# Tuning O_2 enzymatic reduction: Roles of methionine-rich domains and electrochemical metalation of active centers

Vita Saska, Paolo Santucci, Anne de Poulpiquet, Didier Gasparutto, Umberto Contaldo, Ievgen Mazurenko, Elisabeth Lojou

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Vita Saska, Paolo Santucci, Anne de Poulpiquet, Didier Gasparutto, Umberto Contaldo, et al. Tuning O_2 enzymatic reduction: Roles of methionine-rich domains and electrochemical metalation of active centers. Bioelectrochemistry, 2025, 166, pp.109051. 10.1016/j.bioelechem.2025.109051. hal-05173110

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# Tuning O₂ enzymatic reduction: Roles of methionine-rich domains and electrochemical metalation of active centers☆

Vita Saska<sup>a</sup>, Paolo Santucci<sup>a</sup>, Anne de Poulpiquet<sup>a</sup>, Didier Gasparutto<sup>b</sup>, Umberto Contaldo<sup>a</sup>, Ievgen Mazurenko<sup>a, *,</sup> Elisabeth Lojou<sup>a, *</sup>

## ARTICLE INFO

### Keywords:

CueO

Catalysis

Methionine domain

Metalation

Electrochemistry

MD simulations

## ABSTRACT

CueOs are multicopper oxidases which feature a methionine rich domain (Met-rich) covering the first electron acceptor and proposed to play roles in enzymatic catalysis. However, how these domains impact catalysis is still a matter of debate. In this work, we compare O₂ reduction at gold electrodes according to the Met-rich content of two CueOs from *Escherichia coli* and *Hafnia alvei* bacteria. The extent of electrocatalysis is shown to depend on the type of CueO adsorbed on the gold surface, with activity being nearly zero when using the CueO protein containing the largest Met-rich domain. To clarify the role of Met-rich domains, CueO engineering was additionally performed by deleting Met-rich domains or exchanging them between the two proteins to create chimeric enzymes. Using a combination of electrochemistry, surface plasmon resonance and molecular dynamic simulation, we demonstrate that the Met-rich domain induces steric hindrance that affects the electron transfer process. In addition, a progressive and potential dependent increase in the catalytic currents is observed. We demonstrate that this activation process is associated with the incorporation of copper cofactors in the immobilized proteins by electrogenerated Cu⁺. This electrochemically induced CueO metalation paves the way for the large-scale production of multicopper oxidases (MCO) followed by in vitro maturation, as high copper concentrations are toxic in vivo.

## 1. Introduction

The superfamily of multicopper oxidases (MCOs) encompasses a large number of enzymes, including well known laccases, bilirubin oxidases, ascorbate oxidases and metalloxidases. Four copper sites form the active centers of MCOs: a mononuclear CuT1, where substrates bind and oxidize and a trinuclear copper cluster (TNC), distant less than 1.5 nm from the CuT1 favoring electron transfer rate and allowing bound O₂ to be reduced to H₂O. The cuproxidases CueOs belong to the MCO superfamily [1–4]. Their cuprous oxidase activity is a distinguishing feature of these enzymes, setting them apart from other MCOs and granting them a physiological role in copper homeostasis [5,6]. A methionine-rich (Met-rich) domain that specifically covers the first electron acceptor, the CuT1 site, is the main characteristic of CueOs which may be a determinant of enzyme activity [7]. The role of the Met-rich domain of CueO in relation with the cuprous oxidase activity is

however still under investigation. We recently studied its impact on cuprous oxidase activity coupling in vitro activity in solution and electrochemical activity of *Escherichia coli* CueO (*EcCueO*) immobilized on carbon nanotube-based electrodes. It was demonstrated that the Met-rich domain is not required for cuprous oxidase activity but acts as a facilitator of Cu⁺ recruitment from the strongly copper chelated forms [8]. This conclusion was allowed thanks to the compared activity of different Cu⁺-complexes, from free electrogenerated forms to chelated ones. It was reinforced by the comparison of the activity displayed by another CueO from *Hafnia alvei* (*HaCueO*) which presents a much larger Met-rich domain (Fig. 1) [9].

The involvement of Met-rich domains in CueO interaction with electrodes for driving O₂ reduction (ORR) has also been investigated. However, the conclusions from these studies vary significantly, with the Met-rich domain reported as being bypassed, facilitating or decreasing the electron transfer rate. Adachi et al. investigated the effect of

<footnote><sup>a</sup> Aix Marseille Univ, CNRS, Laboratoire de Bioénergétique et Ingénierie des Protéines (BIP), Institut de Microbiologie de la Méditerranée, Marseille, France</footnote>

<footnote><sup>b</sup> Univ. Grenoble Alpes, CEA, CNRS – UMR5819 SyMMES / Lab. Chimie Reconnaissance Etude des Assemblages Biologiques – 17 rue des Martyrs 38054 Grenoble cedex 9, France</footnote>

<footnote>* This article is part of a Special issue entitled: 'GFB symposium 2025' published in Bioelectrochemistry.</footnote>

<footnote>* Corresponding authors.
E-mail addresses: imazurenko@imm.cnrs.fr (I. Mazurenko), lojou@imm.cnrs.fr (E. Lojou).</footnote>
elimination of helix regions in the Met-domain on direct electrochemical O₂ reduction (DET) by *EcCueO* [10]. They studied at mesoporous carbon electrodes recombinant native CueO and its variants, which lack the helix covering the electron-donating substrate-binding site. They proposed that electrons are transferred from the electrode to the CuT1 site via a negatively charged region near the site, bypassing the helix. Schwaneberg et al. compared the bioelectrocatalytic behavior of wild type (WT) *EcCueO*, a Met-rich truncated CueO (ΔMet), and a methionine-to-serine substituted CueO adsorbed on a carbon nanotube (CNT) network [11]. The Met-rich domain was proposed to play a key role in rapid immobilization of CueO on electrodes. The hydrophobic interactions between methionine residues in the Met-rich motif and the CNTs were proposed to be the main driving force for enzyme binding in a preferred orientation for DET. In a study dedicated to ORR by *Aquifex aeolicus* McoA, a CueO-like enzyme adsorbed on a carbon electrode, Moro et al. noted that a reductive activation was required to reach maximum bioelectrocatalytic activity for ORR [12]. Such a potential dependence was explained based on previous results demonstrating that the enzyme is initially in a resting state that needs to be reduced before becoming active [13].

