Real-Time, Label-free Electrical Transduction of Catalytic Events in a Single-Protein Redox Enzymatic Junction

Abstract

Single-enzyme catalysis offers a promising approach for unravelling the dynamic behaviour of individual enzymes as they undergo a reaction, revealing the complex heterogeneity that is lost in the averaged ensembles. Here we demonstrate real-time, label-free monitoring of the electrical transduction of single-protein enzymatic activity for two redox enzymes, cytochrome P450cam and glutathione reductase, trapped in an electrochemically controlled nanoscale tunnelling junction immersed in the aqueous enzymatic mixture. The conductance switching signal observed in individual transients of the electrical current flowing through the single-protein junction shows that the tunnelling conductance is modulated by the enzymatic reaction; subtle changes of the enzyme redox state occurring during the chemical catalysis process result in fluctuations of the enzyme junction conductivity, which are captured as a switching signal. At the applied electrochemical reducing potential for electrocatalysis, the transient oxidation of the trapped enzyme in every catalytic cycle opens an additional redox-mediated electron tunnelling channel in the single protein junction that results in a temporary current jump, contributing to the observed conductance switching features. The latter is experimentally assessed via electrochemically controlled conductance measurements of the single-protein junction. The statistical analysis of the switching events captured over long time periods results in average frequencies that correlate well with the reported catalytic turnover values of both enzymes obtained in standard bulk assays. The single-enzyme experiments reveal the acute heterogenous behaviour of enzymatic catalysis and the quantification of single enzyme turnover frequencies.