Separation of Electron-Transfer and Coupled Chemical Reaction Components of Biocatalytic Processes Using Fourier Transform ac Voltammetry Barry D. Fleming, Jie Zhang, and Alan M. Bond* School of Chemistry, Monash University, Clayton, Victoria 3800, Australia Stephen G. Bell and Luet-Lok Wong Inorganic Chemistry Laboratory, University of Oxford, Oxford OX1 3QR, U.K. The underlying electron-transfer and coupled chemical processes associated with biologically important catalytic reactions can be resolved using a combination of Fourier transform ac voltammetry with an analysis of the sepa- rated dc and ac components. This outcome can be achieved because the response associated with generation of the catalytic current is essentially confined to the steady- state dc component, whereas the electron-transfer step is dominant in the fundamental and higher harmonics. For the mediated oxidation of glucose with glucose oxi- dase, it was found that the underlying reversible redox chemistry of the mediator, ferrocenemonocarboxylic acid, as detected in the third and higher harmonics, was totally unaffected by introduction of the catalytic process. In contrast, for the catalytic reduction of molecular oxygen by cytochrome P450, slight changes in the P450 redox process were detected when the catalytic reaction was present. Simulations of a simple catalytic reaction scheme support the fidelity of this novel FT ac voltammetric approach for examining mechanistic nuances of catalytic forms of electrochemical reaction schemes. Enzymes are known to catalyze a wealth of important reactions within mammals, insects, plants, yeasts, and bacteria. For example, cytochrome P450 enzymes, which have been isolated from all the above sources, catalyze hydroxylations, epoxidations, sulfoxida- tions, dehalogenations, and many other reactions. 1 Many of these enzymatic reactions involve redox processes, whereby the enzyme may undergo very fast electron-transfer processes. In the elec- trochemical context, a simple example of a catalytic process is summarized by eqs 1 and 2 where a reversible electron-transfer process that takes place at an electrode (eq 1) is coupled with an irreversible homogeneous chemical reaction (eq 2) that takes place in the solution phase adjacent to the electrode surface. Reaction of the nonelectroactive species, Z, with the oxidized species, B, results in the regeneration of the reduced species, A. If Z is in a significant concentration excess relative to B, then pseudo-first-order kinetics can be assumed. 2 Voltammetric studies of biocatalytic processes have been used to probe the nature of the fundamental electron-transfer properties of the enzyme active site, including effects of substrate binding. Such processes, or related ones, have also been exploited in development of electrochemically based bioreactors or biosen- sors. 3 The electroactive components in these catalytic processes can be freely diffusing in the solution phase, or they can be confined to the electrode surface. The classical case of an electrochemical biosensor application is associated with the mediated oxidation of glucose with glucose oxidase (GOx). Typically, a water-soluble ferrocene derivative such as ferrocenemonocarboxylic acid (FMCA) is used as a mediator. 4 This mediated reaction can be described 5,6 by a reaction scheme of the type where GOx (ox) and GOx (red) are the oxidized and reduced forms of GOx, respectively. * Corresponding author. Fax: +61 3 9905 4597. E-mail: alan.bond@ sci.monash.edu.au. (1) Ortiz de Montellano, P. R., Ed. Cytochrome P450: Structure, Mechanism & Biochemistry, 2nd ed.; Plenum Press: New York, 1995. (2) Bard, A. J.; Faulkner, L. R. Electrochemical methods, 2nd ed.; John Wiley & Sons: New York, 2001. (3) Armstrong, F. A.; Wilson, G. S. Electrochim. Acta 2000, 45, 2623-2645. (4) Cass, A. E. G.; Davis, G.; Francis, G. D.; Hill, H. A. O. H.; Aston, W. J.; Higgins, I. J.; Plotkin, E. V.; Scott, L. D. L.; Turner, A. P. F. Anal. Chem. 1984, 56, 667-671. (5) Bartlett, P. N.; Tebbutt, P.; Whitaker, R. G. Prog. React. Kinet. 1991, 16, 55-155. (6) Bond, A. M. Broadening Electrochemical Horizons; Oxford University Press: New York, 2002. A h B + ne - (1) Z + B f A (2) 2FMCA h 2FMCA + + 2e - (3) GOx (red) + 2FMCA + f GOx (ox) + 2FMCA + 2H + (4) glucose + GOx (ox) f gluconolactone + GOx (red) (5) Anal. Chem. 2005, 77, 3502-3510 3502 Analytical Chemistry, Vol. 77, No. 11, June 1, 2005 10.1021/ac048151y CCC: $30.25 © 2005 American Chemical Society Published on Web 04/23/2005