CueOs were also studied at bare gold surface with the aim of catalyzing O₂ reduction. Such immobilization may result in low ORR catalytic current compared to immobilization on carbon-based electrodes or even thiol-modified gold electrodes. This decrease in current is likely due to unfavorable orientation or flattening caused by direct metal-protein interactions. Additionally, sub-monolayer coverage on a bare gold electrode has been hypothesized to lack sufficient steric protection against enzyme denaturation [14]. Sugimoto et al. examined the effect of potential of adsorption of *EcCueO* on a bare gold electrode and showed the highest DET activity occurred with adsorption at around the point of zero charge [15]. Two metalloxidases CueO-like from hyperthermophilic bacteria, McoP from *Pyrobaculum aerophilum* [16] and AaMcoA [17] were reported to remain active when immobilized on a gold surface. In the former enzyme, ORR was attributed to the structural rigidity typical of hyperthermophilic bacteria. In the latter case, it was

proposed that enzyme orientation for efficient and stable direct electron transfer was insured by the Met-rich domain close to the active site [17]. Indeed, an enzyme variant in which the Met-rich domain adopted a conformation that reduced its distance to CuT1 exhibited significantly higher ORR catalytic performance, likely due to the decreased distance between CuT1 and the electrode. Schwaneberg et al. investigated *EcCueO* WT and the ΔMet variant [11]. Weak O₂ reduction could be observed in the ΔMet variant compared to the WT protein, suggesting a role of the Met-rich domain in the ORR. Finally, Chumillas et al. highlighted an increase of the catalytic current for ORR upon consecutive cycles, with a faster activation when more negative potentials were applied [18]. It was proposed that thiol bonds were formed between sulfur containing aminoacids and the electrode.

In the current work, we aim to clarify the impact of Met-rich domains on direct O₂ reduction by studying CueOs with different conformation and chemical composition of the Met-rich domain. Taking profit of the comprehensive characterization of *EcCueO* and *HaCueO* on CNT surfaces to assess their cuprous oxidase activity, we extend the investigation to ORR with the enzyme immobilized on thiol-modified and bare gold electrodes [8,9]. The distinct electrochemical behavior of the two enzymes strongly suggests a role of the Met-rich domain, as further confirmed by studies on proteins lacking the Met domain and on chimeric variants in which Met-domains were exchanged between WT CueOs. By combining electrochemistry, surface plasmon resonance and molecular dynamic simulations, we discuss the steric effect induced by the Meth-rich domain on the electron transfer process. In addition, we rationalize the gradual and potential dependent increase in catalytic currents and demonstrate that it results from CueO metalation by electrogenerated Cu⁺.

## 2. Material and methods

### 2.1. *Chemicals and materials*

LB Broth (Miller's), Ampicillin sodium salt, isopropyl-β-D-

<figure><img src="image_2.png" /><figcaption>**Fig. 1.** Structures of *EcCueO* (PDB entry 3OD3) and *HaCueO* (MD model) showing the difference in the Met-rich domains (highlighted in orange and purple respectively). CuT1 and TNC are represented as dark blue and light blue spheres respectively. Bottom: Methionine-rich domain sequence alignment between *EcCueO* and *HaCueO*. The colors follow the ESPript similarity scheme: identical residues are highlighted with a red background; residues with similar physicochemical properties are shown in red font on a white background; regions containing equivalent or similar residues are enclosed within blue frames. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)</figcaption></figure>
thiogalactopyranoside (IPTG), DNase I, 3-(N-morpholino)propane-
sulfonic acid (MOPS), 2,2'-Azino-bis(3-ethylbenzothiazoline-6-sulfonic
acid (ABTS), ethylenediaminetetraacetic acid disodium salt dihydrate
(purity >99 %) (EDTA), NaOH and copper standard solution for Atomic
Absorption Spectrometry (AAS), 1 g L⁻¹ in 2 % HNO₃, were obtained
from Thermo Fisher Scientific. CuSO₄, ZnSO₄, MgSO₄, NaCl, K₂HPO₄,
KH₂PO₄, CH₃COONa, biotin, 1,3-bis[tris(hydroxymethyl)methylamino]
propane(BisTris), N-methyl-2-pyrrolidone (NMP), cysteamine 95 % and
6-mercaptohexanoic acid (6-MHA) were obtained from Sigma Aldrich
(Merck, Germany). Protease inhibitors cocktail cOmplete Mini EDTA-
free was from Roche. Amino-functionalized CNTs (CNT-NH₂) were ob-
tained from DropSens (Spain). 1 mg mL⁻¹ CNT-NH₂ dispersions were
prepared in NMP by sonicating for four hours. H₂SO₄ 25 % from Labkem
(Spain), acetic acid glacial and EtOH 96 % were from CARLO ERBA
Reagents (Germany). All chemicals for protein purification were high
purity products. All solutions were prepared using ultrapure MilliQ
water (18.2 MΩ cm). Sodium acetate (NaOAc) buffer solutions were
prepared by mixing CH₃COONa and acetic acid in an appropriate ratio
to obtain the desired pH and a final concentration of 0.1 M. 40 mM
MOPS buffer pH 7.0 was prepared by dissolving MOPS in MilliQ water
with adjustment of pH to 7.0 with 4 M NaOH. Potassium phosphate
buffers (PBS) were prepared by dissolving K₂HPO₄ and KH₂PO₄ in
MilliQ water in various ratios for desired pH values with addition of
NaCl when necessary.

2.2. Plasmid construction for met-rich chimeric CueOs

The plasmids used in this study are listed in Table S3. The chimera
vector was constructed by amplifying the Met-rich domain of *cueO* gene
of *E. coli* from the plasmid pAV97 using primers JB613 and JB614. The
primers JB619 and JB620 were used to amplify the backbone vector,
containing partial *cueO* from *H. alvei*, from the plasmid pAV182 allowing
complementary ends. Finally, the plasmid pJB310 was obtained by
ligation of the two DNA fragments with 5× InFusion Snap assembly kit
following supplier recommendations. The plasmid pJB312 was similarly
produced using primers JB615 and JB616 to obtain the Met-rich domain
of *cueO* gene of *H. alveii* from the plasmid pAV182. The backbone
plasmid was amplified from pAV97 with primers JB617 and JB618. The
plasmids pJB310 and pJB312 were sequenced to verify the construction.
The sequence primers are listed in Table S4.

2.3. Protein expression and purification

E. coli DH5α cells transformed with the desired plasmid were grown
in fresh LB broth (Miller's) supplemented with ampicillin (Amp) at a
final concentration of 100 µg mL⁻¹. The culture was incubated overnight
at 37 °C with agitation (150 rpm) until the optical density at 600 nm
(OD₆₀₀) reached approximately 0.6. Induction of gene expression was
initiated by adding 0.1 mM IPTG, followed by incubation for 24 h at
25 °C with agitation (150 rpm) until an OD₆₀₀ of 5–6 was reached in the
presence of 0.5 mM CuSO₄.

