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Article

Fungal Laccases with High and Medium Redox Potential: Is the T1 Center Potential a Key Characteristic of Catalytic Efficiency in Heterogeneous and Homogeneous Reactions?

by
Olga Morozova
*,
Maria Khlupova
,
Irina Vasil’eva
,
Alexander Yaropolov
and
Tatyana Fedorova
*
A. N. Bach Institute of Biochemistry, Research Center of Biotechnology, Russian Academy of Sciences, Leninsky Ave. 33, 119071 Moscow, Russia
*
Authors to whom correspondence should be addressed.
Int. J. Mol. Sci. 2025, 26(15), 7488; https://doi.org/10.3390/ijms26157488
Submission received: 8 July 2025 / Revised: 30 July 2025 / Accepted: 31 July 2025 / Published: 2 August 2025
(This article belongs to the Special Issue Advanced Research on Enzymes in Biocatalysis)
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Abstract

Catalytic and bioelectrocatalytic properties of four white rot fungal laccases (Trametes hirsuta, ThL; Coriolopsis caperata, CcL; Steccherinum murashkinskyi, SmL; and Antrodiella faginea, AfL) from different orthologous groups were comparatively studied in homogeneous reactions of electron donor substrate oxidation and in a heterogeneous reaction of dioxygen electroreduction. The ThL and CcL laccases belong to high-redox-potential enzymes (E0T1 = 780 mV), while the AfL and SmL laccases are medium-redox-potential enzymes (E0T1 = 620 and 650 mV). We evaluated the efficiency of laccases in mediatorless bioelectrocatalytic dioxygen reduction by the steady-state potential (Ess), onset potential (Eonset), half-wave potential (E1/2), and the slope of the linear segment of the polarization curve. A good correlation was observed between the T1 center potential of the laccases and their electrocatalytic characteristics; however, no correlation with the homogeneous reactions of electron donor substrates’ oxidation was detected. The results obtained are discussed in the light of the known data on the three-dimensional structures of the laccases studied.

Graphical Abstract

1. Introduction

The emerging environmentally friendly technologies employing biocatalysts rather than traditional chemical catalysts has attracted increasing attention. Laccases (p-diphenol/oxygen oxidoreductase, EC 1.10.3.2), belonging to the blue multicopper oxidase (MCO) family, are promising biocatalysts capable of biotransforming various compounds. Laccases are non-specific enzymes that catalyze the single-electron oxidation of numerous organic compounds, such as monophenols, polyphenols, and aromatic amines, as well as certain inorganic compounds, with the concomitant reduction in molecular oxygen to water [1,2]. However, despite extensive research, the molecular basis of their functional diversity still remains unclear. In particular, no current method can reliably predict laccase activity towards complex substrates [3]. This presents a challenge to the rational design of laccases for various technological applications.
Fungal laccases comprise three cupredoxin-type domains with a Greek-key β-barrel topology. The active site of laccases includes four copper ions, typically classified as T1, T2, and T3 copper centers. The T2 and T3 centers form a trinuclear copper cluster (TNC) [4]. The T1 center is located in the third domain, while the T2/T3 cluster is between the first and third domains. The T1 center is localized at a distance of ~12–13 Å from TNC and is connected to it by a conserved His-Cys-His triad involved in electron transfer [5]. Based on the T1 center redox potential, laccases are subdivided into high- (E0T1 = 720–790 mV), medium- (E0T1 = 460–720 mV), and low-potential (E0T1 ≤ 460 mV) enzymes [2,6]. The T1 center is thought to accept electrons from reducing substrates; the electrons are transferred to TNC where molecular oxygen is reduced to water [4,7].
Laccases are promising biocatalysts which can be used in various applications [8]. They can serve as electrocatalysts for the dioxygen reduction reaction [6,8]. Direct (mediatorless) bioelectrocatalysis was first demonstrated for the Polyporus versicolor laccase adsorbed on a carbon black electrode [9]. The adsorbed laccase catalyzed dioxygen electroreduction at slightly acidic pH values in the absence of a redox mediator. The potential on the electrode in the dioxygen atmosphere was set close to the oxygen equilibrium potential. The mechanism of this bioelectrocatalytic reaction was studied by Tarasevich et al. [10], who demonstrated that the reduction of dioxygen to water occurs without the intermediate formation of hydrogen peroxide.
Recently, numerous studies have demonstrated direct bioelectrocatalysis by laccases from various sources [11,12,13,14,15], as well as by other redox enzymes such as peroxidases [16,17], hydrogenases [18,19], PQQ dehydrogenases [20,21,22], nitrite reductase [23], bilirubin oxidase [24], and cellobiose dehydrogenases [25,26]. During direct bioelectrocatalysis, enzyme-modified electrodes can act as either electron donors or acceptors, depending on the biocatalyst used.
In the case of MCOs, namely laccases and bilirubin oxidases, the electrode acts as an electron donor in bioelectrocatalytic dioxygen reduction. Using the MCO-modified carbonaceous electrodes, electrons are transferred from the electrode first to the T1 center, and then to the T2/T3 cluster where O2 reduction occurs [4,7,27]. Sekretareva et al. [28] considered the possibility of electron transfer from the graphite electrode directly to the T2/T3 cluster of adsorbed bilirubin oxidase, and showed that due to the high reorganization energy of the fully oxidized TNC, electron transfer from the electrode to the TNC does occur through the T1 center.
Electrocatalytic studies of laccases adsorbed on the electrode surface greatly contribute to understanding their action mechanism and estimating their efficiency in heterogeneous reactions. In general, controlling the electrode potential enables the rate of interfacial electron transfer to be changed. The electrocatalytic properties of laccases in heterogeneous dioxygen reduction depend on the enzyme T1 center’s redox potential and structure, electrode material, the enzyme orientation on its surface, and the activity of an immobilized enzyme [29,30,31,32,33,34,35].
In this work, we compared four fungal laccases from different orthologous groups to establish the relationship between the redox potential of the enzyme’s T1 center and its catalytic efficiency in homogeneous and heterogeneous reactions. To this end, we chose the laccases from the basidial white rot fungi Coriolopsis caperata (CcL), Trametes hirsuta (ThL), Antrotiella faginea (AfL), and Steccherinum murashkinskyi (SmL).

