4818 zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA Langmuir zyxwvu 1996,11, 4818-4822 zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLK Characterization of Mercaptoethylamine-Modified Gold Electrode Surface and Analyses of Direct Electron Transfer to Putidaredoxin Ling Sang Wongt and Vincent L. Vilker**t Department zyxwvuts of Chemical Engineering, University of California, Los Angeles, California 90024-0159 William T. Yap and Vytas Reipa Biotechnology Division, National Institute of Standards and Technology, Gaithersburg, Maryland 20899-0001 Received March 6, 1995. In Final Form: August 18,1995@ Direct electron transfer to putidaredoxin (Pdx) in solution from bare gold and mercaptoethylamine- modified gold electrodes was studied by cyclic voltammetry. The current response of zyxw Pdx at a bare gold electrode was irreversible and highly unstable. The modified electrode offered improved, but still impersistent, quasi-reversible current response. Surface-enhanced Raman spectroscopy revealed that mercaptoethylamine adsorbed on gold was in mixed trans and gauche conformations at 0.0 V (Ag/AgCl) initially. As the potential was changed anodically or cathodically over the range of zyxw +0.4 to -0.8 V, the adsorbate population became largely gauche in conformation. A link between the conformation change and deteriorationof zyxwvuts Pdx current response at the electrodewas suggested. Simulationsof the electrochemical system yielded an estimated Pdx diffusion coefficient D= 1.4 x lo-' cm2/s and the apparent heterogeneous electron transfer rate constant k" zyxwvutsrqp = 1.0 x 10-4 c d s . Introduction Studies of electron transfer (ET) in biological systems encompass fundamental problems such as molecular recognition between redox partners, conformation and dynamics of inter- and intramolecular ET, and determi- nation of thermodynamic and kinetic parameters of the ET process. Voltammetric study of redox proteins at solid electrodes has received wide attention in the last two decades due to improved functional electrodes for efficient coupling to solution pr~teins.l-~ This method has the advantage of permitting the study of a redox protein in the absence ofits physiologicalpartner. Effects of solution temperature, ionic strength, and pH on the formal potential and heterogeneous ET rate constant can also be determined by voltammetry. The success has also ben- efitted biosensor development and commer~ialization.~ We are interested in direct ET to putidaredoxin (Pdx) from a solid electrode because Pdx is critical for electron transport and catalytic turnover for cytochrome P-45OCam monooxygenase (EC 1.14.15.1, CPY101).6 P-45OC,, has been arguably the most important model system for the ubiquitous, general class of P-450 enzymes.' The potential + Current address: Biotechnology Division, National Institute of Standards and Technology, Gaithersburg, MD 20899. @ Abstract published in Advance ACS Abstracts, November 1, 1995. (1) Armstrong, F. A.; Butt, J. N.; Sucheta, A. Methods in Enzymology; Academic Press: San Diego, 1993; Vol. 227, pp 479-500. (2) Kinnear, K. T.; Monbouquette, H. G. Langmuir 1993,9, 2255. (3) King, B. C.; Hawkridge, F. M.; Taniguchi, I. InRedorMechanisms and Interfacial Properties ofMoleculesofBiologica1 Importance; Schultz, F. A,, Taniguchi, I., Eds.; The Electrochemical Society: Pennington, (4) Guo, L. H.; Hill, H. A. 0. Advances in Inorganic Chemistry; Academic Press: New York, 1991; Vol. 36, pp 341-375. (5) Cass, A. E. G., Ed. Biosensors: a practical approach; IRL Press: London. 1990. 1993; pp 56-62. (6) Lipscomb, J. D.; Sligar, S. G.; Namtvedt, M. J.; Gunsalus, I. C. (7) Omura, T., Sato, R., Fujii-Kuriyama, Y., Eds. Cytochrome P-450; J. Biol. Chem. 1976,251, 1116. Kodansha: Tokyo, 1993. technological importance of P-450 enzymes has been recognized for a long time, but it has not materialized on a wider basis partly due to their requirements of expensive biological reducing sources and multiple redox partnem8 A mercaptoethylamine-modified gold electrode is found to be effective for direct electron transfer to putidaredoxin in aqueous sol~tion.~ This implies that perhaps expensive biological electron sources for P-450cam can be replaced by direct current. The modified surface is not yet optimized for rapid ET to Pdx, however. To improve the performance of the modified electrode, a better understanding of the properties of the modifier film is a prerequisite. While long chain alkanethiols and their derivatives adsorbed on gold have been studied extensive1y,l0-l2 shorter alkyl chain compounds like mercaptoethylamine have not. The aim of this paper is to provide some fundamental understand- ing about the properties ofthe modified surface that effects ET to Pdx. Cyclic voltammograms of Pdx on bare and mercaptoethylamine-modified gold electrodes were com- pared. Kinetic parameters (heterogeneous ET rate con- stant and diffusion coefficientof Pdx) were extracted from data by simulation. Effects of changing potentials on the modifier film were investigated by surface-enhanced Raman spectroscopy (SERS). The relationship between the electrochemicalresponse of Pdx and the film properties obtained from SERS will be discussed. Experimental Section Materials. Putidaredoxin (Pdx) was purified from an Es- cherichia coli DH5a clone which was kindly supplied by Professor J. A. Peter~0n.l~ Growth and purification protocols are detailed (8) Schubert, F.; Scheller, F.; Mohr, P. Pharmazie 1985, zy 40, 233. (9) Wong, L. S.; Vilker, V. L. J. Ebctroanal. Chem., in press. (10)Rowe, G. K.; Creager, S. E. Langmuir 1994, 10, 1186. (11) Garrell, R. L.; Chadwick, J. E. Colloids Surf., A 1994, 93, 59. (12) Prime, K. L.; Whitesides, G. M. Science 1991,252, 1164. (13) Peterson, J. A.; Lorence, M. C.;Amameh, B. J. Biol. Chem. 1990, 265,6066. This article not,subject to US. Copyright. Published 1995 by the American Chemical Society