The cells were subsequently harvested by centrifugation at 5000 rpm, washed with 50 mM PBS with 150 mM NaCl at pH 7.4, and resuspended at 4 °C in the same buffer containing a protease inhibitor cocktail, 0.02 mg L⁻¹ DNase I, and 4 mM MgSO₄. Cell lysis was performed using a French Press (Thermo Scientific) at 4 °C, applying 1000 psi for two cycles. Cell debris was removed by ultracentrifugation at 40000 rpm for 45 min at 4 °C. The resulting supernatant was manually filtered through a 0.45 µm cutoff membrane and subsequently loaded onto a Strep-Tactin®XT 4Flow® 5 mL column (IBA Lifesciences) at a flow rate of 1.5 mL min⁻¹ (PreCol pressure = 0.3 MPa, ΔCol = 0.13 MPa) using a 50 mL superloop. The column had been pre-equilibrated with PBS containing 150 mM NaCl at pH 7.4. Non-adsorbed proteins were removed by washing the column with 50 mM PBS containing 150 mM NaCl at pH 7.4, while CueO was eluted using 50 mM PBS supplemented with 5 mM biotin at pH 7.4.

The eluted CueO was concentrated using an Amicon® Ultra Cen-
trifugal Filter (Millipore) with a molecular weight cutoff of 30 kDa. The
concentrated protein was subsequently desalted in 40 mM MOPS buffer
(pH 7.0) using a Sephadex® G-25 (Cytiva) 5 mL column.

An in vitro copper incorporation step was performed for at least one aliquot of each CueO preparation by dialyzing the protein for 14 h at 4 °C in 40 mM MOPS buffer (pH 7.0) supplemented with five equivalents of CuSO₄ under aerobic conditions. Excess copper was subsequently removed by desalting in 40 mM MOPS buffer (pH 7.0) using a Sephadex® G-25 (Cytiva) 5 mL column. The desalted CueO was then concentrated to a final concentration of 100–300 µM using an Amicon® Ultra Centrifugal Filter (Millipore) with a 30 kDa molecular weight cutoff. The purified CueO preparation was aliquoted, flash-frozen in liquid nitrogen, and stored at –80 °C. The apo-enzymes were expressed without the addition of copper to the culture medium and the in vitro copper incorporation step was not performed during the purification.

Concentrations of enzymes were determined by UV–vis spectroscopy (Cary 60 UV–vis spectrophotometer, Agilent Technologies) from the absorbance at λ = 280 nm using extinction coefficients calculated with ExPASy ProtParam (ε (EcCueOchimHaMet) = 63,035 M⁻¹ cm⁻¹, ε (HaCueOchimEcMet) = 80,565 M⁻¹ cm⁻¹) [19]. The presence of CuT1 was confirmed from the absorption spectra at λ<sub>CuT1</sub> = 610 nm, ε = 5000 M⁻¹ cm⁻¹ [20,21] and a characteristic shoulder of binuclear CuT3 was observed at λ<sub>CuT3</sub> = 330 nm [22].

2.4. Solution-based enzymatic assays

Enzymatic activity of CueOs in solution was determined with Cary 60 UV-vis spectrophotometer (Agilent Technologies) by measuring the absorption increase in the presence of ABTS in 0.1 M NaOAc buffer pH 5 at λ = 420 nm, T = 30 °C.

2.5. Inductively coupled plasma optical emission spectroscopy (ICP-OES)

To prove the completion of CuT1 and TNC active sites with Cu atoms,
ICP-OES was applied using an iCAP-7000 ICP-OES analyzer (Thermo
Scientific). Protein amounts were 150 and 75 µg for each CueO prepa-
ration. The average values reported in Table S1 are based on at least four
replicates for each concentration.

2.6. Circular dichroism spectroscopy (CD)

Circular dichroism (λ range: 250–190 nm) was used for the charac-
terization of secondary structure of enzymes. Measurements were
recorded on Jasco J-815 spectrophotometer at 25 °C (interval: 0.2 nm;
pitch: 0.2 nm; DIT: 1 s, bandwidth: 1 nm; scanning speed: 20 nm min⁻¹).
Enzyme stocks were defrosted on ice and centrifuged. After that,
desalting into 10 mM phosphate buffer pH 7.0 was performed on Zeba
Spin Desalting columns (7 K MWCO). Final concentrations of samples
were measured on Cary 60 UV-vis spectrophotometer (Agilent Tech-
nologies). Samples were placed in a quartz cuvette with a path length of
0.1 cm. Each spectrum was accumulated 3 times. Data convolution
processed in BESTSEL [23].

2.7. Electrochemistry

Cyclic voltammetry (CV) and chronoamperometry (CA) were per-
formed using a potentiostat from Autolab PGSTAT30 controlled by 2.1
Nova software (Metrohm) in a 3-electrode electrochemical cell. Poly-
crystalline gold electrode was used as the working electrode. For
comparative purpose, glassy carbon electrode modified with amine-
functionalized carbon nanotubes (CNT-NH₂) was alternatively used.
Mercury sulfate Hg/Hg₂SO₄ (sat. 1 M K₂SO₄) was the reference elec-
trode, and a Pt wire served as auxiliary electrode. Potential range was
converted to Ag/AgCl (sat. 3 M KCl) by addition of +430 mV. The cell
was thermostated at 30 °C and saturated with O₂ by continuous
bubbling throughout the experiments. 0.1 M NaOAc buffer pH 5 was used as the electrolyte unless otherwise specified.

## 2.8. Preparation of electrodes

The gold disc electrodes (from BioLogic, geometric surface area $A_{geom}$ = 0.02 cm²) were polished for 5 min using alumina slurry with particle size 1, 0.3 and 0.04 µm (ESCIL, Lyon France) in the order of decreasing particle size on a fabric polishing pad (PRESI), ultrasonicated in a water-alcohol solution (EtOH:H₂O = 1:1) for 5 min and electrochemically cleaned in a 0.05 M H₂SO₄ by cyclic voltammetry till stable and reproducible voltammograms are obtained (20 cycles). The electrodes were then washed with Milli-Q water, dried and immersed in 5 mM cysteamine or 6-MHA in EtOH overnight for SAM formation or directly used for enzyme immobilization. 5 µL of 0.5 µM CueOs (unless otherwise specified) diluted from stock in 40 mM MOPS pH 7.0 were drop-casted on the surface of Au and stored for 15 min at 4 °C. The electrode was then rinsed with Milli-Q water and used immediately. The electroactive area was measured from the peak area of gold oxide reduction at 0.9 V vs Ag/AgCl in 0.05 M H₂SO₄, assuming the conversion coefficient of 390 µC cm⁻² [24]. Glassy carbon electrode (GC electrode, geometric surface area of $A_{geom}$ = 0.071 cm²) was polished on wet PRESI adhesive pad (4000), ultrasonicated in 1:1 H₂O:EtOH mixture for 5 min, rinsed with MilliQ and EtOH, then dried. 5 µL of 1 mg mL⁻¹ CNT-NH₂ suspension were drop-casted on clean GC electrode and dried in vacuum. All the currents are reported to the electroactive surface area for gold electrodes and geometric surface area for GC electrodes. All electrochemical experiments were performed at least in triplicate.

