Electrostatic interactions

4.1.4.2.1 Laccases

Although in most studies laccase orientation has been performed via the supramolecular orientation strategy, another approach relies on electrostatic interaction. This approach was proposed for Cerrena unicolor laccase (pI = 3.95) immobilized on CNPs364 or

SW-CNTs458 _{bearing sulfonate functionalities in a silicate sol-gel matrix. This surface}

chemistry induced better reproducibility and at least twice higher currents. However, the increased performances observed in this case are more likely favored by the hydrophilicity of the structured matrix, rather than due to better orientation on negative charges, since for the same laccase a better orientation on positive charges was confirmed by PM-IRRAS studies.428 _{Electrostatic interactions could also drive an enhanced immobilization for an}

118 affinity tag at the C-ter close to the T1.459 In contrast, QCM studies performed with different

laccases showed that the nature of the charges did not influence the enzyme coverage.428,459

Studies performed with various laccases highlighted that electrode surface chemistry has to be customized according to the specific structure of each enzyme. Ulstrup et al. compared the influence of electrode surface charge for 3 different laccases: Coprineus cinereus (Cc-laccase), Streptomyces coelicolor (Sc-laccase), Myceliophtora thermophila (Mt- laccase), and Mv-BOD.233 The differences in electrocatalytic behavior were notable, and

could be easily correlated with the residues surrounding the T1 Cu atom. Mv-BOD and Cc- laccase, who share a positive charge area around the T1 center, displayed the highest activity on carboxylate-terminated SAMs. On the contrary, Sc-laccase showed the best activity on alkyl-terminated thiols, consistently with a hydrophobic pocket around the T1 Cu. Mt-laccase, the only one bearing a negative patch around the active site, was also the only one not to display a catalytic current.233

4.1.4.2.2 Bilirubin oxidases

A considerably different behavior between most laccases and Mv-BOD was observed: contrary to laccases, adsorbing Mv-BOD on molecules with a single aromatic ring or presenting a hydrophobic end lowered catalytic currents.17,233 When a complete structure of

Mv-BOD was obtained,129 _{this could be explained by the presence of a narrow hydrophilic}

substrate binding site close to T1, with basic amino acid residues positively charged at acidic and neutral pH. The differences in terms of amino-acid sequence between bacterial BODs and fungal BODs also resulted in different behaviors.434

Due to the difficulty to rationalize how DET with Mv-BOD could be obtained on carbon surfaces, which generally bear lots of functionalities and have a porous structure,460

119 extended to carbon or more complex architectures: KB,461 GO, ECR-sputtered CNFs431 and

CNTs.152,216,338,437,462,463 Here also DET was proved because the catalysis started at a potential

that could be attributed to the T1 whatever the chemical functions at the electrode surface,151

even when the surface was modified with redox active groups.152,338,458 _{Studies globally}

showed the importance of the electrode charges,151,437,463 catalytic currents being low for

positive charges, and higher for negative charges (most studies were performed at neutral or near-neutral pH). This proved that amino-acids near the T1 are more important than the global charge of the BOD whose isoelectric point is 4.1. Especially the adequacy of carboxylic functions17,151,220,432 _{has been highlighted. On carbon presenting carboxylate functions on}

aromatic protrusion,17 kinetic analysis148 evidenced that the enzyme adopted a narrow cone of

orientations. This can be partly attributed to an increase of hydrophilicity, or a better

wettability of the electrode431,437,462 rather than to enhanced enzyme loadings. As a matter of

fact, similar enzyme loadings have been calculated for different electrode surface charges.216

However, the interactions favoring a functional immobilization could be more complex than simply electrostatic. Indeed, some studies have suggested that for optimal immobilization of Mv-BOD, the anchoring molecules need to possess both aromatic properties and negative charges.432,461 This is consistent with previous studies where Mv-BOD

adsorbed on MW-CNTs modified with different compounds, all bearing carboxylic functions, showed no special difference with unmodified CNTs, except when the compound was Pyrroloquinoline quinone (PQQ). For this latter the catalytic currents almost doubled.152,319,338

Consistently also, Mv-BOD was perfectly oriented on MW-CNTs bearing carboxy- naphthalene moieties. DET currents reached 4 ± 0.4 mA.cm-2 in a stirred oxygenated solution,

and no increase was observed upon ABTS addition.216 On the contrary, when Mv-BOD was

immobilized on CNTs bearing positively charged amino groups, O2 reduction was observed at

120 TNC.216 In that case, addition of ABTS led to high MET currents ( 2.5 mA.cm-2). This is

also consistent with DET at T1 requiring negative surface charges at the electrode.

Quite contradictory results were however recorded, with Mv-BOD showing a better orientation than Tv-laccase on anthracene-modified MW-CNTs. Upon addition of ABTS only 11% increase in current densities were observed, while 59% increase was recorded for Tv- laccase.266,267,464,465

Mv-BOD immobilized on PG modified with naphthyl-2-carboxylate moieties was compared with platinum. The limiting current density, jlim, was 10 mA.cm-2 for the platinized

PG electrode, with an estimated platinum concentration of 2150 pmol.cm-2. For Mv-BOD, jlim

was 0.7 mA.cm-2_{, which represents a much better efficiency since the enzyme concentration}

was supposed to be lower than 10 pmol.cm-2. In addition, the reaction overpotential for

oxygen reduction was much smaller at the Mv-BOD naphthyl-2-carboxylate modified PG electrode than at Pt under all experimental conditions.17

Although electrostatic interactions have enabled Efficient immobilization of different MCOs, the specificity of the electrode modification towards immobilization of each particular enzyme is of crucial importance. A few examples underline it. The presence of COO- on the

electrode surface did not improve the bioelectrocatalytic activity for a CueO, whose T1 is surrounded by acidic residues, while an improvement was recorded with positively charged modification.461 Two different BODs, the bacterial Bp-and the fungal Mv-BODs, sharing only

36% sequence homology had also very different behaviors on CNTs with different surface charges.434 The comparison of DET/MET currents, and modeling with Armstrong’s

model,148,239 _{showed that Mv- was correctly oriented on negative surfaces (as already}

demonstrated), while Bp- was much better on positive. This result was consistent with their respective positive and negative charges surrounding the T1 in the considered pH range. This

121 difference was also observed for naphtoate-CNTs, which were favorable for Mv-BOD but detrimental for Bp-BOD.466