Advancing Techniques for Investigating the Enzyme–Electrode Interface

Abstract

ConspectusEnzymes are the essential catalytic components of biology and adsorbing redox-active enzymes on electrode surfaces enables the direct probing of their function. Through standard electrochemical measurements, catalytic activity, reversibility and stability, potentials of redox-active cofactors, and interfacial electron transfer rates can be readily measured. Mechanistic investigations on the high electrocatalytic rates and selectivity of enzymes may yield inspiration for the design of synthetic molecular and heterogeneous electrocatalysts. Electrochemical investigations of enzymes also aid in our understanding of their activity within their biological environment and why they evolved in their present structure and function. However, the conventional array of electrochemical techniques (e.g., voltammetry and chronoamperometry) alone offers a limited picture of the enzyme–electrode interface.How many enzymes are loaded onto an electrode? In which orientation(s) are they bound? What fraction is active, and are single or multilayers formed? Does this static picture change over time, applied voltage, or chemical environment? How does charge transfer through various intraprotein cofactors contribute to the overall performance and catalytic bias? What is the distribution of individual enzyme activities within an ensemble of active protein films? These are central questions for the understanding of the enzyme–electrode interface, and a multidisciplinary approach is required to deliver insightful answers.Complementing standard electrochemical experiments with an orthogonal set of techniques has recently allowed to provide a more complete picture of enzyme–electrode systems. Within this framework, we first discuss a brief history of achievements and challenges in enzyme electrochemistry. We subsequently describe how the aforementioned challenges can be overcome by applying advanced electrochemical techniques, quartz-crystal microbalance measurements, and spectroscopic, namely, resonance Raman and infrared, analysis. For example, rotating ring disk electrochemistry permits the simultaneous determination of reaction kinetics and quantification of generated products. In addition, recording changes in frequency and dissipation in a quartz crystal microbalance allows to shed light into enzyme loading, relative orientation, clustering, and denaturation at the electrode surface. Resonance Raman spectroscopy yields information on ligation and redox state of enzyme cofactors, whereas infrared spectroscopy provides insights into active site states and the protein secondary and tertiary structure. The development of these emerging methods for the analysis of the enzyme–electrode interface is the primary focus of this Account. We also take a critical look at the remaining gaps in our understanding and challenges lying ahead toward attaining a complete mechanistic picture of the enzyme–electrode interface.

Disparity of Cytochrome Utilization in Anodic and Cathodic Extracellular Electron Transfer Pathways of Geobacter sulfurreducens Biofilms

“…The secondary benefit of using an ITO substrate is that it is optically transparent, allowing for in situ spectroscopic experiments to be carried out. 11,19,33 To grow biofilms, IO−ITO electrodes were immersed in a G. sulf urreducens medium (featuring acetate to be oxidized by the bacteria) in oxygen-free conditions and poised at ∼0.3 V vs SHE for 4 days. 30 SEM images acquired after a typical initial growth in anodic mode show G. sulfurreducens cells attached to the IO−ITO surface (Figure 2a and 2b).…”

Recent advances in surface chemistry of electrodes to promote direct enzymatic bioelectrocatalysis

“…Advanced methods to get new insights into enzyme amount, conformation and activities distribution are required especially coupling electrochemistry to spectroscopies or microscopies [62,63]. Ultimately, control of enzyme binding on electrodes will help the development of single-enzyme electrochemistry [64].…”

Material–Microbe Interfaces for Solar-Driven CO2 Bioelectrosynthesis

“…Due to recent rapid developments in the field of bioinorganic hybrid materials for solar-to-chemical synthesis, it has become important to investigate the electron transfer mechanisms associated with such systems. Several advanced spectroscopic characterization techniques have been adapted to gain insight into such systems [83,84].…”