## 2.9. Surface plasmon resonance (SPR)

An Autolab SPRINGLE SPR instrument (Eco Chemie, the Netherlands) was used for recording CueO adsorption kinetics on bare gold disks (ø 25 mm, SPR.BK7.AU.10 from Kinetic Evaluation Instruments, The Netherlands). 10 mM potassium phosphate buffer +25 mM NaCl pH 7.4 (PBS) was used as a running buffer and for the stabilization of the surface. After stabilization, 50 µL of enzyme aliquots were injected on gold in 100 pM-10 µM range of concentrations. Positions of gold sensor on hemicylinder prism were changed for every protein concentration, as regeneration of the surface was not allowed. The SPR signal was monitored at room temperature (25 °C). Coverage of gold surfaces by enzymes was calculated from change of resonance angle according to $122 mdeg = 1 ng mm^{-2}$ (Autolab Springle).

## 2.10. Molecular dynamics and protein modeling

Molecular dynamics (MD) simulations were performed using YASARA software [25] as previously described [9]. Briefly, all proteins were simulated for 500 ns using the AMBER14 force field [26], with a time step 2.5 fs for bonded interactions and 5.0 fs for nonbonded interactions, under standard conditions (298 K and 1 atm) in the NPT ensemble. To monitor structural fluctuations during the simulation, the volume of the Met-rich domain was calculated every 250 ps using a built-in YASARA command. Additionally, the shortest distance from the CuT1 site to the protein surface, generated using a solvent probe radius of 3 Å, was determined. The coordinates of the closest surface point to the CuT1 site were found, and both this distance and the corresponding surface residue at these coordinates were recorded every 250 ps.

Molecular graphics and structural analyses were performed using UCSF ChimeraX 1.6.1 (University of California, USA). For *EcCueO*, we utilized the crystallographic structure with a fully resolved Met-rich domain (PDB entry 3OD3). While we previously deposited the crystallographic structure of *HaCueO* (PDB entry 8R4F), a significant portion of its Met-rich domain was not resolved. Therefore, for illustration and analysis of *HaCueO*, we used a representative structure obtained by averaging snapshots over the 500 ns of molecular dynamics simulation

[9]. Copper atoms in all structures are represented as spheres with van der Waals radii of 2.0 Å. The structures of chimeras were manually constructed by swapping the Met-rich domains between *EcCueO* and *HaCueO* using YASARA software, followed by energy minimization to ensure realistic bond lengths and angles.

Molecular lipophilicity surface mapping was performed using ChimeraX by calculating the hydrophilic/hydrophobic potential at each grid point as the sum of atomic contributions to the n-octanol/water partition coefficient. Non-protein atoms were excluded from the calculation. The grand average of hydropathicity (GRAVY index) was calculated by summing the corresponding hydropathy scores of all protein residues on the Kyte-Doolittle scale and confirmed using the ExPASy ProtParam tool [19].

# 3. Results and discussion

## 3.1. Direct O₂ reduction by *EcCueO* and *HaCueO* WT proteins on self-assembled-monolayer (SAM) modified gold electrodes

The comparative direct O₂ reduction was initially investigated at polycrystalline gold electrodes modified with self-assembled monolayers (SAMs) of various thiols, onto which *EcCueO* or *HaCueO* were adsorbed (Fig. 2). Cysteamine and 6-MHA were selected to induce positively and negatively charged surfaces, respectively. Both proteins exhibited catalytic activity for ORR when immobilized on the positively charged SAM, generating currents of –37 ± 15 and –10 ± 3 µA cm⁻² at a potential of 0.2 V vs Ag/AgCl for *EcCueO* and *HaCueO* respectively, with a similar onset potential of +470 mV vs Ag/AgCl. This onset potential closely resembles that previously reported for both enzymes on CNT-based electrodes [8,9] and corresponds to electron transfer via the CuT1 site. In contrast, no enzymatic activity was observed on the negatively charged 6-MHA SAM gold electrode for either protein. This marked difference that depends on the charge of the electrochemical interface aligns with our previous findings for the same CueOs adsorbed onto carbon-modified electrodes [9]. Specifically, positively charged CNTs promoted catalysis, while negatively charged ones allowed only weak catalysis. This distinct behavior was attributed to the enzyme's orientation, driven by electrostatic interactions between the negative local charge near CuT1 and the electrode surface [8,9]. On SAM modified gold electrodes, electron transfer for ORR by CueOs is thus also controlled by the environment of CuT1 whatever the composition of the Met-rich domain. However, a lower current is observed with *HaCueO* on positively charged SAM electrode compared to *EcCueO*, which is not the case on positive CNT-NH₂ (Fig. S1). We previously reported that while both enzymes exhibit a similar patch of negatively charged residues near the CuT1 site, the charges in *HaCueO* are distributed more homogeneously across the surface [9]. This may induce a larger distribution of orientation of *HaCueO* on more planar gold surface compared to CNT film, hence a lower current for direct ORR.

## 3.2. Direct O₂ reduction by *EcCueOs* WT and ΔMet on bare gold electrodes

We further aimed to evaluate whether the Met-rich domain has a greater impact on enzymatic O₂ reduction on bare gold electrode, which lack dominant surface charges. To this end, we first compared the *EcCueO* WT with its ΔMet variant (Fig. 3).

*EcCueO* WT and ΔMet exhibited a catalytic current for ORR with an onset potential for both enzymes at +450 mV, consistent with the value obtained on SAM-based electrodes.

A particular feature requires attention. An increase in catalytic current with continuous CV cycling is observed especially for the *EcCueO* WT, leveling off after few cycles at 5 mV s⁻¹ (Fig. 3A). This behavior has been previously reported for this protein by Chumillas et al. [18], and was observed by our group with another CueO protein, *Thermus thermophilus* CueO [27]. Chumillas et al. attributed the activation process to
<figure><img src="image_3.png" /><figcaption>**Fig. 2.** CVs for direct O₂ reduction by (A) *EcCueO* WT and (B) *HaCueO* WT on cysteamine (green curve) or 6-MHA (red curve) modified gold electrodes. Insets are zooms of CVs on 6-MHA in the presence (red curves) or in the absence of enzymes (grey curves). 0.1 M NaOAc buffer pH 5.0, *T* = 30 °C, O₂ sat., scan rate 5 mV s⁻¹. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)</figcaption></figure>

<figure><img src="image_4.png" /><figcaption>**Fig. 3.** CV consecutive scans of ORR by *EcCueOs* adsorbed on bare gold electrode. (A) *EcCueO* WT and (B) *EcCueO* ΔMet (black curves). CVs on bare gold electrodes in the absence of enzymes are shown in grey. Insets: normalized currents of 10 consecutive CV cycles of CueOs activity at *E* = 0.25 V vs Ag/AgCl. Error bars correspond to standard deviation of 3 replicates. 0.1 M NaOAc buffer pH 5.0, *T* = 30 °C, O₂ sat., scan rate 5 mV s⁻¹. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)</figcaption></figure>

bond formation between the gold surface and sulfur-containing residues. Although they did not mention specifically the Met-rich domain, the implicated residues likely belong to this domain. Notably, we observed in the current work that the catalytic current tends to stabilize within 2 cycles in the case of *EcCueO* ΔMet, which may support the hypothesis of progressive orientation of CueO WT through the Met-rich domain (Fig. 3B). However, in this configuration, the distance between CuT1 and the electrode should also increase, making it difficult to explain the observed increase in the catalytic current.