2. Results and Discussion

The most studied and well-characterized laccases are produced by white rot fungi belonging to the order Polyporales. The T1 center of many laccases, mostly represented by the Polyporaceae family, has a high redox potential, which enables the efficient oxidation of numerous substrates and makes laccases promising biocatalysts. However, the catalytic properties of laccases may greatly vary, likely due to differences in their amino acid sequences, in particular, in positions critical for substrate binding. Indeed, the genomes of white rot Polyporales may contain up to nine non-allelic copies of the genes encoding laccase isoenzymes [36]. The phylogenetic analysis of laccase amino acid sequences has shown the presence of orthologous groups [36,37]. The enzymes from the same group have similar physicochemical, biochemical, and catalytic properties [38].
In the present work, we analyzed the laccases of the basidial white rot fungi from two clades of the Polyporales order: the Core (CcL and ThL) and Residual (SmL and AfL) polyporoid clades [39]. Their physicochemical properties are presented in Table 1. While all the enzymes contain four copper ions and give UV-visible absorption spectra typical of laccases, they have different carbohydrate contents, isoelectric points, and T1 center redox potentials. The CcL and ThL from the Core polyporoid clade are high-redox-potential enzymes (E0T1 = 780 mV), whereas the AfL and SmL from the Residual polyporoid clade are medium-redox-potential enzymes (E0T1 = 620 and 650 mV).
All four laccases adsorbed on MWCNT-electrodes catalyzed the dioxygen electroreduction in the pH range of 3.0–5.5. Dioxygen electroreduction produced water without forming hydrogen peroxide. The analytical method using peroxidase and ABTS showed no hydrogen peroxide in the reaction medium at the potentials corresponding to the diffusion-limited current. It should be noted that all the enzymes exhibited no electrocatalytic activity at pH 7.0. Linear voltammograms (Figure 1) were recorded at a potential scan rate of 1 mV s−1, as at this scan rate the dioxygen reduction occurred under pseudo-steady-state conditions. In general, the polarization curve of the dioxygen electroreduction reaction includes three segments: (1) a kinetic segment characterizing the catalytic properties of an electrocatalyst; (2) a kinetic diffusion segment; (3) a diffusion-controlled segment describing the dependence of the current on the substrate mass transfer [42]. The efficiency of electrocatalysis can be estimated by the steady-state potential of an electrode (Ess), the onset potential (Eonset), the half-wave potential (E1/2), as well as by the slope of the polarization curve linear segment close to the steady-state potential (Δi/ΔE), which is related to the exchange current density. These parameters are commonly used to assess the activity of electrocatalysts. It should be noted that the definitions of the Eonset vary from publication to publication [43]. We used the definition of the onset potential as a potential value corresponding to 5% of the diffusion-limited current [44].
In our work, we evaluated the efficiency of direct bioelectrocatalysis using all four parameters. Figure 1 shows linear voltammograms of dioxygen electroreduction on the MWCNT-electrodes with adsorbed enzymes recorded at pH 5.5. Table 2 summarizes the electrocatalytic characteristics of the enzymes. Still, one should bear in mind that efficient bioelectrocatalysis requires the maximum filling of the electrode surface with an enzyme in an orientation that ensures the involvement of all immobilized molecules in the bioelectrocatalytic process. The surface concentrations (Γ) of adsorbed laccases on the MWCNT-electrodes were in the range of 1.58–1.86 pmol cm−2 (Table 2).
As shown in Table 2, the Eonset and Ess potentials of the electrodes modified with the high-redox-potential CcL and ThL were the closest to the equilibrium oxygen potential for O2/H2O (890 mV at pH 5.5 in the air). The medium-redox-potential SmL and AfL had more negative Ess, Eonset, and E1/2 values, indicating a lower electrocatalytic activity in dioxygen reduction.
For most practically important electrochemical reactions, the current depends exponentially on the potential range shift:
η = a + b · l g i
where η—the cathode overpotential of dioxygen electroreduction; a = − R T α n F l n i 0 ; b = 2.3 R T α n F ; i—the current; i0—the exchange current; R—the universal gas constant; F—the Faraday constant; T—the absolute temperature; n—the number of electrons transferred; and α—the charge transfer coefficient, varying from 0 to 1. However, the initial (kinetic) segment of the polarization curve displays a linear dependence between the overvoltage and the current:
η = R T i n F i 0
The slope of the linear segment of the polarization curve (Δi/ΔE) correlates with the exchange current, which also characterizes the electrocatalyst activity. The steeper the slope is, the greater is the exchange current, and, consequently, the higher the activity of an electrocatalyst. As can be seen from Table 2, the Δi/ΔE values for the high-redox-potential CcL and ThL are greater compared to the medium-redox-potential SmL and AfL. The correlation between the laccase T1 center potential and the electrocatalytic characteristics (Ess, Eonset, E1/2, and Δi/ΔE) was linear with R2 = 0.84–0.92 (Supplementary Figure S1). This indicates the contribution of the T1 center redox potential to the efficiency of mediatorless bioelectrocatalysis.
We analyzed the dependence of E1/2 on the pH value (Figure 2). For all the laccases studied, we observed a linear decrease in the E1/2 value with a slope of 10–35 mV/pH in a pH range of 3.0–4.5. For the ThL and SmL, there was a deviation from the linear dependence at pH > 4.5.
The current model of bioelectrocatalysis [28] states that, in the case of MCOs, the electrode replaces the reductant substrate acting as an electron donor. Consequently, the mechanism of electrocatalytic reaction (the electron transfer from the electrode through the T1 center to the T2/T3 cluster) is expected to resemble homogeneous catalysis. Therefore, in the present work, we examined the catalytic properties of laccases in homogeneous oxidation reactions of substrates with different values of redox potentials. To rule out the influence of non-enzymatic reactions, which usually accompany the enzymatic oxidation of organic substrates (donors of hydrogen atoms), we used electron donor substrates such as K4Fe(CN)6, K4Mo(CN)8, and ABTS (Table 3). It should be noted that the KM values for oxygen in laccase-catalyzed reactions were in the range of 150–200 μM [45], which allowed us to carry out kinetic studies in air-saturated buffer solutions.
