Proton-Coupled Electron Transfer of Flavodoxin Immobilized on Nanostructured Tin Dioxide Electrodes: Thermodynamics versus Kinetics Control of Protein Redox Function Yeni Astuti, † Emmanuel Topoglidis, † Paul B. Briscoe, ‡ Andrea Fantuzzi, ‡ Gianfranco Gilardi, ‡ and James R. Durrant* ,† Contribution from the Departments of Chemistry and Biological Sciences, Imperial College London, South Kensington, London SW7 2AZ, U.K. Received January 20, 2004; E-mail: j.durrant@imperial.ac.uk Abstract: In this paper, we report a spectroelectrochemical investigation of proton-coupled electron transfer in flavodoxin D. vulgaris Hildenborough (Fld). Poly-L-lysine is used to promote the binding of Fld to the nanocrystalline, mesoporous SnO2 electrodes. Two reversible redox couples of the immobilized Fld are observed electrochemically and are assigned by spectroelectrochemistry to the quinone/semiquinone and semiquinone/hydroquinone couples of the protein’s flavin mononucleotide (FMN) redox cofactor. Comparison with control data for free FMN indicates no contamination of the Fld data by dissociated FMN. The quinone/ semiquinone and semiquinone/hydroquinone midpoint potentials (Eq/sq and Esq/hq) at pH 7 were determined to be -340 and -585 mV vs Ag/AgCl, in good agreement with the literature. Eq/sq exhibited a pH dependence of 51 mV/pH. The kinetics of these redox couples were studied using cyclic voltammetry, cyclic voltabsorptometry, and chronoabsorptometry. The semiquinone/quinone reoxidation is found to exhibit slow, potential-independent but pH-sensitive kinetics with a reoxidation rate constant varying from 1.56 s -1 at pH 10 to 0.0074 s -1 at pH 5. The slow kinetics are discussed in terms of a simple kinetics model and are assigned to the reoxidation process being rate limited by semiquinone deprotonation. It is proposed that this slow deprotonation step has the physiological benefit of preventing the undesirable loss of reducing equivalents which results from semiquinone oxidation to quinone. Introduction The chemical nature of the binding site of the redox cofactor in biological redox proteins is critical in determining the redox function of the cofactor. Differences in the protein environment can result in the same redox cofactor performing a broad range of functions, including electron or atom transfer, substrate activation and or conversion, and ligand binding. 1,2 The acid/ base properties of the binding site are a key factor in influencing this functionality. Many biological redox reactions involve the uptake or release of protons from the cofactor and/or its protein environment. It is well established that such protonation/ deprotonation events can have a strong influence on the thermodynamics of the reaction and result in a pH-dependent reaction free energy. 3 In addition to this widely established thermodynamic control of redox function, attention is increas- ingly focused on the importance of protonation/deprotonation events in influencing the kinetics of the overall redox reaction, 4-6 and thereby physiological function. One of the most ubiquitous redox cofactors employed by nature is the isoalloxazine ring or “flavin”, the prosthetic group of the large class of flavoproteins. This redox cofactor can undergo both one- and two-electron reduction, resulting in the formation of the singly reduced semiquinone and doubly reduced hydroquinone states. One or both of these reduction steps are typically directly coupled to protonation of the isoalloxazine ring. This range of redox properties allows flavoproteins to fulfill a diverse range of biochemical functions, including their functions in organic molecule hydroxylation in Class II cyto- chrome P450’s and O 2 consumption in glucose oxidase. 2 One class of flavoproteins, flavodoxins (Fld), are small (15-20 kDa) electron-transferring proteins that have the flavin mononucleo- tide (FMN) as the redox cofactor noncovalently bound to a single polypeptide. Flavodoxins are widely distributed among various types of microorganisms where they can replace ferredoxins as electron mediators for a number of biological transformations. 2,7-14 The redox functions of Fld proteins are † Department of Chemistry. ‡ Department of Biological Sciences. (1) Chapman, S. K.; Simon, D.; Munro, A. W. Struct. Bonding 1997, 88, 39- 70. (2) Muller, F. Topics in Current Chemistry; Springer, Berlin, 1981; Vol. 108, pp 71-108. (3) Krishtalik, L. I. Biochim. Biophys. Acta 2003, 1604, 13-21. (4) Brzezinski, P. Biochemistry 1996, 35, 5611-5615. (5) Rappaport, F.; Lavergne, J. Biochim. Biophys. Acta 2001, 1503, 246-259. (6) Hirst, J.; Duff, J. L. C.; Jameson, G. N. L.; Kemper, M. A.; Burgess, B. K.; Armstrong, F. A. J. Am. Chem. Soc. 1998, 120, 7085-7094. (7) Dubourdieu, M.; le Gall, J.; Favaudon, V. Biochim. Biophys. Acta 1975, 376, 519-32. (8) Simondsen, R. P.; Tollin, G. Mol. Cell. Biochem. 1980, 33, 13-24. (9) Odom, J. M.; Peck, H. D., Jr. Annu. ReV. Microbiol. 1984, 38, 551-592. (10) Thorneley, R. N. F.; Deistung, J. Biochem. J. 1988, 253, 587-595. (11) Mayhew, S. G.; Tollin, G. Chemistry and Biochemistry of FlaVoenzymes III; CRC Press: Florida, 1992; Vol. 3, pp 389-426. (12) Ludwig, M. L.; Luschinsky, C. L. Chemistry and Biochemistry of FlaVoenzymes III; CRC Press: Florida, 1992; Vol. 3, pp 427-467. (13) Setif, P. Biochim. Biophys. Acta 2001, 1507, 161-179. Published on Web 06/05/2004 10.1021/ja0496470 CCC: $27.50 © 2004 American Chemical Society J. AM. CHEM. SOC. 2004, 126, 8001-8009 9 8001