3.2.3. Bilirubin Oxidase

Bilirubin oxidase (BOD) is another well-known blue multicopper oxidase class containing four copper centers (T1, T2/T3), catalyzing the dioxygen reduction reaction (ORR) into water in four-electron direct bioelectrocatalysis [75,96]. The blue T<sup>1</sup> center has a copper atom located closed to the protein surface for accepting electrons from the natural electron donor (bilirubin) or an electrode. The external electrons are relayed via a short ligand-bound intramolecular peptide bridge to the T2/T<sup>3</sup> center, where ORR takes place. Target BODs are mainly from *Myrothecium verrucaria* (*Mv*BOD) [26,29,75,143–145], *Trachyderma tsunodae* (*Tt*BOD) [146], *Bacillus pumilus* (*Bp*BOD) [147], and *Magnaporthe oryzae* (*Mo*BOD) [148]. Single-crystal gold electrodes modified with -NH2, -COOH, -OH, and -CH<sup>3</sup> terminated SAMs have been studied and show that negatively charged SAMs are favorable for proper *Mv*BOD orientations due to the highly positively charged region close to the T<sup>1</sup>

center [42]. E◦ values of T<sup>1</sup> (0.69 V vs. NHE) and T2/T<sup>3</sup> (0.39 V vs. NHE) in *Tt*BOD have been observed under aerobic conditions [146], but E◦ of the T2/T<sup>3</sup> centers drops to 0.36 V vs. NHE in the resting state of the enzyme, indicative of an IET pathway from T<sup>1</sup> to T2/T<sup>3</sup> triggered by the ORR process.

Characterization of enzyme loading and conformation on the electrode is crucial [149]. Lojou and associates studied the adsorption of *Mv*BOD on both positively (NH<sup>3</sup> <sup>+</sup>) and negatively (COO– ) charged SAM-modified electrodes with no significant difference in enzyme loading as disclosed by surface plasmon resonance (SPR) spectroscopy [26]. DET and MET processes were found to dominate on the COO- and NH<sup>3</sup> <sup>+</sup> surfaces, respectively, consistent with the positively charged surroundings of the T<sup>1</sup> Cu [26]. Polarization-modulated infrared reflection absorption spectroscopy (PMIRRAS) showed strong electrostatic interactions between the negatively charged SAMs and the positively charged *Mv*BOD surface near the T<sup>1</sup> Cu [26]. PMIRRAS and SPR ellipsometry were jointly used to demonstrate the effect of electrostatic interactions on the enzyme adsorption, catalytic performance, and stability of *Mv*BOD at four different pH values (Figure 6) [75]. The dipole moment of the enzyme is distinct at different pH levels, showing a direction towards the T<sup>1</sup> center in neutral or slightly acidic electrolytes but significantly shifted in the strongly acid electrolyte (Figure 6a). This accords with the different charge distributions at the T<sup>1</sup> Cu center. PMIRRAS spectra showed the amide I (ca. 1680 cm−<sup>1</sup> ) and amide II (ca. 1550 cm−<sup>1</sup> ) peaks, related to vibrational C=O and N-H modes in *Mv*BOD, respectively, see Figure 6b. The wavelengths of the two peaks remained unchanged upon enzyme immobilization, which suggests that the secondary structure of the *Mv*BOD is retained. In addition, amide I/amide II ratios were similar at different pH levels, further indicating that the orientations of *Mv*BOD were independent of pH. − −

Γ **Figure 6.** (**a**) *Mv*BOD structure and dipole moments in the pH range 3.6 to 7.5. CuT<sup>2</sup> /T<sup>3</sup> contains three copper atoms marked in blue and CuT<sup>1</sup> with a single copper atom marked in gold; (**b**) PMIRRAS signals of *Mv*BOD-modified bioelectrodes with 6-MHA SAM; (**c**) Enzyme coverage (Γ) and enzyme layer thickness at different pH recorded by SPR ellipsometry; (**d**) Cartoon illustration of the charge distributions of *Mv*BOD at different pH levels, with the CuT<sup>1</sup> center, as well the 6-MHA and 4-ATP SAM-modified electrode surfaces. The neutral electrode surface is indicated with green stars. Reproduced with permission from [75]. Copyright 2018, American Chemical Society.

Enzyme loading, critical to catalytic activity, declines as pH increases (Figure 6c [75]). The authors demonstrated that *Mv*BOD on a negatively charged 6-MHA SAM-modified electrode does not form a saturated monolayer at pH 7.5 but possibly more than a single monolayer at pH 3.6. The electrostatic interactions between enzyme and SAMs thus not only strongly affect the enzyme adsorption, the dipole moment of the enzyme, and the charge around the T<sup>1</sup> center, but they also determine the enzyme orientation and catalytic rate (Figure 6d). Gholami and associates immobilized *Mv*BOD on a gold microfilm by electropolymerization of TCA [150]. Molecular dynamics simulation has provided a more comprehensive understanding of *Mv*BOD in DET-type bioelectrocatalysis [151]. In particular, *Mv*BOD showed various orientations reflecting wide charge distributions, representing a "back-on" and "lying-on" state on positively and negatively charged electrodes, respectively.