Upon oxidation of the high-redox-potential substrate K4Mo(CN)8 (E0 = 782 mV [46]), the catalytic efficiency of laccases (kcat/KM) at pH 5.5 decreased as follows: CcL > AfL > ThL > SmL (Table 3). In the case of ABTS (E0 = 682 mV [47]) and K4Fe(CN)6 (E0 = 412 mV [48]) oxidation, the laccases exhibited a different catalytic efficiency: AfL ≥ CcL > ThL > SmL and CcL > SmL ≥ AfL > ThL, respectively. A number of studies have shown that in homogeneous reactions, the efficiency of laccase-catalyzed oxidation of various organic substrates (hydrogen atom donors), expressed in lg(kcat/KM), correlates with the difference between the potentials of the laccase T1 center and the substrate (ΔE = E0T1 − E0S) [49,50,51]. Our results demonstrated the lack of such a correlation in the oxidation of electron donor substrates (Supplementary Figure S2).
Glazunova et al. [52] also showed the absence of this correlation in the laccase-catalyzed oxidation of hydrogen atom donor substrates using CcL, ThL, SmL, and AfL. However, as shown in another study [53], when the high-redox-potential laccases from T. hirsuta and Trametes versicolor (E0T1 = 780 and 800 mV) were compared to low-redox-potential laccases from Rhus vernicifera and Melanocarpus albomyces (E0T1 = 410 and 460 mV), the correlation of oxidation efficiency of inorganic substrates (electron donors) with the ΔE value was revealed. Yet, no such correlation was observed in the case of phenolic organic substrates (hydrogen atom donors) [53]. Certain high-redox-potential laccases were shown to have differences in the efficiency of the oxidation of phenolic substrates (hydrogen atom donors) and K4Fe(CN)6 [54]. In the present work, the high-redox-potential CcL had K4Mo(CN)8 and K4Fe(CN)6 oxidation efficiency an order of magnitude higher than ThL, with comparable ABTS oxidation efficiency. The medium-redox-potential AfL demonstrated an efficiency of K4Mo(CN)8 and ABTS oxidation an order of magnitude higher than SmL, with comparable K4[Fe(CN)6] oxidation efficiency (Table 3).
The absence of a correlation between ΔE = E0T1 − E0S and the efficiency of substrate oxidation in homogeneous reactions might arise from the protein structures of different laccases. Indeed, a number of studies have shown that the efficiency of laccase catalysis in homogeneous reactions depends not only on the T1 center potential, but also on other enzyme characteristics, such as the protein architecture, hydrophobicity, and charge of the substrate-binding pocket [52,55,56,57].
Glazunova et al. [41] demonstrated that the secondary coordination sphere of the T1 center of fungal laccases consists of seven loops, designated as S1–S7. Loops S2, S3, and S7 are quite conservative, whereas S1, S4, S5, and S6 show a high degree of variability. Figure 3 shows the location of these loops relative to the T1 center copper ion, using the example of SmL (PDB 5E9N). The amino acid composition of these loops in CcL, ThL, SmL, and AfL, as well as laccases from the corresponding orthologous groups, is shown in Figure 4. The amino acid sequence identity of laccases within the same orthologous group is about 80–99%, while the sequence identity between different orthologous groups mostly does not exceed 70%.
Molecular dynamics simulations reported in [55] demonstrated that the amino acid residues Asp209 and His458 (Figure 4) in the conserved S2 and S7 loops of the T. versicolor laccase (PDB 1GYC), respectively, play an important role in substrate binding. These amino acid residues are critically important since they form hydrogen bonds, salt bridges, or π-π stacking interactions upon substrate binding. The electron transfer may involve the nitrogen atom of Nε2 His458. Moreover, the ionization potential, shape, and binding affinity of the substrate largely determine the enzyme KM value for a specific substrate [55].
The His458 (H458) residue in loop S7 is highly conserved in all laccases since it is involved in coordinating the T1 center copper ion. The residue at position 209 in the conserved S2 loop is Asp (D209) in the case of ThL and CcL, and SmL and Glu (E209) in the case of AfL (Figure 4). The isoelectric point of Glu is shifted to a more alkaline region (pI 3.22) compared to Asp (pI 2.77). Protonation of the amino acid residue at position 209 affects the mobility of the loops in the substrate-binding pocket [55].
Variable loops S1, S4, S5, and S6 of the T1 copper ion environment differ in length and amino acid composition in laccases from different orthologous groups (Figure 4). A number of studies [55,56,57] have shown that the structure of the S1 loop, which exhibits high flexibility and significant conformational variability, affects the substrate binding near the T1 center. The S1 loop can adopt three main conformations (hook-like, expanded, and vertical pseudothree-centered arch-like conformations) upon binding to the phenolic substrate catechol [57]. The length and amino acid composition of this loop affect substrate binding and the KM value. Residues Asn267 (N267) and Phe268 (F268) in the S4 loop of the T. versicolor laccase (PDB 1GYC) also have a major effect on substrate binding near the T1 center [55]. As can be seen from Figure 4, Asn267 (N267) is highly conserved. At the same time, the laccases studied in this work contained different amino acid residues at position 268: ThL–Phe (F268), CcL and SmL–Ser (S268), and AfL–Asp (D268). Differences in the amino acid composition and length of variable loops S1, S4, S5, and S6 of the four laccases studied apparently affect the charge and hydrophobicity of the loops that form the substrate-binding pocket near the T1 center. As can be seen from Figure 5, the accessibility of the variable loops to the solvent varies, with the most significant differences observed for loops S1 and S5.
The total charge of the loops which form the substrate-binding pocket near the T1 center varies in laccases from different orthologous groups (Figure 4). Loop S1 has a charge of 0, +1, −1, and −2 in CcL, ThL, AfL, and SmL, respectively. The total charge of loop S4 in the AfL is −2; it is 0 in the other three laccases. Loop S5 in CcL and SmL has a positive charge (+1) and it is uncharged in ThL and AfL.
Apart from the potential of the T1 center of laccases, the structure and composition of the loops that form the substrate-binding pocket significantly affect the efficiency of homogeneous catalysis by laccases. This can explain the lack of correlation between the potential of the T1 center of the enzymes studied and their catalytic properties in the homogeneous reactions of electron donor substrate oxidation. In case of heterogeneous catalysis, the effect of the substrate-binding pocket structure on the electrocatalytic characteristics of laccases seems to be negligible, while the T1 center redox potential is a major factor that determines the efficiency of direct bioelectrocatalysis.

