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 surface lysines 72, 73, and 79 were adsorbed on carboxyalkanethiol self-assembled monolayers (SAMs) on gold, and the thermodynamics and kinetics of the heterogeneous protein-electrode electron-transfer (ET) reaction were determined by voltammetry. The reaction thermodynamics were also measured for the same species freely diffusing in solution. The selected lysine residues surround the heme group and contribute to the positively charged domain of cytc involved in the binding to redox partners and to carboxyl-terminated SAM-coated surfaces. The E degrees' (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 the corresponding 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 significant changes in the E degrees' values, which do not simply follow the Coulomb law. The results suggest that the species-dependent orientation of the protein (and thereby of the heme group) toward the negatively charged SAM influences the electrostatic interaction and the resulting E degree' change. Moreover, these charge suppressions moderately affect the kinetics of the heterogeneous ET acting on the reorganization energy and the donor-acceptor distance. The kinetic data suggest that none of the studied lysines belong to the interfacial ET pathway.