Comprehensive Study of the Enzymatic Catalysis of the Electrochemical Oxygen Reduction Reaction (ORR) by Immobilized Copper Efflux Oxidase (CueO) From Escherichia coli

Abstract

In recent years, enzymatic fuel cells have experienced a great development promoted by the availability of novel biological techniques that allow the access to a large number of enzymatic catalysts. One of the most important aspects in this area is the development of biocatalysts for the oxygen reduction reaction (ORR). Laccases from the group of enzymes called blue multi-cooper oxidases have received considerable attention because of their ability to catalyze the electrochemical oxygen reduction reaction to water when immobilized on metallic or carbonaceous electrode materials. In this paper we report a comprehensive study of the electrocatalytic activity of the enzyme Copper efflux oxidase (CueO) from Escherichia coli immobilized on different electrode materials. The influence of the electrode substrate employed for protein immobilization was evaluated using glassy carbon, gold or platinum electrodes. Gold and platinum electrodes were modified using different self-assembled monolayers (SAM) able to tune the electrostatic interaction between the protein and the substrate, depending on the nature of the terminal functional group in the SAM. The effects of protein immobilization time, electrode potential, solution pH and temperature, protein and O2 concentration have been carefully investigated. Finally, direct electron transfer (DET) was investigated in the presence of the following inhibitors: fluoride (F−), chloride (Cl−) and azide (N3-).

Interplay between Orientation at Electrodes and Copper Activation of Thermus thermophilus Laccase for O2 Reduction

“…The favored DET process on the positive SAM and the absence of any DET on the negative one confirm the hypothesis based on the structure examination. Our results also emphasize the very similar electrochemical behavior of Tt Lac and E. coli CueO 24 as a function of the electrode surface charges.…”

“…On the opposite face of the molecule a net positive patch is present, inducing a significant dipole moment (878 D at pH 5, 965 D at pH 7) pointing oppositefrom the Cu T1. As a result, it is expected that immobilizing the enzyme on negatively charged interfaces will repel the Cu T1 surrounding, thus disfavoring DET, in a similar way as reported for E. coli CueO 24.…”

Inhibition in multicopper oxidases: a critical review

“…226,237 Chronoamperometry is commonly used to evaluate the resistance of an MCO-modified cathode to common halide inhibitors. 176,231,[238][239][240] For example, by injecting increasing concentrations of NaCl or NaF into the electrochemical cell over time, both Beneyton et al and Vaz-Dominguez et al were able to demonstrate that their MCO-modified electrodes were highly resistant to Cl − inhibition, but not to F − inhibition (Fig. 5A and B).…”

“…Chronoamperometry is used to visualize the timedependent response of the catalytic current to changes in the experimental conditions, such as variations in substrate concentration, inhibitor concentration, temperature and pH. 29,42,230,231 CV can be used to measure the current response of immobilized enzymes to a scanning potential applied to the electrode. 226,232 The shape of the CV (sigmoidal in the simplest case) can be indicative of electron transfer events taking place at the enzyme-electrode interface.…”

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

“…Metal modification is expected to prevent change in enzyme conformation induced by certain amino acids interaction with the bare metal [6]. Self-assembled-monolayers (SAMs), most often based on Au-S bonds, on metal surfaces are ideal tools to understand the role of surface hydrophobicity or charges on the efficiency of the electrocatalysis as highlighted by studies on copper efflux oxidase [7], cellobiose dehydrogenase [8], or human sulfite oxidase [9]. The additional advantage is the possibility to couple electrochemistry with analytical surface methods, such as quartz crystal microbalance (QCM), surface plasmon resonance (SPR), ellipsometry, surface enhanced infrared absorption (SEIRA), polarization modulation-infrared reflection-adsorption spectroscopy (PMIRRAS), surface-enhanced Raman spectroscopy (SERS) etc.…”