J. Langmuir 1992, 8, 2560-2566. (64) Becka, A. M.; Miller, C. J. J. Phys. Chem. 1992, 97, 6233-6239. (65) Shimazu, K.; Hashimoto, Y.; Kawaguchi, T.; Toda, K. J. Electroanal. Chem. 2002, 534, 163-169. (66) Kakiuchi, T.; Usui, H.; Hobara, D.; Yamamoto, M. Langmuir 2002, 18, 5231-5238. (67) Iwami, Y.; Hobara, D.; Yamamoto, M.; Kakiuchi, T. J. Electroanal. Chem. 2004, 564, 77-83.

Redox Reaction of Adsorbed Cytochrome c

Conclusion The charge inversion of the single positive charge of surface lysine residues by mono-CDNP-derivatization of cyt c leads to the decrease in the rate of ET for all CDNP derivatives adsorbed on MHA-modified Au(111) electrodes. The magnitude of the decrease in ket depends on the position of the CDNP substitution. The CDNP modification of lysine residues around the heme edge significantly decreases the rate, demonstrating the importance of local charge environment in determining the rate of ET. For other lysine residues outside of the interaction domain of cyt c with the SAM, the effect of CDNP derivatization is explained by the model in which the dipole moment vector of CDNP-cyt c determines the adsorbed orientation and the ET rate. The ratio of the ket of mCDNPc to that of

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native cyt c is correlated to the change in the dipole moment vector of cyt c due to the CDNP modification. Acknowledgment. This work was supported by a Grant-in-Aid for Scientific Research (No. 14205120) and a Grant-in-Aid for Exploratory Research (No. 15655008) (T.K.). This work was partly supported by the Casio Science Promotion Foundation (S.I.). Supporting Information Available: Procedure for the separation of mCDNPc’s from the four major fractions. This material is available free of charge via the Internet at http://pubs.acs.org. LA047447W