Electron Transfer Properties and Hydrogen Peroxide, Electrocatalysis of Cytochrome c Variants at Positions 67 and 80

Replacement of the axial Met80 heme ligand in electrode-immobilized cytochrome c with a noncoordinatingAla residue and alteration of the hydrogen bonding network in the region nearby following substitution ofTyr67 were investigated as effectors of the thermodynamics and kinetics of the protein-electrode electrontransfer (ET) and the heme-mediated electrocatalytic reduction of H2O2. To this end, the voltammetry of theMet80Ala, Met80Ala/Tyr67His, and Met80Ala/Tyr67Ala variants of yeast iso-1-cytochrome c chemisorbedon carboxyalkanethiol self-assembled monolayers was measured at varying temperature and hydrogen peroxideconcentration. The thermodynamic study shows that insertion of His and Ala residues in place of Tyr67results mainly in differences in protein-solvent interactions at the heme crevice with no relevant effects onthe 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, mostlikely 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 H2O2 reduction atthe electrode, whereas wild-type cytochrome c does not. H2O2 electrocatalysis occurs with an efficientmechanism likely involving a fast catalase-like process followed by electrocatalytic reduction of the resultingdioxygen at the electrode. Comparison of Met80Ala/Tyr67His with Met80Ala/Tyr67Ala shows that the presenceof a general acid-base residue for H2O2 recognition and binding through H-bonding in the distal heme siteis a key requisite for the reductive turnover of this substrate.