Electron Transfer Properties and Hydrogen Peroxide Electrocatalysis of Cytochrome cVariants at Positions 67 and 80.

Replacement of the axial Met80 heme ligand in electrode-immobilized cytochrome cwith 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 H2O2. To this end, the voltammetry of the Met80Ala, Met80Ala/Tyr67His, and Met80Ala/Tyr67Ala variants of yeast iso-1-cytochrome cchemisorbed 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 Eo′ 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 H2O2reduction at the electrode, whereas wild-type cytochrome cdoes not. H2O2electrocatalysis 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 H2O2recognition and binding through H-bonding in the distal heme site is a key requisite for the reductive turnover of this substrate. [ABSTRACT FROM AUTHOR]