Effects of mutational (Lys to Ala) surface charge changes on the redox properties of electrode-immobilized cytochrome c

Untrimethylated yeast iso-1-cytochrome c (cytc) and its single and multiple Lys to Ala variants at the surfacelysines 72, 73, and 79 were adsorbed on carboxyalkanethiol self-assembled monolayers (SAMs) on gold, andthe thermodynamics and kinetics of the heterogeneous protein-electrode electron-transfer (ET) reaction weredetermined by voltammetry. The reaction thermodynamics were also measured for the same species freelydiffusing in solution. The selected lysine residues surround the heme group and contribute to the positivelycharged domain of cytc involved in the binding to redox partners and to carboxyl-terminated SAM-coatedsurfaces. The E°¢ (standard reduction potential) values for the proteins immobilized on SAMs made of 11-mercapto-1-undecanoic acid and 11-mercapto-1-undecanol on gold were found to be lower than those for thecorresponding diffusing species owing to the stabilization of the ferric state by the negatively charged SAM.For the immobilized proteins, Lys to Ala substitution(s) do not affect the surface coverage, but induce significantchanges in the E°¢ values, which do not simply follow the Coulomb law. The results suggest that the speciesdependentorientation of the protein (and thereby of the heme group) toward the negatively charged SAMinfluences the electrostatic interaction and the resulting E°¢ change. Moreover, these charge suppressionsmoderately affect the kinetics of the heterogeneous ET acting on the reorganization energy and the donoracceptordistance. The kinetic data suggest that none of the studied lysines belong to the interfacial ET pathway.