5.2.5. When Aggregates or Partially Unfolded Enzymes Operate in Bioelectrocatalysis

We clearly stated in the introduction of this review that the secondary and tertiary structure of enzymes control their activity. Any change in the conformation was thus expected to modify the activity, yielding inactive states upon denaturation. We otherwise underlined that aggregated enzymes, for example in the form of CLEAs, could maintain the native conformation showing a stable, although often lower, enzymatic activity. Efforts to maintain stable enzyme electroactivity are mainly directed toward enzymes with unchanged conformations. However, and contrary to these strategies, examples of aggregates or partially unfolded forms of proteins have been reported to promote bioelectrocatalytic properties. Aggregated proteins immobilized on nanostructured carbon-based electrodes may enabled direct electrocatalysis showing enhanced stability compared to non-precipitated proteins [266,267]. Pyranose oxidase was immobilized on a CNT-based electrode via three different procedures: (i) covalent attachment (CA), (ii) enzyme coating (EC), (iii) enzyme precipitate coating (CLEA). After 34 days at room temperature, CLEA retained 65% of initial electroactivity toward glucose oxidation, while CA and EC maintained 9.2% and 26% of their initial activities, respectively. LAC aggregates were formed by computational design through production of a new dimeric interface that induces self-assembly of the protein into crystalline-like assemblies [268]. Combined with CNT incorporated in the aggregates, the bioelectrode showed high direct electroenzymatic rate for O<sup>2</sup> reduction with enhanced thermostability. LAC from *T. versicolor* was precipitated in the presence of Cu2+ yielding flower-like particles [207]. The Cu/LAC nanoflowers were integrated in carbonaceous nanomaterials and tested as catalysts for O<sup>2</sup> bioelectroreduction. A wellshaped direct electroenzymatic signal was observed, demonstrating the functionality of the electron pathway through CuT1 in the flower particles (Figure 12). The bioelectrode delivered 72% initial current after 6 days of daily 2 h discharge, and the H2/O<sup>2</sup> fuel cell constructed based on the Cu/LAC flower-based biocathode maintained 85% initial power after 15 days of operation.

**Figure 12.** LAC assembled through Cu-flower like particles displays direct and stable electrocatalysis of O<sup>2</sup> reduction. (**A**) SEM image of a Cu/LAC nanoflower, (**B**) Catalytic reduction of O<sup>2</sup> (red curve) by LAC in Cu nanoflowers on CNTs, (**C**) Operational stability. Adapted with permission from [207].

It was also recently shown that the large sub-unit of a NiFe HASE was able to sustain H<sup>2</sup> oxidation by itself, showing that an incomplete structure still allows catalysis [269]. As another illustration, LAC from *Aspergillus* sp. was immobilized on Fe2O<sup>3</sup> NPs [270]. Careful spectroscopic analysis, including FT-IR, fluorescence and EPR, demonstrated a partial unfolding of the protein which tends to a higher exposure of the CuT1. A large direct catalytic current in the range of 3 mA/cm<sup>2</sup> was observed, with an electrochemical CV shape denoting fast ET. However, it should be stressed that heterogeneity of enzyme population certainly exists at the electrochemical interface, and methods are now required to distinguish whether the electrochemical activity is actually linked to the unfolded proteins or to the small fraction of proteins remaining correctly folded.
