Using Scanning Electrochemical Microscopy (SECM) to Measure the Electron-Transfer Kinetics of Cytochrome c Immobilized on a COOH-Terminated Alkanethiol Monolayer on a Gold Electrode Katherine B. Holt* Department of Chemistry, UniVersity College London, 20 Gordon Street, London WC1H 0AJ, United Kingdom ReceiVed NoVember 7, 2005 Cytochrome c was electrostatically immobilized onto a COOH-terminated alkanethiol self-assembled monolayer (SAM) on a gold electrode at ionic strengths of less than 40 mM. Scanning electrochemical microscopy (SECM) was used to simultaneously measure the electron transfer (ET) kinetics of the bimolecular ET between a solution-based redox mediator and the immobilized protein and the tunneling ET between the protein and the underlying gold electrode. Approach curves were recorded with ferrocyanide as a mediator at different coverages of cytochrome c and at different substrate potentials, allowing the measurement of k BI ) 2 × 10 8 mol -1 cm 3 s -1 for the bimolecular ET and k° ) 15 s -1 for the tunneling ET. The kinetics of ET was also found to depend on the immobilization conditions of cytochrome c: covalent attachment gave slightly slower tunneling ET values, and a mixed CH 3 /COOH-terminated ML gave faster tunneling ET rates. This is consistent with previous studies and is believed to be related to the degree of mobility of cyt c in its binding configuration and its orientation with respect to the underlying electrode surface. Introduction Cytochrome c (cyt c) is an electron-transfer protein found to be loosely associated with the inner membrane of mitochondria, where it transfers electrons between the membrane-bound cyt c reductase protein (complex III) and the cyt c oxidase (complex IV). Cyt c possesses excess positive charge due to the presence of lysine residues around its exposed heme edge, which allows it to complex with its partner proteins at anionic surface sites in a configuration that is ideal for efficient electron transfer. It is believed that cyt c is loosely bound to the negatively charged inner membrane by electrostatic attraction, allowing some degree of mobility along the membrane surface as it shuttles between the docking sites of its partner proteins. Extensive studies of the electrochemistry of cyt c are found in the literature because of its ease of preparation and stability. 1,2 Although a few studies of the direct electrochemistry of cyt c exist, 3 the protein typically adsorbs and denatures on nonmodified electrode surfaces leading to deactivation. For this reason, self- assembled monolayers (SAM) on gold electrodes are often used to provide surfaces onto which the protein will adsorb without deactivation, retaining electrochemical activity. 4 In particular, negatively charged COOH-terminated SAMs are used because at low ionic strengths the positively charged cyt c will electrostatically bind to the SAM, resulting in an adsorbed layer of electroactive protein. 5-10 The addition of a “zero length” carbodiimide coupling agent can result in the formation of amide covalent linkages between the lysines of the cyt c and the COOH termini of the SAM. 11 The ET kinetics of cyt c adsorbed on COOH-terminated SAMs of varying chain length has been studied using techniques such as cyclic voltammetry (CV), 6,7,9,11 electrochemical impedance spectroscopy (EIS), 6 and time- resolved UV-vis subtractive reflectance spectroscopy. 7,12 The effect on ET of solution pH, 13 temperature, 6 monolayer com- position, 5,9,13 and amino acid substitution on the protein has been determined, 7,9 as well as the binding technique (electrostatic vs covalent). 10,11 Values of k° were found to be dependent on the chain length of the alkanethiol. 4 In general, it is found that ET becomes slower with increasing negative charge on the surface of the SAM, as observed on increasing the pH of the solution from pH 7 to above 8. 13 An increase in the ET rate was observed on forming a mixed ML of COOH/OH-terminated alkanethiols due to a reduction in the surface negative charge. 5 Most electrochemical studies of cyt c adsorption on SAMs have used CV to extract the standard tunneling rate constant, k°, for ET between the protein and the underlying electrode. Values of k° have been determined by measuring the peak separation between the reduction and oxidation peaks using Laviron’s method. 14 However, there are experimental difficulties in extracting rate constants in this manner in the low ionic strength solutions required for the electrostatic adsorption of cyt c. For relatively fast ET, the peak separation will be too small to give a reliable indicator of k°, requiring that faster scan rates be used to increase the separation. In low ionic strength (high resistance) solutions, this creates problems with iR compensation, and the RC characteristics of many systems means that high enough scan rates cannot be reached. Additionally, when cyt c is not uniformly adsorbed on the surface different binding configurations * Corresponding author. E-mail: k.b.holt@ucl.ac.uk. (1) Hill, H. A. O.; Guo, L. H.; McLendon, G. In Cytochrome c: A Multidisciplinary Approach; Scott, R. A., Mauk, A. G., Eds.; University Science Books: Sausalito, CA, 1996; pp 317-333. (2) Hill, H. A. O. Coord. Chem. ReV. 1996, 151, 115-123. (3) Marken, F.; Paddon, C. A.; Asogan, D. Electrochem. Commun. 2002, 4, 62-66. (4) Fedurco, M. Coord. Chem. ReV. 2000, 209, 263-331. (5) El Kasmi, A.; Wallace, J. M.; Bowden, E. D.; Binet, S. M. Linderman J. Am. Chem. Soc. 1998, 120, 225-226. (6) Song, S.; Clarke, R. A.; Bowden, E. F.; Tarlov, M. J. J. Phys. Chem. 1993, 97, 6564-6572. (7) Imabayashi, S.; Mita, T.; Kakiuchi, T. Langmuir 2005, 21, 2474-2479. (8) Petrovic, J.; Clark, R. A.; Yue, H.; Waldeck, D. H.; Bowden, E. F. Langmuir 2005, 21, 6308-6316. (9) Wei, J. J.; Liu, H.; Niki, K.; Margoliash, E.; Waldeck, D. H. J. Phys. Chem. B 2004, 108, 16912-16917. (10) Jin, W.; Wollenberger, U.; Karget, E.; Schunck, W.-H.; Scheller, F. W. J. Electroanal. Chem. 1997, 433, 135-139. (11) Collinson, M.; Bowden, E. F. Langmuir 1992, 8, 1247-1250. (12) Wu, L.-L.; Huang, H.-G.; Li, J.-X.; Luo, J.; Lin, Z.-H. Chem. Lett. 1998, 1137-1138. (13) Imabayashi, S.; Mita, T.; Kakiuchi, T. Langmuir 2005, 21, 1470-1474. (14) Laviron, E. J. Electroanal. Chem. 1979, 101, 19-28. 4298 Langmuir 2006, 22, 4298-4304 10.1021/la0529916 CCC: $33.50 © 2006 American Chemical Society Published on Web 03/29/2006