J. Phys. Chem. B 2003, 107, 9947-9949
9947
Coupling to Lysine-13 Promotes Electron Tunneling through Carboxylate-Terminated Alkanethiol Self-Assembled Monolayers to Cytochrome c Katsumi Niki,†,‡ W. Reef Hardy,‡ Michael G. Hill,‡ H. Li,§ James R. Sprinkle,| Emanuel Margoliash,§,⊥ Kyoko Fujita,†,# Ryutaro Tanimura,†,# Nobufumi Nakamura,# Hiroyuki Ohno,# John H. Richards,† and Harry B. Gray*,† Beckman Institute, California Institute of Technology, Pasadena, California 91125, Department of Chemistry, Occidental College, Los Angeles, California 90041, Department of Biological Sciences, UniVersity of Illinois at Chicago, Chicago, Illinois 60607, Department of Biological and Exercise Sciences, Northeastern Illinois UniVersity, Chicago, Illinois 60625, Department of Biochemistry, Molecular Biology and Cell Biology, Northwestern UniVersity, EVanston, Illinois 60208, and Department of Biotechnology, Tokyo UniVersity of Agriculture and Technology, Koganei, Tokyo 184-8588, Japan ReceiVed: May 20, 2003
Electrochemistry of surface-modified cytochrome c (cyt c) bound electrostatically to carboxylate-terminated alkanethiol self-assembled monolayers (SAM) reveals highly anisotropic electronic coupling across the protein/ monolayer interface. Substitution of a lysine residue with alanine at position 13 in recombinant rat cyt c (RC9-K13A) lowers the interfacial electron transfer (ET) rate more than 5 orders of magnitude, whereas ET is only slightly affected by replacement of lysine-72 or lysine-79 with alanine. The results clearly show that lysine-13 is directly involved in coupling the protein to the SAM carboxylate terminus. Interfacial ET rates for both yeast iso-1 cyt c and the mutant RC9-K13R indicate that arginine-13 couples the protein to the carboxylate interface less well than lysine-13.
Mitochondrial cytochrome c (cyt c) functions as an electron carrier both in vivo and in vitro. Extensive chemical modification of the -amino groups of lysines,1 differential protection experiments,2 and site-directed mutagenesis studies3,4 have revealed that six or seven lysine residues surrounding the cyt c heme crevice play an important role in binding interactions and electron transfer (ET) with most of its redox partners.5-9 Intraprotein electronic coupling between these residues and the heme center is of interest in the context of the relative efficiencies of different through-bond electron-tunneling pathways.10,11 We are investigating these pathways using electrochemistry at chemically modified surfaces. It is well-known that cyt c binds electrostatically to carboxylic acid-terminated alkanethiols that self-assemble onto gold (HOOC-SAMs) (Figure 1).12-19 Electrochemically triggered redox reactions through these SAMs yield ET data as a function of both driving force and distance; analysis of the data allows an estimate of the intramolecular coupling from the heme edge to specific terminal lysine residues.20 Extensive work on this system has identified several key steps in the HOOC-SAM-mediated ET process:19,20 the initial step is nonspecific association between the protein and the carboxylateterminated surface, followed by rotational diffusion on the SAM to reach the proper configuration to facilitate the ET event. (This type of mechanism was first proposed by Andrew et al. for intra* To whom correspondence should be addressed. Phone: (626)395-6500. Fax: (626)449-4159. E-mail:
[email protected]. † California Institute of Technology. ‡ Occidental College. § University of Illinois at Chicago. | Northeastern Illinois University. ⊥ Northwestern University. # Tokyo University of Agriculture and Technology.
Figure 1. Illustration of the cytochrome c/HOOC-SAM interface. The proposed Lys-13/carboxylate interaction that couples the electrode to the heme is highlighted in red. Other surface lysine residues are shown in yellow.
ET in the cyt c/cyt b5 complex, in which both cyt c and its redox partner are able to rotate about a specific surface domain.21) When intermolecular ET is limited by rotational diffusion, the rate becomes independent of the path length between the heme center and the electrode. Previous work suggests that the lysine residue at position 13 (Lys-13) of cyt c couples the heme to the carboxylic acid terminus of the HOOC-SAMs.22,23 In particular, substitution of Lys-13 with alanine in recombinant rat cyt c (RC9) lowers the electrode kinetics over 5 orders of magnitude. In the present work, we have prepared a series of mutants (RC9-K13A, K13E/ E90K, K72A, and K79A) in which potentially important lysine
10.1021/jp035392l CCC: $25.00 © 2003 American Chemical Society Published on Web 08/22/2003
9948 J. Phys. Chem. B, Vol. 107, No. 37, 2003
Figure 2. Cyclic voltammograms for RC9-K13E/E90K immobilized on a HOOC(CH2)2S/gold-bead electrode. Data were collected in 10 mM phosphate, pH 7. Scan rates: 20, 50, and 100 mV/s.
