2.2.1. Formal Potential Shift of Electrode-Active Sites

The negative shift of the formal potential of the electron-donating site for the substrate oxidation process (vice versa for the substrate reduction) by mutation is very effective in reducing the thermodynamic overpotential in the intramolecular electron transfer in redox enzymes with multiredox sites (Figure 1). The formal potentials of metallic cofactors such as hemes and copper clusters are predominantly controlled by coordinated amino acid residues. Particularly, the point mutation of the axial ligands of hemes and blue copper clusters can easily tune their formal potentials. The direction of the potential shift depends in part on the electron-donating/accepting characteristics of the mutated amino acid side chains [100,101]. Briefly in general, an axial ligand with a relatively strong electron-donating character shifts the formal potential of the cofactor in the negative potential direction due to the stabilization of the oxidized state of the cofactor and vice versa. The formal potentials of redox enzymes are also greatly interfered with by conformational changes caused by mutated amino acid residues.

– FDH is among the accepted model enzymes to investigate the effects of protein engineering on DET-type bioelectrocatalysis. The enzyme is a heterotrimeric enzyme composed of a FAD-containing large catalytic subunit, a three-heme *c* (called hemes 1*c*, 2*c*, and 3*c*) from the N-terminus-containing cytochrome subunit, and a small subunit and proceeds with a DET-type reaction by transferring electrons from FAD, heme 3*c*, and heme 2*c* to an electrode in this order [102–104]. The sixth axial ligand (methionine 450) of heme 2*c* was then replaced by glutamine with electron-donating characteristics to shift the formal potential of heme 2*c* in the direction of the negative potential. The FDH variant (M450Q\_FDH) provided DET-type bioelectrocatalytic waves for the oxidation of fructose at a half-wave potential of approximately 50 mV, more negative than that of the recombinant native enzyme, as shown in Figure 4A [103,104]. A drastic change in the limiting catalytic current was not observed. This suggests that the rate constant of the intramolecular electron transfer from FAD to heme 2*c* in the enzyme is sufficiently large compared with the catalytic reaction rate constant at the catalytic center (FAD); the latter predominantly determines *k*c,DET in Equation (5).

type bioelectrocatalysis of "substrate oxidation " ( **Figure 4.** Illustrative drawing of the effect of mutations on steady-state bioelectrocatalytic waves in the DET-type bioelectrocatalysis of "substrate oxidation". (**A**) Negative potential shift in the formal potential of the electron-donating site of the DET-type redox enzyme reactions, in which the intramolecular electron transfer is not the rate-determining process in the enzymatic catalytic reaction. (**B**) Positive potential shift in the formal potential of the electron-donating site of the DET-type redox enzyme reactions, in which the intramolecular electron transfer is the rate-determining process in the enzymatic catalytic reaction, and (**C**) downsizing without any change in the intrinsic enzymatic activity.

*Γ Γ* Δ Δ *Γ* Δ On the other hand, such replacement is also effective for the axial ligand of the electron-accepting site (type I blue copper site) to negatively shift the formal potential. Replacement of the axial ligand methionine 467 in the type I copper site of BOD with glutamine (M467Q\_BOD) caused a large negative shift (approximately 0.23 V) in the half-wave potential of the DET-type bioelectrocatalytic waves. This means an increase in the overpotential in the intramolecular electron transfer in the enzyme. Fortunately, in this case, the catalytic limiting current density increased compared with that of the recombinant native BOD, as shown in Figure 4B (note here that the catalytic wave in Figure 4B is illustrated for substrate oxidation) [105]. Most probably, the catalytic rate constant of the dioxygen reduction at the type II/III catalytic site is sufficiently large compared with the rate constant of the intramolecular electron transfer from the electron-accepting type I site to the dioxygen-reducing type II/III site; the intramolecular electron transfer is the rate-determining step to determine *k*c,DET in Equation (5). In addition, the electron transfer kinetics seems to obey the linear free energy relationship (LFER); the increase in the formal potential difference between type I and II/III sites (that is, the driving force of the reaction) results in an increase in the intramolecular electron transfer rate constant.