3. Materials and Methods

3.1. Materials

All commercially available chemicals were of high purity and were used without further purification, namely, 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS), K4Fe(CN)6, K4Mo(CN)8 (Sigma-Aldrich, Steinheim, Germany), citric acid, Na2HPO4, and NaOH (Riedel-de Haën, Seelze, Germany). The multi-walled carbon nanotubes “Taunit M” (Nano-TechCentre Ltd., Tambov, Russia) (outer diameter 10–30 nm; length ≥ 2 μm; specific surface area ≥ 270 m2/g) were used without any modification. Flexible graphite foil (thickness 0.2 mm) was purchased from (Unichimtek, Klimovsk, Moscow region, Russia). All the solutions were prepared using water purified with a Simplicity® Water Purification System (Merck KGaA, Darmstadt, Germany).

3.2. Laccases

The mycelial cultures of the basidiomycete strains used in this study were received from the Collection of the Komarov Botanical Institute, Russian Academy of Sciences (St. Petersburg, Russia): T. hirsuta strain LE-BIN 072, C. caperata strain LE-BIN 0677, S. murashkinskyi LE-BIN 1963, and A. faginea LE-BIN 1998. Laccases CcL (Lac2; GenBank #AGE13770.1), ThL (LacA; GenBank #AIZ72721.1), SmL (Lac2; GenBank #AFI41889.1), and AfL (LacA; GenBank #ALE66817.1) were isolated from culture liquids of basidial fungi as described in [41,58]. All the enzymes were homogeneous according to SDS electrophoresis. The amino acid sequence of laccase isozymes was confirmed by MALDI-TOF/TOF MS/MS using a BrukerUltraflex II MALDI TOF/TOF mass spectrometer (BrukerDaltonics, Bremen, Germany) [41]. The PDB codes of CcL, ThL, SmL, and AfL structures in Protein Data Bank are 4JHV, 3FPX, 5E9N, and 5EHF, respectively.

3.3. Fabrication of Laccase-Modified MWCNT Electrodes

Strips of graphite foil (0.5 × 2 cm) were used as a current collector. A total of 100 μL of MWCNT dispersion in ethanol (1 mg mL−1) was applied to the surface of carbon foil without a binder and dried in air. The electrode working surface was 0.25 cm2. An enzyme solution was applied to the MWCNT-electrode, and after 30 min the electrodes were washed several times with 0.1 M Na–citrate–phosphate buffer to remove the loosely binding enzyme. The maximum enzyme adsorption on the MWCNT’s surface was achieved under these conditions.
The surface concentration of laccases adsorbed on the MWCNT-electrodes (Γ, pmol cm−2) was evaluated based on the difference between the protein concentrations in the solution before and after enzyme adsorption. Protein concentration was determined as described in [59] by the difference in the optical density of the protein solution at 228.5 and 234.5 nm using bovine serum albumin (BSA) as a protein concentration standard.

3.4. Kinetic Studies of the Laccases

Kinetic measurements were performed using a Shimadzu UV 1240 mini spectrophotometer (Shimadzu Europa GmbH, Duisburg, Germany). Reactions were carried out at 23 °C in 0.1 M Na–citrate–phosphate buffer at pH 5.5. For each laccase, the range of substrate concentrations and a working enzyme concentration were selected. The following molar extinction coefficients of the ABTS, K4Mo(CN)8, and K4Fe(CN)6 oxidation products were used for calculations: ε420 = 36,000 M−1cm−1 [60], ε388 = 1400 M−1cm−1 [61], and ε420 = 1040 M−1cm−1 [62], respectively. The kinetic parameters of the reactions were calculated using the Lineweaver Burk plots. The initial reaction rates were determined at least in triplicate.

3.5. Electrochemical Measurements

Electrochemical measurements were performed using a BAS CV-50W voltammetric analyzer (Bioanalytical Systems Inc., West Lafayette, IN, USA) and a single-compartment, three-electrode cell. A platinum wire and an Ag/AgCl electrode (BAS) served as counter and reference electrodes, respectively. MWCNT-covered graphite foil with adsorbed laccase served as a working electrode. Three working electrodes were fabricated for each enzyme. Linear voltammograms were recorded using a stationary electrode in air-saturated 0.1 M Na–citrate–phosphate buffer solution. All potentials are given vs. normal hydrogen electrode (NHE). Results are presented as the mean ± standard deviation. Statistical comparisons are performed using one-way ANOVA followed by Tukey’s post hoc test (p < 0.05).

3.6. Enzyme Structure Analysis

Laccase amino acid sequence alignment was carried out using ClustalW. The solvent accessible surface area was calculated using service PISA (via CCP4 v9.0.010) (protein interfaces, surfaces, and assemblies) [63].

4. Conclusions

We examined the direct bioelectrocatalytic reduction of dioxygen on MWCNT-electrodes modified with high-redox-potential laccases from Coriolopsis caperata and Trametes hirsuta as well as medium-redox-potential enzymes from Steccherinum murashkinskyi and Antrotiella faginea. We revealed a correlation between the redox potential of the T1 center and catalytic efficiency of the laccases studied in heterogeneous (electrochemical) reactions. No correlation was observed in the homogeneous reactions of electron donor substrate oxidation. This can be explained by differences in the secondary coordination sphere of the T1 copper ion, namely, the amino acid composition of the conservative and variable loops responsible for substrate binding. Therefore, for practical applications of laccases, their structure and catalytic properties should preliminary be studied in detail.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/ijms26157488/s1.

