Direct Electron Transfer Kinetics in Horseradish Peroxidase Electrocatalysis Rafael Andreu,* ,² Elena E. Ferapontova, ‡ Lo Gorton, § and Juan Jose Calvente* ,² Departamento de Quı ´mica Fı ´sica, UniVersidad de SeVilla, 41012-SeVilla, Spain, School of Chemistry, UniVersity of Edinburgh, Edinburgh EH9 3JJ, United Kingdom, and Department of Analytical Chemistry, Lund UniVersity, SE-221 00 Lund, Sweden ReceiVed: July 7, 2006; In Final Form: October 23, 2006 The study of direct electron transfer between enzymes and electrodes is frequently hampered by the small fraction of adsorbed proteins that remains electrochemically active. Here, we outline a strategy to overcome this limitation, which is based on a hierarchical analysis of steady-state electrocatalytic currents and the adoption of the “binary activity” hypothesis. The procedure is illustrated by studying the electrocatalytic response of horseradish peroxidase (HRP) adsorbed on graphite electrodes as a function of substrate (hydrogen peroxide) concentration, electrode potential, and solution pH. Individual contributions of the rates of substrate/enzyme reaction and of the electrode/enzyme electron exchange to the observed catalytic currents were disentangled by taking advantage of their distinct dependence on substrate concentration and electrode potential. In the absence of nonturnover currents, adoption of the “binary activity” hypothesis provided values of the standard electron-transfer rate constant for reduction of HRP Compound II that are similar to those reported previously for reduction of cytochrome c peroxidase Compound II. The variation of the catalytic currents with applied potential was analyzed in terms of the non-adiabatic Marcus-DOS electron transfer theory. The availability of a broad potential window, where catalytic currents could be recorded, facilitates an accurate determination of both the reorganization energy and the maximum electron-transfer rate for HRP Compound II reduction. The variation of these two kinetic parameters with solution pH provides some indication of the nature and location of the acid/base groups that control the electronic exchange between enzyme and electrode. Introduction Enzymatic redox centers are embedded into a protein matrix, which plays a key role in catalysis, and it also helps to isolate the redox center from adventitious reactants. The low electronic conductivity of the surrounding amino acid chains makes electron-transfer rates prohibitively slow in most orientations around the redox center. In spite of these difficulties, direct (mediatorless) electron transfer (DET) between several electrode surfaces and more than 40 redox enzymes has been observed already, 1 paving the way for the development of “third genera- tion” biosensors. 2 Beyond its elegant simplicity, analysis of the current generated in DET experiments can be exploited, under either nonturnover or catalytic conditions, to gain further insight into the mechanisms of electron transfer in biological systems. 3 Horseradish peroxidase (HRP) is a glycosylated plant per- oxidase (MW ≈ 44 kDa) that catalyzes the reduction of hydrogen peroxide by a variety of organic and inorganic cosubstrates. 4 It contains one ferriprotoporphyrin IX as the heme prosthetic group. HRP has found widespread use as a component of clinical diagnostic kits and biosensors. 5 The basic catalytic scheme involves three steps: (i) oxidation of the native ferric enzyme by hydrogen peroxide to form an oxyferryl Fe IV dO group and a porphyrin π cation radical, denoted as the Compound I intermediate, (ii) reduction of the cation radical of Compound I by a one-electron donor to give Compound II, and (iii) reduction of the oxyferryl group by a second one- electron donor molecule to revert the enzyme back to its resting ferric state. The last two reduction steps are accompanied by the uptake of two protons and the removal of a water molecule. Detailed molecular structures of these catalytic intermediates have become available recently from X-ray diffraction measure- ments. 6 Direct electron transfer of HRP adsorbed on a carbon electrode was first reported by Yaropolov et al. 7 in 1978 and, since then, it has also been observed on a variety of electrode materials. 8 However, most of these studies involve an inter- conversion between the Fe(III) and Fe(II) oxidation states of the enzyme, as can be easily inferred from the location of the DET current at rather negative potential values (typically e -0.1 V vs AgCl|Ag at pH ) 7). 8,9 Alternatively, formal potentials of the HRP Compound I/Compound II and Compound II/Fe(III) redox couples take rather positive values (close to 0.7 V vs AgCl|Ag at pH ) 7), 10,11 thus allowing for a highly efficient H 2 O 2 reduction mechanism operating at small overvoltages. Nonturnover currents associated with HRP Compound I and Compound II have not been reported thus far, and evidence for DET comes from the observation, in the presence of adsorbed HRP, of H 2 O 2 reduction currents at positive potential values that are consistent with the involvement of Compound I and Compound II as catalytic intermediates. The lack of detectable nonturnover signals is also found with other electrocatalytic enzymes, as in the case of laccases adsorbed on carbon, 12 and it is likely to be caused by the small fraction of adsorbed enzymes that meet the two basic requirements to act as a DET bioelectrocatalyst: (a) an adequate orientation that facilitates * Corresponding authors: Phone: +34-954557177. Fax: +34-954557174. E-mail: fondacab@us.es; pacheco@us.es. ² Universidad de Sevilla. ‡ University of Edinburgh. § Lund University. 469 J. Phys. Chem. B 2007, 111, 469-477 10.1021/jp064277i CCC: $37.00 © 2007 American Chemical Society Published on Web 12/19/2006