Nanostructured gold surfaces are expected to improve the DET current density of BOD [29]. However, the apparent and real current densities normalized to the geometric and real gold surface area, respectively, should be distinguished. In comparison to FDH on 1 nm AuNPs [44], this is particularly important when the gold nanostructures are of larger size than BOD. Pankratov and associates investigated the size effect of sub-monolayer AuNPs (diameter: 20, 40, 60, and 80 nm, significantly larger than *Mv*BOD) on the bioelectrocatalytic performance of *Mv*BOD [152]. Although the apparent ORR current density increased with increasing size from 20 to 80 nm (proportionally to the real surface area), similar values (15 ± 3 uA cm−<sup>2</sup> ) for the real current density were obtained. This can be explained by the fact that the ET rate constant is independent of the AuNP size in this case, with similar values of 10.3 ± 0.5 s−<sup>1</sup> and 10.7 ± 0.3 s−<sup>1</sup> for the *Mv*BOD-based bioelectrode with and without AuNPs, respectively. Siepenkoetter and associates immobilized *Mv*BOD on NPG using diazonium grafting coupled with an MPA SAM [28]. NPG electrodes with average pore sizes between 9 and 62 nm were finally investigated. The maximum apparent ORR current density was achieved with 10 and 25 nm pores, slightly larger than the enzyme and likely due to a compromise between the real gold surface area and enzyme loading, but similar real current densities were registered for these pore sizes.

## 3.2.4. Laccase

The laccases (Lac) constitute a third important member class of the multicopper oxidases, containing T<sup>1</sup> and T2/T<sup>3</sup> centers [27,153,154]. Similar to BOD, T<sup>1</sup> and T2/T<sup>3</sup> Cu centers act as electron acceptance and ORR centers, respectively. E◦ of the T<sup>1</sup> center from tree Lac is lower than those from fungal Lac, varying in the range 300 to 800 mV vs. NHE [155]. Bioelectrocatalysis of fungal Lac has been extensively investigated [156]. SAMs with specific functional groups favor productive orientations of Lac at the electrode surface. Thorum and associates reported that the overpotential of ORR of Lac from *Trametes versicolor* (*Tv*Lac) could be decreased by employing an anthracene-2-methanethiol SAM on gold. The aromatic anthracene presumably penetrates into the hydrophobic pocket close to the T<sup>1</sup> center, facilitating DET [157]. Climent and associates investigated the DET-type catalytic activity of three different Lacs (*Coprinus cinereus* (*Cc*Lac), *Myceliophthora thermophila* (*Mt*Lac), and *Streptomyces coelicolor* (*Sc*Lac)) [38]. *In situ* STM enabled single-molecule understanding of enzyme–electrode electronic interactions and the IET process on well-defined Au(111) surfaces with various SAMs. MPA SAMs with the carboxyl terminal group was best for *Cc*Lac, while alkyl and amino SAMs were most suitable for *Sc*Lac. No catalytic signal was found for *Mt*Lac on any SAMs. As for *Ax*CuNiR, single-molecule in situ STM contrasts were observed only in the presence of enzyme substrate, nitrite, and dioxygen, respectively. Traunsteiner and coworkers exploited DET-type bioelectrocatalysis of *Tv*Lac using diluted MPA SAMs and a linker molecule of thiolated veratric acid (tVA) that could approach the T<sup>1</sup> site [158]. Optimal catalytic activity was shown when the tVA and MPA SAMs were mixed uniformly, whereas the catalytic activity decreased dramatically due to the aggregation of tVA. Molecular dynamics simulations also showed that the positively charged SAMs were more favorable for the DET of *Tv*Lac, with a narrow orientation distribution.

A recent study evaluated *Thermus thermophilus* (*Tt*Lac), exhibiting a methionine rich domain. Figure 7 shows the detailed three-dimensional structure and electrostatic charge distributions of the surface amino acid residues of *Tt*Lac [69]. The T<sup>1</sup> center, used for transferring electrons to an external electrode, showed a negatively charged zone. Gold electrodes modified with negatively (-COO−), uncharged (-OH), and positively charged groups (-NH<sup>3</sup> <sup>+</sup>) were adopted, with the highest catalytic current on the positively charged electrode and with no or only weak catalytic signals on the negatively charged and uncharged electrodes, respectively [69]. Nanostructured materials have been developed recently to optimize the Lac orientation. NPG modified with 4-ATP SAM could increase the catalytic performance and stability at high temperatures [159]. Cristina and associates reported that AuNPs (particle size: 5 nm, comparable to the size of the enzyme) served as electronic bridges, thus promoting DET with a heterogeneous ET rate constant over 400 s−<sup>1</sup> [54]. Nanostructured electrodes consisting of low-density graphite (LDG) and gold nanorods (AuNRs, average length: 31 ± 6 nm, width: 5 ± 1 nm) have, finally, been prepared to orient Lac for improved ORR [27]. −

**Figure 7.** (**a**) Model structure of *Tt*Lac (PDB 2XU9) showing the hairpin domain (magenta), the T<sup>1</sup> and T2 /T<sup>3</sup> Cu centers (blue spheres) and all Met sulfurs (yellow spheres); (**b**) Electrostatic potentials at the surface of *Tt*Lac in the same orientation as in the top panel at pH 5: positive charges in blue, negative charges in red, and neutral in white. The positive end of the dipole moment vector is shown as a yellow stick. Reproduced with permission from [69]. Copyright 2020, American Chemical Society.