### 3.3. Evidence of progressive *EcCueO* metalation

A similar evolution of the CV curves may result from various distinct phenomena beyond progressive adsorption/reorientation. For instance, such an increase in catalytic current may instead be induced by progressive protein activation in the course of CV cycling, as proposed for

*McoA* from *Aquifex aeolicus* [12]. To evaluate whether the activation process was potential-controlled, we ran a chronoamperometric experiment at different applied potentials with *EcCueO* WT adsorbed on the gold electrode. Fig. 4A displays the evolution of the catalytic current with applied potential starting from *E*<sub>app</sub> = +0.43 V down to *E*<sub>app</sub> = +0.18 V vs Ag/AgCl. It can clearly be observed that the catalytic current starts increasing at potentials lower than +0.28 V. Having in mind the cuprous oxidase activity of CueOs, we evaluated the effect of CuSO<sub>4</sub> addition on the activation process. We noticed that the addition of nanomolar amounts of CuSO<sub>4</sub> into the electrolyte at +0.18 V further enhances the activation process (Fig. 4B), while maintaining the same potential dependence as previously described. Furthermore, the value of the catalytic current reached after activation persists even after transferring the electrode to a copper-free buffer (Fig. 4C). This persistence, along with the relatively slow activation kinetics, distinguishes this process from CueO cuprous oxidase activity, where the catalytic current
<figure><img src="image_5.png" /><figcaption>**Fig. 4.** Chronoamperometric measurements of ORR by *EcCueO* WT adsorbed at the bare gold electrode (A) at different applied potentials, (B) with addition of 100 nM CuSO₄ (τ = 5 min) at different applied potentials and (C) with addition of 100–1000 nM CuSO₄ (τ = 5 min) and transfer to Cu²⁺-free buffer (τ = 30 min) at E<sub>appl</sub> = 0.18 V vs Ag/AgCl. 0.1 M NaOAc buffer pH 5.0, T = 30 °C, O₂ sat. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)</figcaption></figure>

rises instantaneously upon CuSO₄ addition, remains constant in its presence, and disappears as soon as the electrode is placed in a copper-free buffer [8]. Moreover, addition of EDTA, known to readily coordinate metal ions, to the electrolyte before CuSO₄ addition induces the stabilization of the current (Fig. S2A). The addition of Zn²⁺ does not induce any changes in the activation process, which is only enhanced after Cu²⁺ addition, strongly suggesting that the activation process is specific to copper (Fig. S2B). Last but not least, the CV of CuSO₄ at the bare gold electrode shows that the onset of Cu²⁺ reduction occurs at ca. + 0.3 V vs Ag/AgCl (Fig. S2C), which matches the potential required for the reductive activation of the protein. Considering the potential at which the activation starts being visible, we thus postulate that it is the electrogenerated Cu²⁺ which induces the activation process.

How can we explain this copper-dependent activation process and what is the copper source? One notable feature of CueO is the presence of additional copper-binding sites within the Met-rich domain. However, these sites likely exhibit low copper-binding affinity, as evidenced by studies on biomimetic Met-containing peptides [28]. In agreement, we recently showed that the Met-rich domain is only acting as transient domain, facilitating Cu⁺ recruitment thanks to these multiple Cu-binding sites [8,9]. However, copper in both redox states can also bind to additional surface-exposed sites on the protein, as observed in several CueO crystal structures obtained in the presence of excess copper ions [29,30]. Accordingly, copper content measured by ICP indicates a copper amount ranging between 4 and 5 atoms per protein (Table S1) indicating partial occupancy of the additional sites. Weak binding, high solvent accessibility together with close location of these Cu-sites to the electrode surface may induce Cu²⁺ release and further Cu⁺ electro-generation. Based on this, we hypothesize that the Cu²⁺ release from the additional binding sites, either into the sample solution or triggered by protein immobilization and further CV cycling, and its subsequent electrochemical reduction to Cu⁺ underlies the observed protein activation.

Additionally, we observed that although *EcCueO* ΔMet does not undergo large activation during continuous cycling (Fig. 3B), it can be activated just as rapidly and to the same extent as the WT after Cu²⁺

addition (Fig. S3). *EcCueO* ΔMet has 17 % lower copper content than WT, as determined by ICP-OES (Table S1). Notably, the ΔMet variant lacks the additional copper-binding sites observed in X-ray crystallography within the Met-rich domain, therefore supporting the conclusion that the initial source of Cu²⁺ originates from these binding sites. We also studied the *EcCueO* variant that cannot coordinate copper at the main substrate-binding Cu5 site (ΔCu5) and thus lacks any cuprous oxidase activity but keeps ORR activity [8]. This variant was much less activated upon Cu²⁺ addition, although about 10 % activation was still obtained (Fig. S3). Two seemingly contradictory conclusions can be deduced from these observations: 1) the Cu5 site is more important for the observed activation process than the entire Met-rich domain; 2) this process should target other sites than Cu5, since a residual activation is still observed in the ΔCu5 mutant. As reported, the CuT2 and CuT3 sites in CueOs could be largely depleted of copper depending on the culture growth conditions [31]. We thus believe that the electrogenerated Cu²⁺ could fill these vacant sites in the TNC, thereby increasing the amount of active enzymes and enhancing the catalytic current.

To confirm this hypothesis, we isolated copper-free enzymes, apo-*EcCueO* WT and apo-*EcCueO* ΔCu5. Both proteins contained only traces of copper (Table S1) and appeared colorless, confirming the absence of the CuT1. When immobilized on the gold electrode, both apo-enzymes exhibited a low initial current (< 0.3 µA cm⁻²), consistent with the lack of active copper centers. However, upon the addition of Cu²⁺, both proteins showed a rapid and pronounced activation (Fig. S3C). Notably, apo-*EcCueO* ΔCu5 reached only ca. 2 µA cm⁻², whereas the amplitude of the apo-*EcCueO* WT activation was higher and the current reached 6 µA cm⁻², comparable to that observed for the pre-metalated *EcCueO* WT (Fig. 3). This difference aligns with the previously reported by us ~2-fold lower intrinsic activity of *EcCueO* ΔCu5 compared to the WT [8]. These results confirm that the metalation doesn't concern the Cu5 but rather TNC and CuT1, as full occupancy of CuT1/TNC copper sites is required to produce an active holoenzyme from the apo form (Scheme 1). From a more practical perspective, we will further demonstrate that this electrochemical method can be broadly applied to all CueOs to achieve complete metalation of immobilized enzymes.
<figure><img src="image_6.png" /><figcaption>**Scheme 1.** Proposed mechanism for in situ electrochemical metalation of CueO.</figcaption></figure>

