Quantifying the Characteristic Turnover Frequency of an Individual Redox Enzyme via Real-Time Electrical Transduction of Catalytic Events in a Label-Free Single-Protein Junction

Abstract

Understanding redox enzymatic reactions remains a fundamental challenge in biochemistry, as these processes involve intricate dynamics that are inherently difficult to dissect using conventional bulk techniques. Single-enzyme catalysis offers a promising approach for unraveling the dynamic behavior of individual enzymes as they undergo a reaction, revealing the complex heterogeneity that is lost in the averaged ensemble. Here, we demonstrate real-time, label-free monitoring of the electrical transduction of single-protein enzymatic activity for two redox enzymes, cytochrome P450camand glutathione reductase, trapped in an electrochemically controlled nanoscale tunneling 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 tunneling conductance is modulated by the enzymatic reaction; subtle changes in 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 tunneling channel in the single-protein junction that results in a temporary tunneling current jump, contributing to the observed conductance switching feature. The latter is experimentally corroborated 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 heterogeneous behavior of enzymatic catalysis and the quantification of single-enzyme turnover frequencies. This work demonstrates a new nanoscale platform for nonlabeled electrical detection of single-enzyme activity in a redox enzymatic process.