Author Contributions

Conceptualization, A.Y. and T.F.; methodology O.M.; formal analysis, A.Y., O.M., and T.F.; investigation, O.M., M.K., and I.V.; writing—original draft preparation, O.M. and A.Y.; writing—review and editing, T.F. and A.Y. All authors have read and agreed to the published version of the manuscript.

Funding

This research received no external funding.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

The original contributions presented in this study are included in the article. Further inquiries can be directed to the corresponding authors.

Acknowledgments

The authors would like to thank Galina Shumakovich for her valuable assistance in setting up the electrochemical experiments and interpreting the results.

Conflicts of Interest

The authors declare no conflicts of interest.

Abbreviations

The following abbreviations are used in this manuscript:
MCOMulticopper oxidase
TNCTrinuclear copper cluster
MWCNTMulti-walled carbon nanotube
NHENormal hydrogen electrode
ABTSDiammonium salt of 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)
ThLTrametes hirsuta laccase
CcLCoriolopsis caperata laccase
SmLSteccherinum murashkinskyi laccase
AfLAntrodiella faginea laccase
TvLTrametes versicolor laccase
ToLTrametes ochracea laccase
CtLCoriolopsis trogii laccase
CgLCoriolopsis gallica laccase
SoLSteccherinum ochraceum laccase
AcL Antrodiella citronella laccase