### 3.4. Comparative direct O₂ reduction by *EcCueO* and *HaCueO* WT and ΔMet on bare gold electrodes

Direct O₂ reduction by *HaCueO* proteins at bare polycrystalline gold electrodes was then compared to ORR by *EcCueO* to evaluate the impact of the conformation and chemical composition of the Met-rich domain on enzyme adsorption/orientation and, consequently, electron transfer kinetics. In contrast to catalysis by *EcCueO* WT, CV measurements of ORR by *HaCueO* WT reveal a weak catalytic response (Fig. S4). Nevertheless, continuous cycling shows the similar trend in activation process as described with *EcCueO*, suggesting the same copper-dependent activation process should occur (Fig. S4). A valid comparison of *HaCueO* and *EcCueO* enzymes requires that both have the same degree of metalation. Thus, we took advantage of the electrochemically-assisted metalation described above to activate the enzymes by chronoamperometry in the presence of 100 nM CuSO₄. Fig. 5A shows a typical CV response for ORR by the two proteins at three different concentrations (500 nM, 1 µM and 5 µM) after the bioelectrode is transferred back to a copper-free electrolyte. Direct O₂ reduction occurs on *EcCueO* WT with minor changes in the catalytic current with increasing enzyme concentrations. In contrast, a rather weak catalytic response can be obtained with *HaCueO* WT regardless of the enzyme concentration used for adsorption.

Since *HaCueO* is active on SAM modified gold electrode, this distinct

behavior suggests the adsorption takes place in a different manner on bare gold. One main difference between *EcCueO* and *HaCueO* being the length of the Met-rich domain, different electrocatalytic activity may be attributed to adsorption through the Met-rich domain that would control the distance between the electrode surface and the CuT1, hence the electron transfer for catalysis. The longer Met-rich domain in the case of *HaCueO* compared to *EcCueO* would have greater negative impact on the electron transfer. Accordingly, ORR occurs using *HaCueO* ΔMet (Fig. 5B) while it is only enhanced by about 10 % using *EcCueO* ΔMet. We will discuss that point further below. To be noted, the current magnitude with *HaCueO* ΔMet is also 3 times lower than *EcCueO* ΔMet, a ratio similar to that one obtained on positive SAM (Fig. 2), where adsorption is taking place without the involvement the Met-rich domain.

Apart from a difference in electron transfer kinetics, we evaluated by SPR whether different amounts of enzymes immobilized on the surface could explain the different electrocatalytic activity of *EcCueO* compared to *HaCueO*. As can be seen on Fig. 6, enzyme coverage on the gold surface increased as CueO concentration raised from 0.5 to 1 µM, especially in the case of *HaCueO*, leveling off for the highest concentration. Nevertheless, similar coverage range within 4–8 pmol cm⁻² calculated for all the CueOs cannot explain the drastic difference obtained in ORR catalysis.

### 3.5. Construction of chimeric CueOs to confirm the involvement of the met-rich domain in direct O₂ reduction on gold electrode

To further investigate the size effect of the CueO Met-rich domain on ORR catalysis at a bare gold electrode, we designed chimeric proteins. For this purpose, Met-rich domains were swapped between *EcCueO* WT and *HaCueO* WT, producing two new chimeric mutants: *EcCueO* with the Met-rich domain from *HaCueO*, named *EcCueO*<sup>chimHaMet</sup>, and *HaCueO* with the Met-rich domain from *EcCueO* named *HaCueO*<sup>chimEcMet</sup> (Fig. 7).

Circular dichroism spectra of both chimeric CueO showed that the structure is maintained, suggesting that exchanging the Met-rich domain does not affect the secondary structure (Fig. S5). Both preparations showed the typical blue coloration, indicating the presence of the CuT1 center. This was confirmed by the UV-visible spectra that reveal the characteristic peak around 610 nm and shoulder around 330 nm, corresponding to CuT1 and Cu-T3 absorbance, respectively (Fig. S5). The ICP-OES copper content quantification shows an average of 3.5 ± 0.1 copper atoms per CueO in the case of *EcCueO*<sup>chimHaMet</sup>, which is approximately one less copper atom than the *EcCueO* WT (4.7 ± 0.2) (Table S1). *HaCueO*<sup>chimEcMet</sup> exhibits 3.1 ± 0.2 copper atoms per CueO, which agrees with the less than four copper atoms measured in *HaCueO*

<figure><img src="image_7.png" /><figcaption>**Fig. 5.** (A) CVs for ORR at bare gold electrodes by *EcCueO* WT (left) and *HaCueO* WT (right) at different concentrations of immobilized enzyme 0.5 µM (black curves), 1 µM (red curves) and 5 µM (green curves); (B) CV for ORR by *EcCueO* ΔMet (left) and *HaCueO* ΔMet (right) with immobilized CueO at a concentration of 500 nM. CVs at the bare gold electrode in the absence of enzymes are in grey. 0.1 M NaOAc buffer pH 5.0, *T* = 30 °C, O₂ sat., scan rate 5 mV s⁻¹. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)</figcaption></figure>
<figure><img src="image_8.png" /><figcaption>**Fig. 6.** Typical SPR sensorgrams displaying interaction of 1 µM CueOs with gold surface (left) and deduced coverage of gold surface by CueOs (right). The enzyme was added at τ = 400 s and washed away at τ = 800 s. Running buffer: 10 mM potassium phosphate buffer +25 mM NaCl pH 7.4, T = 25 °C. A monolayer of enzymes corresponds to Γ = 5.87 pmol cm⁻², average enzyme diameter was set as φ = 6 nm referring to *EcCueO* structure (PDB entry 3OD3). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)</figcaption></figure>

<figure><img src="image_9.png" /><figcaption>**Fig. 7.** Structures of: (Top) *EcCueO* WT (left) and *Ha CueO* WT (right) and (Bottom) chimera mutants *EcCueO*<sup>chimHaMet</sup> (left) and *HaCueO*<sup>chimEcMet</sup> (right).</figcaption></figure>

WT (3.1 ± 0.1) (Table S1). This suggests that the heterologous expres-
sion of *HaCueO*<sup>chimEcMet</sup> into *E. coli* hosting cells works with similar ef-
ficiency as the *HaCueO* WT. Enzymatic activity for both chimeric CueOs
was quantified in solution using ABTS as the electron donor in classical
spectrometric assays (Fig. S6, Table S2). Interestingly, the activities of
chimeras were different from the activities of their respective WT.
*EcCueO*<sup>chimHaMet</sup> activity decreases compared to WT *EcCueO*, while

*HaCueO*<sup>chimEcMet</sup> activity increases compared to WT *HaCueO*. It is well known that ABTS activity by CueOs is lower compared to other MCOs due to the presence of the Met-rich domain covering the CuT1. In agreement, the results obtained with the chimeric proteins highlight that the larger the Met-rich domain lowers the ABTS activity and vice versa.

