Electron Transfer Properties and Hydrogen Peroxide Electrocatalysis of Cytochrome c Variants at Positions 67 and 80 Stefano Casalini, †,‡ Gianantonio Battistuzzi, † Marco Borsari, † Carlo Augusto Bortolotti, † Giulia Di Rocco, † Antonio Ranieri, † and Marco Sola* ,†,§ Contribution from the Department of Chemistry, UniVersity of Modena and Reggio Emilia, Via Campi 183, I-41100 Modena, Italy, and CNR-INFM National Center nanoStructures and bioSystems at Surfaces - S3, Via Campi 213/A, I-41100 Modena, Italy ReceiVed: September 18, 2009; ReVised Manuscript ReceiVed: December 18, 2009 Replacement of the axial Met80 heme ligand in electrode-immobilized cytochrome c with a noncoordinating Ala residue and alteration of the hydrogen bonding network in the region nearby following substitution of Tyr67 were investigated as effectors of the thermodynamics and kinetics of the protein-electrode electron transfer (ET) and the heme-mediated electrocatalytic reduction of H 2 O 2 . To this end, the voltammetry of the Met80Ala, Met80Ala/Tyr67His, and Met80Ala/Tyr67Ala variants of yeast iso-1-cytochrome c chemisorbed on carboxyalkanethiol self-assembled monolayers was measured at varying temperature and hydrogen peroxide concentration. The thermodynamic study shows that insertion of His and Ala residues in place of Tyr67 results mainly in differences in protein-solvent interactions at the heme crevice with no relevant effects on the E o ′ values at pH 7, which for single and double variants range from approximately -0.200 to -0.220 V (vs SHE). On the contrary, both double variants show much lower ET rates compared to Met80Ala, most likely as a consequence of a change in the ET pathways. In the present nondenaturing immobilizing conditions, and with hydrogen peroxide concentrations in the micromolar range, the variants catalyze H 2 O 2 reduction at the electrode, whereas wild-type cytochrome c does not. H 2 O 2 electrocatalysis occurs with an efficient mechanism likely involving a fast catalase-like process followed by electrocatalytic reduction of the resulting dioxygen at the electrode. Comparison of Met80Ala/Tyr67His with Met80Ala/Tyr67Ala shows that the presence of a general acid-base residue for H 2 O 2 recognition and binding through H-bonding in the distal heme site is a key requisite for the reductive turnover of this substrate. Introduction Heterogeneous protein-electrode electron transfer (ET) is the fundamental event for the current and potential responses of hybrid interfaces made of self-assembled redox proteins in an electrochemical environment. These constructs can variably be exploited in nanostructured bioelectronic devices, from field effect-like transistors to biosensors. 1-7 In these systems, replace- ment of large and delicate redox enzymes with small electron- transfer proteins, as are or subjected to a tailored engineering, is an open field of investigation. 8-16 In particular, the advantages offered by modified cytochrome c (cytc hereafter) compared to a larger heme-enzyme as the core constituent of an amperometric biosensor for substrates of industrial and clinical importance (such as O 2 ,O 2 - ,H 2 O 2 , NO, CO, NO 2 - ) include: (i) an easier electrical communication between the redox center and the electrode; (ii) the covalent anchoring of the heme to the protein matrix that would prevent heme loss in hostile environments; (iii) the stability of the protein over a wide range of T and pH; and (iv) the availability of an easily tunable system to optimize performances. The problem of hydrogen peroxide biosensing is of particular interest as this molecule is directly related to the level of oxidative stress of cellular and extracellular fluids/compart- ments. 17 Catalases and peroxidases act as antioxidant protective enzymes as they get rid of hydrogen peroxide utilizing it as an oxidant for useful cellular chemistry or disproportionating it to water and dioxygen, respectively. 18,19 Therefore, their heme site is poised to recognize and turn over hydrogen peroxide. It follows that modification of the heme environment of cyto- chrome c with insertion of some key features of the above sites could lead to an efficient and robust catalytic system for the redox turnover of H 2 O 2 . 20,21 The basic changes would include removal of the sixth axial iron ligand (Met 80) to produce a five-coordinate heme center, enlargement of the distal cavity, and insertion of a distal histidine. 20 This would help H 2 O 2 recognition and binding to the heme center of cytc, and subsequent reduction to water could then be achieved with electrons either provided by a substrate in a conventional peroxidase reaction in solution or delivered through an electrode to an immobilized protein. Here we focus on the latter case, in which the protein would show a “pseudoperoxidase activity”, exploitable for amperometric H 2 O 2 biosensing. 22 In particular, our aim is to understand how the aforementioned basic changes in the heme site needed for activity affect the redox thermo- dynamics of cytochrome c and the rate of heterogeneous ET. Thus, mutations were made at the axial Met80 heme ligand, which was replaced with a noncoordinating Ala residue and at Tyr67 which was substituted with a His and Ala residue (Figure 1). Tyr67 was chosen because its spatial relationship with the heme group in cytc is similar to that of the catalytically relevant * Corresponding author. Tel.: +39 059 2055037. Fax: +39 059 373543. URL: http://155.185.2.170/sitiwebgruppi/Sola/homesolagroup2009.html. E-mail: marco.sola@unimore.it. † University of Modena and Reggio Emilia. ‡ Present address: Istituto per lo Studio dei Materiali Nanostrutturati, ISMN-CNR, Via Gobetti 101, I-40129, Bologna, Italy. § CNR-INFM National Center - S3. J. Phys. Chem. B 2010, 114, 1698–1706 1698 10.1021/jp9090365  2010 American Chemical Society Published on Web 01/08/2010