Even in MET-type bioelectrocatalysis, the redox potential shift of the electron-donating site (for substrate oxidation) can tune the overpotential in the intramolecular electron transfer process. In addition, the intermolecular electron transfer kinetics between the electron-donating site in the enzyme and the mediator seem to be improved in theory by the potential shift mutation of the electron-donating site (for substrate oxidation) on the basis of the concept of LFER, though there is no report on this matter. Therefore, the mutational tuning of the redox potential of the electron-donating site (for substrate oxidation) may also expand the strategy of the mediator selection for MET-type bioelectrocatalysis.

## 2.2.2. Downsizing

As described by Equation (5), an increase in ΓE,eff is essential to getting a large bioelectrocatalytic current density. ΓE,eff can be then increased by downsizing enzymes from which the regions not deeply involved in bioelectrocatalysis are deleted, as shown in Figure 4C.

Downsizing effects were also investigated in FDH. Heme 1*c* of FDH is suggested to be uninvolved in the DET-type reaction, and the downsized FDH without the heme 1*c* region (∆1*c*\_FDH) showed a larger DET-type bioelectrocatalytic current density than the recombinant native FDH (r\_FDH) [104,106]. In addition, a further downsized FDH without the heme 1*c* and 2*c* regions (∆1*c*2*c*\_FDH) also showed DET-type bioelectrocatalytic activity with reduced overpotential due to direct electrical communication between an electrode and heme 3*c* with the most negative formal potential [104,107]. However, whereas ΓE,eff was suggested to be increased, *j*cat of ∆1*c*2*c*\_FDH was as large as that of r\_FDH (smaller than that of ∆1*c*\_FDH), which seemed to be ascribed to a decrease in enzymatic activity (≈ *k*c,DET) of ∆1*c*2*c*\_FDH by excessive deletion [104,107]. Thus, it is important to avoid conformational changes due to deletion and retain enzymatic activity as high as possible. Furthermore, a double mutant of downsizing and the potential shift (M450Q∆1*c*\_FDH) accomplished both an increase in *j*cat and an overpotential reduction [104,108,109].

#### 2.2.3. Surface Amino Acid Mutation

It is desirable to tightly immobilize enzymes on electrodes in both DET- andMET-type bioelectrocatalytic reactions. Cross-linkers such as glutaraldehyde [110], carbodiimide [29], and maleimide [71] are often used for the covalent immobilization of enzymes [111]. The mutation of amino acid residue(s) located on the enzyme surface also enhances the cross-coupling reactions and can control the orientation of the enzyme on a suitable electrode surface. For example, cysteine introduction onto the enzyme surface close to the electrode-active site can increase the enzyme orientations suitable for DET-type reactions at thiol- and maleimide-functionalized electrodes by forming (di)sulfide bonds. Holland et al. reported DET-type bioelectrocatalysis of cysteine-introduced GOD conjugated with maleimide-modified gold nanoparticles on a gold electrode [71]. In addition, Ferapontova et al. revealed that cysteine mutation of HRP was effective for its orientation on gold electrodes to improve DET-type bioelectrocatalytic properties [112].

#### 2.2.4. Fusion Protein

DET-type bioelectrocatalysis is sometimes achieved by introducing an electrode-active domain into a native enzyme. Cellobiose dehydrogenase (CDH) is often used as a model of DET-type fusion enzymes. CDH has two domains: a larger catalytic dehydrogenase domain and a smaller electrode-active cytochrome domain. The domains are linked by a flexible polypeptide [113]. The fused cytochrome domain mediates the electron transfer from the catalytic domain to electrodes, such as a "built-in mediator." Utilizing the fusion protein-engineering methods, DET-type reactions by FAD-GDH [114,115], pyrroloquinoline quinone (PQQ)-dependent GDH [116], and flavodoxin [117] were reported.