O₂ reduction by chimeric CueOs adsorbed on bare gold electrodes
<figure><img src="image_10.png" /><figcaption>**Fig. 8.** Comparison of ORR catalytic current density at E = + 0.25 V vs Ag/AgCl obtained from CV measurements at the bare gold electrode: *EcCueO* WT (black), *EcCueO* ΔMet (red), *HaCueO* WT (blue), *HaCueO* ΔMet (green), *EcCueO*<sup>chimHaMet</sup> (purple), *HaCueO*<sup>chimEcMet</sup> (brown). 0.1 M NaOAc buffer pH 5.0, T = 30 °C, O<sub>2</sub> sat., scan rate 5 mV s<sup>-1</sup>. Error bars correspond to standard deviation of 3 replicates. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)</figcaption></figure>

and electrochemically activated in the presence of Cu²⁺ was then compared to the WT and ΔMet proteins. CV current densities measured at +0.25 V vs Ag/AgCl are compared for all proteins in Fig. 8. Two interesting observations can be made. First, while *HaCueO* WT ORR activity is nearly zero, *HaCueO*<sup>chimEcMet</sup> exhibits ORR with the same magnitude as the *HaCueO* ΔMet. This is a clear indication that the Met-rich domain of *HaCueO* is involved in the slow electron transfer rate at the gold electrode. Accordingly, *EcCueO*<sup>chimHaMet</sup> gives 3 times lower catalytic current compared to *EcCueO* WT, further implying that the Met-rich domain of CueO is involved in the electron transfer at the gold electrode.

### 3.6. How do met-rich domains interfere with the gold electrode to drive the electron transfer process?

We first examine the specific interactions that may occur between the methionine-rich domain and the gold surface. Electrostatic interactions between CueO enzymes and bare gold electrodes can be excluded as a significant factor. Gold surfaces typically exhibit an iso-electric point around pH 5, depending on surface oxidation states [32]. Under our experimental conditions, this implies that the gold surface carries a slight negative charge during protein adsorption and remains neutral during electrochemical measurements. Our previous studies have shown that enzyme reorientation can occur in response to changes in electrostatic interactions between the adsorption phase and subsequent immersion in the electrolyte [33]. If such reorientation occurred under the current conditions, we would anticipate behavior akin to that observed with 6-MHA-modified electrodes—namely, an absence of catalytic current for both *HaCueO* and *EcCueO* proteins—which contradicts our observations. Turning to hydrophobic interactions, our

experiments revealed that the enzyme droplet maintained a well-defined shape upon contacting the gold surface, suggesting moderate hydrophobicity. This observation aligns with literature reports indicating that, while freshly prepared gold surfaces are inherently hydrophilic under ultra-high vacuum (UHV) conditions, upon exposure to ambient air, these surfaces rapidly adsorb airborne contaminants, leading to increased hydrophobicity and contact angles of approximately 50–60° [34]. In agreement, contact angles for freshly electropolished gold surfaces were found to be close to 60° [35]. The molecular lipophilicity potential (MLP) analysis of *EcCueO* and *HaCueO* WT enzymes was performed to search for any hydrophobic interaction with the gold surface (Fig. S7). No specific surface-exposed hydrophobic regions were observed, neither for the whole enzymes nor for the Met-rich domain. Moreover, the distribution of hydrophilic/hydrophobic residues on the surface of both enzymes, including the Met-rich domains, is rather homogeneous. In agreement, the grand average of hydropathicity (GRAVY) indexes are weakly negative (–0.201 for *EcCueO* and – 0.290 for *HaCueO*) showing that proteins are overall slightly more hydrophilic than hydrophobic. These analyses rule out the involvement of hydrophobic interactions that would control the enzyme adsorption for electron transfer process.

Could the affinity of methionine residues for gold surface which is often evocated in publications related to CueO immobilization on gold be the key in CueO adsorption? Contrary to cysteine bearing a reactive thiol SH-group which can form a covalent Au–S bond, in the case of methionine the alkyl protected sulfur is unlikely to form covalent bonds. Rare studies reported the nature and strength of methionine interaction with gold. Interactions as a separate residue indicated a noncovalent bonding character [36,37]. Formation of monolayers of methionine on gold colloids were analyzed by SERS, and adsorption through the sulfur atom was demonstrated, although with a strength of interaction lower than in the case of cysteine residues [38]. Otherwise, helical peptides were bound to gold via multiple interactions with methionine residues [39]. The comparison of adsorption on gold with a peptide with similar secondary structure but without methionines revealed a stronger adsorption via the thioether side chain of methionine. Finally, a molecular dynamic study on a peptide showed that methionine residue has strong affinity, largely contributing to the binding energy of the entire peptide chain to the gold surface [40]. Since SPR measurements made in the present work do not show any difference in enzyme coverage, we can hypothesize that the larger number of methionine residues in the Met-rich domain of *HaCueO* can increase the strength of the interaction, as shown with peptides.

However, assuming that the enzyme interacts with the gold surface via the methionine-pool of the Met-rich domain, the lack of DET for *HaCueO* WT compared to *EcCueO* WT, as well as the increased catalytic current observed in both ΔMet variants remain puzzling. To investigate this, we performed 500 ns molecular dynamics (MD) simulations for all four protein variants and analyzed the trajectories to assess the steric hindrance induced by the Met-rich domain related to the CuT1. First, although the Met-rich domain showed high flexibility during the simulations, its volume remained relatively constant: between 6.3 and 6.6 nm³ for *EcCueO* WT and between 10.2 and 10.6 nm³ for *HaCueO* WT (Fig. S8), i.e. 63% larger volume in *HaCueO*. This difference exceeds the ~44% obtained by just counting the number of extra-residues present in the domain. Such bulkier Met-rich domain in *HaCueO* could induce steric hindrance of the enzyme over the gold surface, decreasing the probability of interfacial electron transfer.

To confirm whether the bulky Met-rich domain increases the distance between the CuT1 site and the protein surface, and thus the electrode surface, we further analyzed the MD trajectories by measuring the shortest distance from the CuT1 site to the protein surface and identifying the surface residue at that point (Fig. 9). In *HaCueO*, the histogram of CuT1-to-surface distances shows that CuT1 is typically 12–15 Å away from the surface, though in some domain conformations it approaches as close as 10 Å. Interestingly, the histogram of *EcCueO*
<figure><img src="image_11.png" /><figcaption>**Fig. 9.** Analysis of the 500 ns MD-trajectories of (from top to bottom) *EcCueO* WT, *HaCueO* WT, *EcCueO* ΔMet and *HaCueO* ΔMet. (Left panel) Histograms showing the distribution of distances from the CuT1 site to the nearest surface point of the enzyme over the course of the simulation. (Right panel) MD-averaged structures of the corresponding proteins, with surface regions closest to CuT1 colour-coded by this average distance: from green (5 Å) to red (15 Å). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)</figcaption></figure>

exhibits a bimodal distance distribution with two maximums appearing at 13–14 Å and 8–10 Å. The closest surface residues in both proteins are located adjacent to, but not within, the Met-rich domain (Fig. 9). Notably, in *EcCueO*, a surface region flanking one side of the domain comes closer to CuT1, explaining the first maximum on the histogram. These MD findings align with the experimental behavior on gold electrodes: the bulkier Met-rich domain in *HaCueO* prevents CuT1 from approaching the electrode closer than ~10 Å, resulting in the almost complete absence of DET. It should be noted that in reality this distance should be even higher since the electrode is not able to penetrate into all the twists of the proteins surface. In contrast, the more compact Met-rich domain in *EcCueO* covers less the CuT1 site, theoretically allowing it to approach the electrode surface several Å closer, thus explaining the presence of DET on gold.