References

  1. Baldrian, P. Fungal laccases—Occurrence and properties. FEMS Microbiol. Rev. 2006, 30, 215–242. [Google Scholar] [CrossRef]
  2. Morozova, O.V.; Shumakovich, G.P.; Gorbacheva, M.A.; Shleev, S.V.; Yaropolov, A.I. Blue laccases. Biochemistry 2007, 72, 1136–1150. [Google Scholar] [CrossRef]
  3. Martin, E.; Dubessay, P.; Record, E.; Audonnet, F.; Michaud, P. Recent advances in laccase activity assays: A crucial challenge for applications on complex substrates. Enzyme Microb. Technol. 2024, 173, 110373. [Google Scholar] [CrossRef]
  4. Solomon, E.I.; Sundaram, U.M.; Machonkin, T.E. Multicopper Oxidases and Oxygenases. Chem. Rev. 1996, 96, 2563–2606. [Google Scholar] [CrossRef]
  5. Jones, S.M.; Solomon, E.I. Electron transfer and reaction mechanism of laccases. Cell. Mol. Life Sci. 2015, 72, 869–883. [Google Scholar] [CrossRef]
  6. Aza, P.; Camarero, S. Fungal Laccases: Fundamentals, Engineering and Classification Update. Biomolecules 2023, 13, 1716. [Google Scholar] [CrossRef]
  7. Bassanini, I.; Ferrandi, E.E.; Riva, S.; Monti, D. Biocatalysis with Laccases: An Updated Overview. Catalysts 2021, 11, 26. [Google Scholar] [CrossRef]
  8. Chaudhary, S.; Singh, A.; Varma, A.; Porwal, S. Recent Advancements in Biotechnological Applications of Laccase as a Multifunctional Enzyme. J. Pure Appl. Microbiol. 2022, 16, 1479–1491. [Google Scholar] [CrossRef]
  9. Berezin, I.V.; Bogdanovskaya, V.A.; Varfolomeev, S.D.; Tarasevich, M.R.; Yaropolov, A.I. Bioelectrocatalysis. The equilibrium oxygen potential in the presence of laccase. Docl. AN SSSR (Rep. USSR Acad. Sci.) 1978, 240, 615–617. [Google Scholar]
  10. Tarasevich, M.R.; Yaropolov, A.I.; Bogdanovskaya, V.A.; Varfolomeev, S.D. Electrocatalysis of a cathodic oxygen reduction by laccase. Bioelectrochem. Bioenerg. 1979, 6, 393–403. [Google Scholar] [CrossRef]
  11. Shleev, S.; Jarosz-Wilkolazka, A.; Khalunina, A.; Morozova, O.; Yaropolov, A.; Ruzgas, T.; Gorton, L. Direct electron transfer reactions of laccases from different origins on carbon electrodes. Bioelectrochemistry 2005, 67, 115–124. [Google Scholar] [CrossRef]
  12. Thuesen, M.H.; Farver, O.; Reinhammar, B.; Ulstrup, J. Cyclic voltammetry and electrocatalysis of the blue copper oxidase Polyporus versicolor laccase. Acta Chem. Scand. 1998, 52, 555–562. [Google Scholar] [CrossRef]
  13. Zhang, L.; Cui, H.; Dhoke, G.V.; Zou, Z.; Sauer, D.F.; Davari, M.D.; Schwaneberg, U. Engineering of Laccase CueO for Improved Electron Transfer in Bioelectrocatalysis by Semi-Rational Design. Chem. Eur. J. 2020, 26, 4974–4979. [Google Scholar] [CrossRef]
  14. Atalah, J.; Zhou, Y.; Espina, G.; Blamey, J.M.; Ramasamy, R.P. Improved stability of multicopper oxidase-carbon nanotube conjugates using a thermophilic laccase. Catal. Sci. Technol. 2018, 8, 1272–1276. [Google Scholar] [CrossRef]
  15. Moro, G.; Brissos, V.; Zanardi, C.H.; Martins, L.O.; Conzuelo, F. Electrochemical investigations of the multicopper oxidase from Aquifex aeolicus under direct electron transfer with carbon electrodes. Electrochim. Acta 2023, 468, 143199. [Google Scholar] [CrossRef]
  16. Yaropolov, A.; Malovik, V.; Varfolomeev, S. Electroreduction of hydrogen peroxide on the electrode with immobilized peroxidase. Dokl. AN SSSR (Rep. USSR Acad. Sci.) 1979, 249, 1399–1401. [Google Scholar]
  17. Ruzgas, T.; Csöregi, E.; Emnéus, J.; Gorton, L.; Marko-Varga, G. Peroxidase-modified electrodes: Fundamentals and application. Anal. Chim. Acta 1996, 330, 123–138. [Google Scholar] [CrossRef]
  18. Morozov, S.V.; Karyakina, E.E.; Zorin, N.A.; Varfolomeyev, S.D.; Cosnier, S.; Karyakin, A.A. Direct and electrically wired bioelectrocatalysis by hydrogenase from Thiocapsa roseopersicina. Bioelectrochemistry 2002, 55, 169–171. [Google Scholar] [CrossRef] [PubMed]
  19. Tsygankov, A.A.; Zorin, N.A.; Starodubov, A.; Khasimov, M.K.; Melnikova, M.S.; Doronin, I.A.; Vasilov, R.G. A simple method for oriented immobilization of HydSL hydrogenase of Thiocapsa bogorovii on carbon electrodes. Int. J. Hydrogen Energy 2023, 48, 39989–39999. [Google Scholar] [CrossRef]
  20. Marchianó, V.; Tricase, A.; Ditaranto, N.; Macchia, E.; d’Ingeo, S.; Di Franco, C.; Scamarcio, G.; Torsi, L.; Bollella, P. High voltage flexible glucose/O2 fully printed hydrogel-based enzymatic fuel cell. J. Phys. D Appl. Phys. 2024, 57, 135503. [Google Scholar] [CrossRef]
  21. Suzuki, Y.; Makino, F.; Miyata, T.; Tanaka, H.; Namba, K.; Kano, K.; Sowa, K.; Kitazumi, Y.; Shirai, O. Essential Insight of Direct Electron Transfer-Type Bioelectrocatalysis by Membrane-Bound D-Fructose Dehydrogenase with Structural Bioelectrochemistry. ACS Catal. 2023, 13, 13828–13837. [Google Scholar] [CrossRef]
  22. Komkova, M.A.; Alexandrovich, A.S.; Karyakin, A.A. Polyazine nanoparticles as anchors of PQQ glucose dehydrogenase for its most efficient bioelectrocatalysis. Talanta 2024, 267, 125219. [Google Scholar] [CrossRef]
  23. Andoralov, V.; Shleev, S.; Dergousova, N.; Kulikova, O.; Popov, V.; Tikhonova, T. Octaheme nitrite reductase: The mechanism of intramolecular electron transfer and kinetics of nitrite bioelectroreduction. Bioelectrochemistry 2021, 138, 107699. [Google Scholar] [CrossRef]
  24. Brocato, S.; Lau, C.; Atanassov, P. Mechanistic study of direct electron transfer in bilirubin oxidase. Electrochim. Acta 2012, 61, 44–49. [Google Scholar] [CrossRef]
  25. Larsson, T.; Lindgren, A.; Ruzgas, T.; Lindquist, S.-E.; Gorton, L. Bioelectrochemical characterisation of cellobiose dehydrogenase modified graphite electrodes: Ionic strength and pH dependences. J. Electroanal. Chem. 2000, 482, 1–10. [Google Scholar] [CrossRef]
  26. Stoica, L.; Ludwig, R.; Haltrich, D.; Gorton, L. Third-Generation Biosensor for Lactose Based on Newly Discovered Cellobiose Dehydrogenase. Anal. Chem. 2006, 78, 393–398. [Google Scholar] [CrossRef]
  27. den Boer, D.; de Heer, H.C.; Buda, F.; Hetterscheid, D.G.H. Challenges in Elucidating the Free Energy Scheme of the Laccase Catalyzed Reduction of Oxygen. ChemCatChem 2023, 15, e202200878. [Google Scholar] [CrossRef]
