5.3.2. Effect of the Electric Field

Compared to other catalytic processes, the special feature of bioelectrocatalysis lies in the electric field imposed at the electrode interface. Although partially shielded by the ions present in the solution, the extent of the electric field can still reach the size of immobilized enzymes, especially in weakly supported electrolytes. It should be expected to have a major impact, but only few studies report the effect of the electric field on enzyme or bioelectrode stability. This lack of information most probably comes from the difficulty to determine which effect among protein leaching, denaturation or reconformation can be responsible for any variation in the electrocatalytic response. The recent use of coupled methods with electrochemistry will certainly lead to major advancements in that field. Adsorption of MCOs on planar gold electrodes was taken as a model for such studies. It was shown using electrochemistry coupled to QCM-D that cycling the potential for O<sup>2</sup> reduction by *M. verrucaria* BOD accelerated mass decrease compared to applying a fixed potential [272]. It was hypothesized that varying the electric field near the surface may distort the adsorbed protein, expelling water and contributing to enzyme denaturation. It was further shown with the same enzyme that continuous cycling induced more stability of the bioelectrode than cycling during a same period but at regular intervals, the electrode being at the open circuit potential (OCP) when not cycling [116]. This result suggests a much lower stability of the bioelectrode when the applied potential was close to the OCP. Considering the potential of zero charge (Epzc) of the electrode, it could be concluded that the strong electric field generated when holding the potential far away from Epzc induced bioelectrode destabilization. Kano's group rationalized these experimental data by modeling the electrostatic interactions between enzymes and electrodes in the electric double layer [275]. Taking the copper efflux oxidase (CueO) enzyme as a model enzyme adsorbed either at bare or butanethiol-modified gold electrodes, the authors demonstrated how the electrical double layer plays a key role in enzyme stability for bioelectrocatalysis. It can be argued that the diameter of the enzyme is much higher than the length of the electrical double layer (5 nm vs. 1 nm at a typical ionic strength for electrocatalysis). However, the domain of CueO facing the electrode is indeed located in the double layer and will face an electric field whose force depends on the applied potential and on the Epzc of the electrode. Hence, at the bare gold electrode, the strong electric field induced by an applied potential much more positive than Epzc of the electrode, in addition to the complementary charges chosen to favor DET orientation, leads to denaturation of the protein.

Heterogeneity of the electric field inside the pores of a porous matrix otherwise affects enzyme stabilization. A model developed in the group of Kano demonstrated the existence of an inner pore region where the electric field remains low, and where weak electrostatic forces help in protein stabilization [238,277,278]. The higher electric field at the entry of the pore should enhance the ET, while inducing less stability [279,280]. This effect known as the "edge effect" was recognized early in the history of bioelectrochemical studies using cytochrome *c* and graphite and may explain efficient bioelectrocatalysis at carbon nanofibers (CNFs) [281–284].

Finally, it should be mentioned the role of the electric field sensed by the enzyme within the course of mediated electrocatalysis against direct electrocatalysis. A relevant example is bioelectrocatalytic oxidation of hydrogen by HASEs. It is known that HASEs are reversibly inactivated at high potentials [285]. In a typical cyclic voltammetry experiment where the enzyme is adsorbed on an electrode, this inactivation results in a decrease in the catalytic current in the forward scan at a potential higher than ca. −0.4 V vs. Ag/AgCl at pH 7. Current recovers when the potential is reversed. It was shown that pushing the potential

to even more positive values induces an irreversible inactivation of the enzyme [286]. Such inactivation process may have consequences on the operation of fuel cells using HASE as anode catalyst, requiring regular steps of reducing voltages to reactivate the enzyme. Alternatively, it was shown that embedding the hydrogenase in a viologen-based hydrogel protected the enzyme from high potential deactivation. In this MET process, the enzyme no longer senses the high potential of the electrode but the potential of the viologen redox couple which is sufficiently negative to prevent HASE inactivation [287–289]. Nevertheless, the use of redox mediators induces other detrimental effects towards enzyme stability, such as production of reactive oxygen species (ROS) that will be discussed in the next section.
