How the Intricate Interactions between Carbon Nanotubes and Two Bilirubin Oxidases Control Direct and Mediated O₂ Reduction

Abstract

Due to the lack of a valid approach in the design of electrochemical interfaces modified with enzymes for efficient catalysis, many oxidoreductases are still not addressed by electrochemistry. We report in this work an in-depth study of the interactions between two different bilirubin oxidases, (from the fungus Myrothecium verrucaria and from the bacterium Bacillus pumilus), catalysts of oxygen reduction, and carbon nanotubes bearing various surface charges (pristine, carboxylic-, and pyrene-methylamine-functionalized). The surface charges and dipole moment of the enzymes as well as the surface state of the nanomaterials are characterized as a function of pH. An original electrochemical approach allows determination of the best interface for direct or mediated electron transfer processes as a function of enzyme, nanomaterial type, and adsorption conditions. We correlate these experimental results to theoric voltammetric curves. Such an integrative study suggests strategies for designing efficient bioelectrochemical interfaces toward the elaboration of biodevices such as enzymatic fuel cells for sustainable electricity production.

Controlling Redox Enzyme Orientation at Planar Electrodes

“…A few recently reported examples demonstrated the ability to transfer the determined molecular basis for DET from a planar to a more complex-structured electrode. The physicochemical properties of the SAM-modified gold electrodes obtained in the case of M. verrucaria BOD were transposed to functionalized nanoporous gold [197], as well as to functionalized carbon nanotubes films [30]. The requirement of an aromatic linker to anchor LAC via the Cu T1 was confirmed on films made of gold nanoparticles [198].…”

“…In the case of ahomogeneous distribution of enzyme orientations we have k min = k 0 exp (−βd 0 ), where βd 0 is a parameter that represents the dispersion of possible orientations the enzyme molecules may adopt while maintaining electron exchange with the electrode. The βd 0 parameter governs the shape of the electrochemical signals as modeled by Armstrong's group [29], and reported in recent papers for the analysis of the ET of immobilized enzymes [30].…”

“…In the case of a homogeneous distribution of enzyme orientations we have kmin = k0exp (−βd0), where βd0 is a parameter that represents the dispersion of possible orientations the enzyme molecules may adopt while maintaining electron exchange with the electrode. The βd0 parameter governs the shape of the electrochemical signals as modeled by Armstrong's group [29], and reported in recent papers for the analysis of the ET of immobilized enzymes [30]. Note that the enzyme orientation on the electrode is not only a key issue for interfacial electron transfer, but is also essential to facilitate the diffusion of substrates and products within the enzyme internal cavity network toward the catalytic site.…”

“…Interestingly, studying the MET/DET ratio appears to be relevant for the evaluation of the distribution of the orientation of the enzymes on the electrode [136]. In addition, being a relative value, the MET/DET ratio is free from errors caused by the variation of adsorbed enzyme amount [30]. This strategy was applied to various enzymes immobilized on thiol-based SAMs and allowed to determine the surface chemistry on the electrode able to promote DET.…”

“…For example, the orientation of laccase from T. versicolor adsorbed on charged SAMs was shown to be controlled by the enzyme's dipole moment and the distribution of charged patches all over the protein surface [173] ( Figure 10). The oriented immobilization on electrode surfaces presenting different charges of two different BODs, one from a fungus (M. verrucaria) and the other from a bacterium (Bacillus pumilus), was shown to be controlled by the variation of their dipole moments as a function of pH [30]. Hence, direct connection of M. verrucaria BOD was obtained upon adsorption at pH 6 on negatively charged surfaces, in relation with a dipole moment pointing towards the T1 Cu, while only mediated catalytic current was obtained on positively charged surfaces [145].…”