  28. Sekretareva, A.; Tian, S.; Gounel, S.; Mano, N.; Solomon, E.I. Electron Transfer to the Trinuclear Copper Cluster in Electrocatalysis by the Multicopper Oxidases. J. Am. Chem. Soc. 2021, 143, 17236–17249. [Google Scholar] [CrossRef] [PubMed]
  29. Pang, H.L.; Liu, J.; Hu, D.; Zhang, X.H.; Chen, J.H. Immobilization of laccase onto 1-aminopyrene functionalized carbon nanotubes and their electrocatalytic activity for oxygen reduction. Electrochim. Acta 2010, 55, 6611–6616. [Google Scholar] [CrossRef]
  30. Opallo, M.; Bilewicz, R. Recent Developments of Nanostructured Electrodes for Bioelectrocatalysis of Dioxygen Reduction. Adv. Phys. Chem. 2011, 2011, 947637. [Google Scholar] [CrossRef]
  31. Wei, W.; Li, P.; Li, Y.; Cao, X.; Liu, S. Nitrogen-doped carbon nanotubes enhanced laccase enzymatic reactivity towards oxygen reduction and its application in biofuel cell. Electrochem. Commun. 2012, 22, 181–184. [Google Scholar] [CrossRef]
  32. Tominaga, M.; Sasaki, A.; Togami, M. Laccase Bioelectrocatalyst at a Steroid-Type Biosurfactant-Modified Carbon Nanotube Interface. Anal. Chem. 2015, 87, 5417–5421. [Google Scholar] [CrossRef]
  33. Dagys, M.; Laurynėnas, A.; Ratautas, D.; Kulys, J.; Vidžiūnaitė, R.; Talaikis, M.; Niaura, G.; Marcinkevičienė, L.; Meškys, R.; Shleev, S. Oxygen Electroreduction Catalysed by Laccase Wired to Gold Nanoparticles via the Trinuclear Copper Cluster. Energy Environ. Sci. 2017, 10, 498–502. [Google Scholar] [CrossRef]
  34. Gentil, S.; Rousselot-Pailley, P.; Sancho, F.; Robert, V.; Mekmouche, Y.; Guallar, V.; Tron, T.; Le Goff, A. Efficiency of site-specific clicked laccase-carbon nanotubes biocathodes towards O2 reduction. Chem. Eur. J. 2020, 26, 4798–4804. [Google Scholar] [CrossRef]
  35. Lim, K.; Sima, M.; Stewart, R.J.; Minteer, S.D. Direct bioelectrocatalysis by redox enzymes immobilized in electrostatically condensed oppositely charged polyelectrolyte electrode coatings. Analyst 2020, 145, 1250–1257. [Google Scholar] [CrossRef] [PubMed]
  36. Savinova, O.S.; Moiseenko, K.V.; Vavilova, E.A.; Chulkin, A.M.; Fedorova, T.V.; Tyazhelova, T.V.; Vasina, D.V. Evolutionary Relationships Between the Laccase Genes of Polyporales: Orthology-Based Classification of Laccase Isozymes and Functional Insight From Trametes hirsuta. Front. Microbiol. 2019, 10, 152. [Google Scholar] [CrossRef] [PubMed]
  37. Moiseenko, K.V.; Maloshenok, L.G.; Vasina, D.V.; Bruskin, S.A.; Tyazhelova, T.V.; Koroleva, O.V. Laccase multigene families in Agaricomycetes. J. Basic. Microbiol. 2016, 56, 1392–1397. [Google Scholar] [CrossRef] [PubMed]
  38. Vasina, D.V.; Mustafaev, O.N.; Moiseenko, K.V.; Sadovskaya, N.S.; Glazunova, O.A.; Tyurin, A.A.; Fedorova, T.V.; Pavlov, A.R.; Tyazhelova, T.V.; Goldenkova-Pavlova, I.V.; et al. The Trametes hirsuta 072 laccase multigene family: Genes identification and transcriptional analysis under copper ions induction. Biochimie 2015, 116, 154–164. [Google Scholar] [CrossRef]
  39. Justo, A.; Miettinen, O.; Floudas, D.; Ortiz-Santana, B.; Sjökvist, E.; Lindner, D.; Nakasone, K.; Niemelä, T.; Larsson, K.-H.; Ryvarden, L.; et al. A revised family-level classification of the Polyporales (Basidiomycota). Fungal Biol. 2017, 121, 798–824. [Google Scholar] [CrossRef]
  40. Glazunova, O.A.; Shakhova, N.V.; Psurtseva, N.V.; Moiseenko, K.V.; Kleimenov, S.Y.; Fedorova, T.V. White-rot basidiomycetes Junghuhnia nitida and Steccherinum bourdotii: Oxidative potential and laccase properties in comparison with Trametes hirsuta and Coriolopsis caperata. PLoS ONE 2018, 13, e0197667. [Google Scholar] [CrossRef]
  41. Glazunova, O.A.; Polyakov, K.M.; Moiseenko, K.V.; Kurzeev, S.A.; Fedorova, T.V. Structure-function study of two new middle-redox potential laccases from basidiomycetes Antrodiella faginea and Steccherinum murashkinskyi. Int. J. Biol. Macromol. 2018, 118, 406–418. [Google Scholar] [CrossRef]
  42. Xia, W.; Mahmood, A.; Liang, Z.; Zou, R.; Guo, S. Earth-Abundant Nanomaterials for Oxygen Reduction. Angew. Chem. Int. Ed. Engl. 2016, 55, 2650–2676. [Google Scholar] [CrossRef]
  43. Batchelor-McAuley, C. Defining the onset potential. Curr. Opin. Electrochem. 2023, 37, 101176. [Google Scholar] [CrossRef]
  44. Zhou, X.; Qiao, J.; Yang, L.; Zhang, J. A Review of Graphene-Based Nanostructural Materials for Both Catalyst Supports and Metal-Free Catalysts in PEM Fuel Cell Oxygen Reduction Reactions. Adv. Energy Mater. 2014, 4, 1301523. [Google Scholar] [CrossRef]
  45. Gorbacheva, M.; Morozova, O.; Shumakovich, G.; Streltsov, A.; Shleev, S.; Yaropolov, A. Enzymatic oxidation of manganese ions catalysed by laccase. Bioorg. Chem. 2009, 37, 1–5. [Google Scholar] [CrossRef]
  46. Taniguchi, V.T.; Malmström, B.G.; Anson, F.C.; Gray, H.B. Temperature dependence of the reduction potential of blue copper in fungal laccase. Proc. Natl. Acad. Sci. USA 1982, 79, 3387–3389. [Google Scholar] [CrossRef] [PubMed]
  47. Bourbonnais, R.; Leech, D.; Paice, M.G. Electrochemical analysis of the interactions of laccase mediators with lignin model compounds. Biochim. Biophys. Acta Gen. Subj. 1998, 1379, 381–390. [Google Scholar] [CrossRef] [PubMed]
  48. O’Reilly, J.E. Oxidation-reduction potential of the ferro-ferricyanide system in buffer solutions. Biochim. Biophys. Acta Bioenerg. 1973, 292, 509–515. [Google Scholar] [CrossRef] [PubMed]
  49. Xu, F. Oxidation of Phenols, Anilines, and Benzenethiols by Fungal Laccases: Correlation between Activity and Redox Potentials as Well as Halide Inhibition. Biochemistry 1996, 35, 7608–7614. [Google Scholar] [CrossRef]
  50. Xu, F.; Kulys, J.J.; Duke, K.; Li, K.; Krikstopaitis, K.; Deussen, H.J.; Abbate, E.; Galinyte, V.; Schneider, P. Redox Chemistry in Laccase-Catalyzed Oxidation of N-Hydroxy Compounds. Appl. Environ. Microbiol. 2000, 66, 2052–2056. [Google Scholar] [CrossRef]
