5.3.1. Reorientation of the Immobilized Enzyme

We stated just before that one key issue in order to enhance the interfacial ET between a redox enzyme and an electrode is the orientation of the enzyme on the solid surface so that the electronic relay on the surface of the protein can be positioned at a distance compatible with fast ET. In many studies reporting enzyme orientation, hydrophobic or electrostatic interactions are used to control the enzyme positioning. These interactions are relevant because they mimic in vivo recognition with the substrate or between redox partners. LAC, as an example, is involved in transformation of many substrates such as phenolic compounds. In accordance, the Cu T1 is engaged in a hydrophobic pocket that can be recognized by the electrode modified by phenyl or anthracenyl groups, leading to an orientation favoring efficient electron transfer [273]. The recognition between HASE and its redox partner cytochrome *c*<sup>3</sup> is another example illustrating electrostatic parameters to be considered in ET. By NMR docking and by resolving the Poisson–Boltzmann equation, it was established that a functional complex is formed thanks to a small negatively charged region on the HASE surface that can interact with the positively charged cytochrome. Such local electrostatic interaction was able to overcome electrostatic repulsion of the overall positive charges of the interacting proteins [274,275]. The same situation arises in bioelectrocatalysis, considering the electrode as the redox partner. Local charges are reflected by charge heterogeneity on the surface of the enzyme and rationalization can be obtained through the calculation of the dipole moment direction. This latter will depend on the pH of the enzyme environment. Hence, it can be expected that an enzyme takes an orientation favorable to ET at one pH but rotates on the electrode when the

pH of surrounding solution is modified. It is the most crucial when electrocatalysis involves proton consumption or production, as it is the case for many electroenzymatic reactions, thus inducing local changes in pH in the weakly buffered solution and potential modification of the catalytic signal.

Evidence of the rotation of BOD was demonstrated on gold electrodes modified by thiol-based SAMs [116]. Both the pH at which the enzyme was adsorbed and the pH of the electrolytic solution were varied. By doing so, not only the charge of the electrode but also the charge of the enzyme varied. Coupling electrochemistry to ellipsometry, SPR, and Phase Modulation Infrared Reflection Absorption Spectroscopy (PMIRRAS), it was shown that the global charge of the protein controls the amount of adsorbed molecules as a function of electrostatic interactions tuned by the pH of adsorption. It was also demonstrated that the local charge in the vicinity of the CuT1 controls the orientation of the enzyme, hence the catalytic current for O<sup>2</sup> reduction. Hence, when the amount of enzyme and its orientation were a priori fixed by adsorption at a fixed pH, the catalytic current recorded in buffers of various pHs nevertheless varied following rotation of the enzyme to adopt the most favorable orientation. It was clearly shown that orientation could be reversibly tuned by changing the pH of the electrolyte, shifting the catalysis from a slow ET rate to a fast one (Figure 13).

**Figure 13.** Reorientation of enzymes on electrode may alter enzyme stability. (**Top**) Tuning the pH of the electrolyte induces rotation of the adsorbed bilirubin oxidase on the electrode surface, hence induces modification in bioelectrocatalysis for O<sup>2</sup> reduction. Adapted with permission from [116]. (**Down**) Scheme of the reorientation of enzymes on an electrode induced by surface coverage. The white sphere represents the active site. Adapted with permission from [115].

Enzyme reorientation, leading to potential bioelectrocatalysis instability, may have another origin. In the course of protein adsorption, protein-protein interactions are increasingly dominating. Hence, while the adsorption of the first proteins in a favorable orientation for ET can be controlled by electrostatic interactions, the interactions between neighboring adsorbed proteins may induce reorientation to decrease the repulsive forces [115] (Figure 13). Coupling electrochemistry to QCM and interferometry for studying O<sup>2</sup> reduc-

tion by BOD on a gold electrode, Blanford et al. clearly demonstrated that an optimum surface coverage exists yielding the highest stable electrocatalytic current [276]. Although proof of enzyme reorientation was not provided, QCM-D allowed to conclude that this optimum was linked to a balance between rate of enzyme adsorption and deformation.
