Direct electrochemistry of proteins

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Abstract
Direct electrochemistry of site-specific mutants of yeast iso-1-cytochrome c (cyt c) and their complexes with bovine cytochrome b5 (cyt b5) has been investigated at edge-plane pyrolytic graphite (EPG) and bis(4-pyridyl)-disulphide-modified gold electrodes. Structure/function relationships have been investigated with the particular aim of clarifying the factors controlling the interactions of proteins at electrode/electrolyte interfaces and the determinants for direct electrochemistry in ternary protein/protein/electrode adducts, e.g. cyt c/cyt b5/EPG. Investigations of the cyt c mutants alone revealed a varietyof electrochemical responses: all the mutants show similar voltammetric reversibility at modified gold electrodes, whereas at EPG electrodes the reversibility follows the order: Asn52Ile–Cys102Thr > Cys102Thr > Asn52Ala–Cys102Thr. Mid-point potentials follow the order: Arg13Ile (+ 60 ± 5 mV vs. standard calomel electrode) > Cys102Thr (+ 40 ± 5 mV) > Lys27Gln (+ 30 ± 5 mV) > Lys72Asp (+ 30 ± 5 mV) > Asn52Ala–Cys102Thr (+ 15 ± 5 mV) > Asn52Ile -Cys102Thr (-10 ± 5 mV). The structural basis for these differences is briefly discussed. When these mutants are bound to cyt b5, the differences in electrochemical respose are greatly enhanced in the ternary cyt c/cyt b5/EPG adducts. A minimal analysis of these differences supports a model of multiple overlapping binding and recognition domains on cyt c which may be finely tuned to allow ternary complex formation so that a single-site variation could modify or abolish direct electrochemistry in the ternary adduct.

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