Cytochrome C Mutants with Enhanced Capabilities in Multi Layer Arrangements

In the field of biosensors it is important to increase the surface concentration of the selected biological recognition elements for a higher sensitivity. Based on cytochrome c, a small heme containing electron carrier protein from the respiratory chain, fully redox active multi layer assemblies have been developed by using the layer by layer technique, where every immobilized protein contributes to the electron transfer with the electrode. This provides the basis for integrating catalytically active enzymes into the layers, giving the opportunity of tuning the sensitivity by the number of layers deposited. This was shown already e.g. for bilirubin oxidase (BOD) and sulfite oxidase (SOX). However, one limiting factor for the effective coupling of proteins in those multi layer systems is the electron self-exchange rate of the cytochrome c molecules. In order to follow the applied potential but also the enhanced conversion at the embedded enzymes, a rather high exchange rate between the immobilized proteins is necessary.

Based on the hypothesis, that the electron transfer happens via hopping between the heme-centers which are embedded in a protein-pocket that is mainly positively charged at neutral pH, we tried to engineer human cytochrome c mutants with increased electron self exchange rates. By replacing some of the positively charged surface lysines around the heme pocket with uncharged alanines via site directed mutagenesis, repulsion between cytochrome c molecules should be decreased and ease the charge transfer.

Six different mutants have been designed, expressed and evaluated. The electrochemical properties of the cytochrome c mutants have been analyzed by cyclic voltammetry at self assembling mono layers (SAMs) of 11-mercapto-1-undecanol (MU) and 11-mercapto-1-undecanoic acid (MUA) on gold. In a second step cytochrome c mutant multi layers have been prepared and analyzed with respect to different rates of the potential sweep in order to elucidate how the systems can follow the driving force.