Figure 3. ET rates vs chain length (n) for cytochromes c immobilized on HOOC(CH2)nSH-SAMs.18 (b) horse heart cyt c, (blue open circle) RC9-K79A, (purple open circle) RC9-K72A, (yellow open circle) RC9K13R, (green filled circle) yeast iso-1 C102S, (red triangle) RC9-K13A, (yellow triangle) RC9-K13E/E90K. Each rate was measured at the formal potential of the protein.
residues have been systematically replaced; comparison of their electrochemical behavior with those of horse heart cyt c and RC9 has confirmed the special role of lysine at position 13. The carboxyl group (OE1) of glutamic acid at position 90 of RC9 is only 3.13 Å from the amino group (NZ) of lysine at position 13, so a mutant in which Lys-13 was replaced with glutamic acid at position 90 (RC9-K13E/E90K) was prepared to minimize the changes in dipolar properties and surface charge distributions from those of RC9. RC9-K72A and RC9-K79A were expressed to study the effects of binding site on the electrode kinetics of cyt c. Electrostatic immobilization of cyt c onto “pretreated” HOOC-SAMs was carried out according to recently developed protocols.24 Cyclic voltammograms were recorded in 10 mM phosphate buffer, pH 7. The protein-electrode ET rate constants through variable-length SAMs were measured at the formal potentials of cyt c and its mutants (∆G° ) 0) by cyclic voltammetry (CV), and were evaluated using Laviron’s formula.25 Cyclic voltammograms for RC9-K13E/E90K are shown in Figure 2. Like RC9-K13A, the formal potential of this protein is about 80 mV more negative than that of RC9. The rate constants for electron tunneling through HOOC(CH2)2SH and HOOC(CH2)5SH SAMs to RC9-K13E/E90K are 1.3 and 0.06 s-1, which are nearly the same as those for the corresponding reactions to RC9-K13A (3.2 and 0.1 s-1) (Figure 3). The configuration of RC9-K13E/E90K on HOOC-SAMs likely is the same as that of RC9, because the two proteins have similar surface-charge distributions and dipolar properties. Conse-
Letters quently, it is probable that the distance between the heme edge of RC9-K13E/E90K and the carboxylic acid-terminus is the same as the analogous spacing for RC9. Strikingly, however, the ET rate constants for both RC9-K13E/E90K and RC9-K13A are over 5 orders of magnitude smaller that that of RC9, whose ET rate is nearly the same as that of horse heart cyt c. A large difference between the linear plot for RC9 and that for K13E/ E90K shown in Figure 326 indicates that Lys-13 couples the carboxylic acid-terminus of HOOC-SAM to RC927 and that the ET process in cyt c is highly directional. Importantly, these results are fully consistent with the attenuated enzymatic activity of cyt c oxidase in the presence of chemically modified Lys-13 cyt c.1 Other potential cyt c ET coupling sites include lysines 72 and 79. Replacement of Lys-72, which interacts with the acidic headgroups of physiological redox partners, by alanine leads only to a small decrease (about 50%) in the electrode reaction rate constant, and no effect at all was observed when Lys-79 was replaced with alanine. Clearly, these positions are not the most favorable ET coupling sites, although they likely are involved in binding to physiological redox partners. Acknowledgment. The authors thank Dojindo Molecular Technology for providing carboxylic acid-terminated alkanethiols. Work at Occidental College was supported by the David and Lucille Packard Foundation’s Initiative for Interdisciplinary Research. Work at Caltech was supported by NIH DK19038, NSF, and the Arnold and Mabel Beckman Foundation. E.M., H.L., and JS acknowledge support