Removing the Met-rich domain drastically reduces the CuT1-to-surface distance for both proteins (Fig. 9). Notably, *HaCueO* ΔMet even shows a slightly shorter distance (5–7 Å) compared to *EcCueO* ΔMet (6–8 Å). This reduction in distance should be reflected in the shape of the catalytic voltammograms. Indeed, both ΔMet variants display a steeper initial current rise than *EcCueO* WT, and an inflection point, indicative of an increased interfacial electron transfer rate constant ($k^0$)

(Fig. 5). Although the low current of *HaCueO* WT prevented reliable blank subtraction, fitting the catalytic curves of *EcCueO* WT, *EcCueO* ΔMet and *HaCueO* ΔMet to a one-electron catalytic irreversible EC' model with infinite dispersion orientations [41] supports this observation (Fig. S9). The primary difference between *EcCueO* WT and *EcCueO* ΔMet lies in the $k^0/k_{cat}$ ratio, which is twice as high for ΔMet. Assuming similar $k_{cat}$ values, this indicates a higher $k^0$ for ΔMet, consistent with MD results showing that CuT1 can approach closer to the electrode surface (Fig. 9). Likewise, *HaCueO* ΔMet exhibits even greater $k^0/k_{cat}$ ratio, reflecting slightly higher T1 accessibility compared to *EcCueO* ΔMet, and confirming the insights obtained from MD.

## 4. Conclusion

In this work we aimed to understand how differences in size and methionine content of the Met-rich domains of two CueOs and their variants affect direct O₂ reduction on bare and SAM-modified gold electrodes. First, we confirmed, that the electrostatic interactions are the primary driving force governing both enzymes orientation and electro-catalytic current on SAM-modified electrodes, consistent with previous results on CNT-based electrodes and independent of Met-rich domain size.

However, when immobilized on bare gold electrodes, both CueOs demonstrated distinct features. Unlike on SAM-modified electrodes, a significant difference emerged between *EcCueO* WT and *HaCueO* WT on bare gold electrodes. Indeed, the latter demonstrated a very low catalytic current density, although the surface coverage and kinetics of adsorption was comparable for all variants, as confirmed by SPR. Pointing at the role of the Met-rich domain, the ΔMet mutant of *HaCueO* recovered catalytic current, and a similar increase was observed for the *EcCueO* ΔMet variant. To further explore this, we constructed two chimeric CueOs by swapping the two Met-rich domains. The electrochemical response of these chimeric enzymes was intermediate between the two WT CueOs, suggesting that the Met-rich domain but also the remainder of the protein structure influence the electrochemical behavior of CueO enzymes on gold surfaces.

Despite previous suggestions of a specific affinity between methionine residues and gold surfaces that would favor adsorption and electron transfer process, our findings indicate that the Met-rich domain hinders rather than facilitates electrocatalysis by CueOs. Indeed, both ΔMet variants outperformed their wild types, while *HaCueO* WT, which contains the highest number of methionine residues, exhibited negligible DET. MD-simulations support a steric explanation: in *HaCueO* the Met-rich domain significantly occludes the CuT1 site, positioning it more than 12–15 Å away from the electrode surface, even when adsorbed through the Met-rich domain, and thus hindering an efficient electron transfer. In contrast, *EcCueO*'s smaller Met-rich domain allows the CuT1 to approach closer, and its deletion in both enzymes further shortens this distance, explaining the improved current densities.

Therefore, while we cannot completely rule out the role of methionine-gold interactions in CueOs orientation, our results highlight that the accessibility of the CuT1 site—correlated with the size and presence of the Met-rich domain—emerges as the primary factor influencing electron transfer efficiency.

A second feature that we analyzed in depth in this work is that the catalytic current of all studied CueOs increased upon continuous cycling or when applying sufficiently negative potentials. Based on the control experiment results, we attributed this feature to the electrochemical metalation of the CueOs, most likely of the TNC, by Cu⁺ species generated at the electrode surface. To our knowledge, this is a first report of electrochemically-assisted metalation of MCOs, potentially paving the way for the large-scale production of MCO followed by in vitro maturation, as high copper concentrations are toxic in vivo.
Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

CRediT authorship contribution statement

**Vita Saska:** Writing – original draft, Methodology, Investigation, Formal analysis. **Paolo Santucci:** Formal analysis. **Anne de Poulpiquet:** Writing – review & editing. **Didier Gasparutto:** Methodology, Formal analysis. **Umberto Contaldo:** Supervision, Methodology, Formal analysis. **Ievgen Mazurenko:** Writing – review & editing, Supervision, Methodology, Investigation, Formal analysis, Conceptualization. **Elisabeth Lojou:** Writing – review & editing, Supervision, Project administration, Funding acquisition, Conceptualization.

Declaration of generative AI and AI-assisted technologies in the
writing process

During the preparation of this work the authors used ChatGPT in
order to improve the readability and language of the manuscript. After
using this tool, the authors reviewed and edited the content as needed
and takes full responsibility for the content of the published article.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have influenced the work reported in this paper.

Acknowledgements

Vita Saska received funding from an Aix-Marseille University grant. Paolo Santucci received funding from the French government under the France 2030 investment plan, as part of the Initiative d'Excellence d'Aix-Marseille Université—A*MIDEX (AMX-21-PEP-001). This work was supported by the National Research Agency (ANR, France) under the grant MetCop (ANR-21-44CE-0024). D.G. is also grateful to the French National Research Agency (Labex ARCANE, ANR-11-LABX-003 and CBH-EUR-GS, ANR-17-EURE-0003) that supported part of this study. We are grateful to Jérôme Becam, Alexandra Vergnes, Laurent Aussel and Benjamin Ezraty (LCB, France) for designing CueOs vectors and *E. coli* strain for protein expression and purification, as well as for fruitful discussions. We thank Marianne Ilbert (BIP, France) for ICP-OES analysis.

Appendix A. Supplementary data

Supplementary data to this article can be found online at https://doi.
org/10.1016/j.bioelechem.2025.109051.

Data availability

Data will be made available on request.

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