from the Edward Mallinckrodt, Jr. Foundation. K.F. thanks the Japan Society for the Promotion of Science (Research Fellowships for Young Scientists) for support. References and Notes (1) Theodorakis, J. L.; Armes, L. G.; Margoliash E. Biochim. Biophys. Acta 1995, 1252, 114-125 and references therein. (2) Bosshard, H. R. In Cytochrome c. A Multidisciplinary Approach; Scott, R. A., Mauk, A. G., Eds.; University Science Books: Sausalito, CA, 1996; pp 373-396. (3) 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. (4) Do¨pner, S.; Hildebrandt, P.; Rosell, F. I.; Mauk, A. G.; von Walter, M.; Buse, G.; Soulimane, T. Eur. J. Biochem. 1999, 261, 379-391. (5) McLendon, G. Acc. Chem. Res. 1988, 21, 160-167. (6) Nocek, J. M.; Zhou, J. S.; DeForest, S.; Priyadarshy, S.; Beratan, D. N.; Onuchic, J. N.; Hoffman, B. M. Chem. ReV. 1996, 96, 2459-2489. (7) (a) Zhen, Y.-J.; Hoganson, C.; Babcock, G. T.; Ferguson-Miller, S. J. Biol. Chem. 1999, 274, 38032-38041. (b) Wang, K.; Zhen, Y.-J.; Sadoski, R.; Grinnell, S.; Geren, L.; Ferguson-Miller, S.; Durham, B.; Millett, F. J. Biol. Chem. 1999, 274, 38042-38050. (8) Flo¨ck, D.; Helms, V. Proteins 2002, 47, 75-85. (9) Leesch, V. W.; Bujons, J.; Mauk, A. G.; Hoffman, B. M. Biochemistry 2000, 39, 10132-10139. (10) Beratan, D. N.; Betts, J. N.; Onuchic, J. N. Science 1991, 252, 1285-1288. (11) (a) Wuttke, D. S.; Bjerrum, M. J.; Winkler, J. R.; Gray, H. B. Science 1992, 256, 1007-1009. (b) Langen, R.; Chang, I.-Jy.; Germanas, J. P.; Richards, J. H.; Winkler, J. R.; Gray, H. B. Science 1995, 268, 17331735. (c) Gray, H. B.; Winkler, J. R. Annu. ReV. Biochem. 1996, 65, 537561. (12) Tarlov, M. J.; Bowden, E. F. J. Am. Chem. Soc. 1991, 113, 18471849. (13) Collinson, M.; Bowden, E. F.; Tarlov, M. J. Langmuir 1992, 8, 1247-1250. (14) Song, S.; Clark, R. A.; Bowden, E. F.; Tarlov, M. J. J. Phys. Chem. 1993, 97, 6564-6572. (15) Kasmi, A. E.; Wallace, J. M.; Bowden, E. F.; Binet, S. M.; Linderman, R. J. J. Am. Chem. Soc. 1998, 120, 225-226. (16) Feng, Z. Q.; Imabayashi, S.; Kakiuchi, T.; Niki, K. J. Electroanal. Chem. 1995, 394, 149-154.
Letters (17) Arnold, S.; Feng, Z. Q.; Kakiuchi, T.; Knoll, W.; Niki, K. J. Electroanal. Chem. 1997, 438, 91-97. (18) Feng, Z. Q.; Imabayashi, S.; Kakiuchi, T.; Niki, K. J. Chem. Soc., Faraday Trans. 1997, 93, 1367-1370. (19) Avila, A.; Gregory, B. W.; Niki, K.; Cotton, T. M. J. Phys. Chem. B 2000, 104, 2759-2766. (20) Niki, K. Electrochemistry 2002, 70, 82-90. (21) Andrew, S. M.; Thomasson, K. A.; Northrup S. H. J. Am. Chem. Soc. 1993, 115, 5516-5521. (22) Niki, K.; Sprinkle, J. R.; Margoliash, E. Bioelectrochemistry 2002, 55, 37-40. (23) Niki, K.; Pressler, K. R.; Sprinkle, J. R.; Li, H.; Margoliash, E. Russ. J. Electrochem. 2002, 38, 74-78.
J. Phys. Chem. B, Vol. 107, No. 37, 2003 9949 (24) Tanimura, R.; Hill, M. G.; Margoliash, E.; Niki, K.; Gray, H. B. Electrochem. Solid-State Lett. 2002, 5, E67-70. (25) Laviron, E. J. Electroanal. Chem. 1979, 101, 19-28. (26) The electrode rate constants for horse heart cyt c and RC9-K13E/ E90K estimated for n ) 0 by linear extrapolation are 8.0 × 106 and 20 s-1. (27) Yeast iso-1 cyt c (C102S) has an arginine at position 13. The mutant RC9-K13R was therefore prepared and investigated electrochemically. Mutant ET rates through HOOC-SAMs are more than 100 times smaller than those of RC9 and are nearly identical with those of native yeast iso-1 cyt c (Figure 3). These results implicate position 13 as the probable ET coupling site for both RC9-K13R and yeast iso-1 cyt c, although a lysine residue at position 11 in yeast iso-1 cyt c also could be involved.