  51. Olbrich, A.C.; Schild, J.N.; Urlacher, V.B. Correlation between the T1 copper reduction potential and catalytic activity of a small laccase. J. Inorg. Biochem. 2019, 201, 110843. [Google Scholar] [CrossRef]
  52. Glazunova, O.A.; Trushkin, N.A.; Moiseenko, K.V.; Filimonov, I.S.; Fedorova, T.V. Catalytic Efficiency of Basidiomycete Laccases: Redox Potential versus Substrate-Binding Pocket Structure. Catalysts 2018, 8, 152. [Google Scholar] [CrossRef]
  53. Frasconi, M.; Favero, G.; Boer, H.; Koivula, A.; Mazzei, F. Kinetic and biochemical properties of high and low redox potential laccases from fungal and plant origin. Biochim. Biophys. Acta Proteins Proteomics 2010, 1804, 899–908. [Google Scholar] [CrossRef] [PubMed]
  54. Shleev, S.V.; Morozova, O.V.; Nikitina, O.V.; Gorshina, E.S.; Rusinova, T.V.; Serezhenkov, V.A.; Burbaev, D.S.; Gazaryan, I.G.; Yaropolov, A.I. Comparison of physico-chemical characteristics of four laccases from different basidiomycetes. Biochimie 2004, 86, 693–703. [Google Scholar] [CrossRef] [PubMed]
  55. Mehra, R.; Muschiol, J.; Meyer, A.S.; Kepp, K.P. A structural-chemical explanation of fungal laccase activity. Sci. Rep. 2018, 8, 17285. [Google Scholar] [CrossRef] [PubMed]
  56. Orlando, C.; Bellei, M.; Zampolli, J.; Mangiagalli, M.; Di Gennaro, P.; Lotti, M.; De Gioia, L.; Marino, T.; Di Rocco, G.; Greco, C.; et al. Comparative analysis of Polyethylene-Degrading Laccases: Redox Properties and Enzyme-Polyethylene Interaction Mechanism. ChemSusChem 2025, 18, e202402253. [Google Scholar] [CrossRef]
  57. Biswas, A.; Radhakrishna, M. Exploring Catechol Binding to Laccase with Insights into Enzyme Dynamics for Biosensing Applications. J. Phys. Chem. B 2025, 129, 3761–3775. [Google Scholar] [CrossRef]
  58. Koroljova-Skorobogat’ko, O.V.; Stepanova, E.V.; Gavrilova, V.P.; Morozova, O.V.; Lubimova, N.V.; Dzchafarova, A.N.; Jaropolov, A.I.; Makower, A. Purification and characterization of the constitutive form of laccase from the basidiomycete Coriolus hirsutus and effect of inducers on laccase synthesis. Biotechnol. Appl. Biochem. 1998, 28, 47–54. [Google Scholar] [CrossRef]
  59. Ehresmann, B.; Imbault, P.; Well, J.H. Spectrophotometric determination of protein concentration in cell extracts containing tRNA’s and rRNA’s. Analyt. Biochem. 1973, 54, 454–463. [Google Scholar] [CrossRef]
  60. Bourbonnais, R.; Paice, M.G. Oxidation of non-phenolic substrates: An expanded role for laccase in lignin biodegradation. FEBS Lett. 1990, 267, 99–102. [Google Scholar] [CrossRef]
  61. Dennis, C.R.; van Zyl, G.J.; Fourie, E.; Basson, S.S.; Swarts, J.C. A kinetic study of the oxidation of the tetrakisoxalatouranate(IV) ion by the octacyanotungstate (V) and the octacyanomolybdate (V) ions in an acidic oxalate buffer medium. Reac. Kinet. Mech. Cat. 2021, 134, 615–627. [Google Scholar] [CrossRef]
  62. Murton, P.D.F.; Timmel, C.R.; Mackenzie, S.R.; Rodríguez-Maciá, P. Blue-light photodegradation of ferricyanide under protein relevant conditions. Dalton Trans. 2025, 54, 4735–4742. [Google Scholar] [CrossRef] [PubMed]
  63. Krissinel, E.; Henrick, K. Inference of Macromolecular Assemblies from Crystalline State. J. Mol. Biol. 2007, 372, 774–797. [Google Scholar] [CrossRef] [PubMed]
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Figure 1. Linear voltammograms of dioxygen electroreduction on the bare (1) and laccase-modified (2) MWCNT-electrodes: (a) C. caperata, (b) T. hirsuta, (c) S. murashkinskyi, and (d) A. faginea. Insert: the linear segment of the polarization curve. Conditions: 0.1 M Na–citrate–phosphate buffer; pH 5.5; electrode working surface: 0.25 cm2; scan rate: 1 mV s−1.
Figure 1. Linear voltammograms of dioxygen electroreduction on the bare (1) and laccase-modified (2) MWCNT-electrodes: (a) C. caperata, (b) T. hirsuta, (c) S. murashkinskyi, and (d) A. faginea. Insert: the linear segment of the polarization curve. Conditions: 0.1 M Na–citrate–phosphate buffer; pH 5.5; electrode working surface: 0.25 cm2; scan rate: 1 mV s−1.
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Figure 2. Plots of the change in E1/2 as a function of pH values for various laccases: 1—CcL; 2—ThL; 3—SmL; 4—AfL.
Figure 2. Plots of the change in E1/2 as a function of pH values for various laccases: 1—CcL; 2—ThL; 3—SmL; 4—AfL.
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Figure 3. The environment of the T1 center copper ion using the example of the SmL (PDB 5E9N). Structurally conserved loops are shown in black, and variable loops are shown in red.
Figure 3. The environment of the T1 center copper ion using the example of the SmL (PDB 5E9N). Structurally conserved loops are shown in black, and variable loops are shown in red.
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Figure 4. Amino acid sequences of the loops that form the substrate-binding pocket of high- and medium-redox-potential laccases from different orthologous groups (SmL 5E9N laccase numbering). Amino acid residues are highlighted depending on their charge and polarity: positively charged, negatively charged, polar uncharged, and non-polar uncharged aromatic residues are shown in blue, red, green, and gray, respectively. The residues of the first coordination sphere of the T1 center copper ion are highlighted in pink. ThL—Trametes hirsuta; TvL—Trametes versicolor; ToL—Trametes ochracea; CcL—Coriolopsis caperata; CtL—Coriolopsis trogii; CgL—Coriolopsis gallica; SmL—Steccherinum murashkinskyi; SoL—Steccherinum ochraceum; AfL—Antrodiella faginea; AcL—Antrodiella citronella.
Figure 4. Amino acid sequences of the loops that form the substrate-binding pocket of high- and medium-redox-potential laccases from different orthologous groups (SmL 5E9N laccase numbering). Amino acid residues are highlighted depending on their charge and polarity: positively charged, negatively charged, polar uncharged, and non-polar uncharged aromatic residues are shown in blue, red, green, and gray, respectively. The residues of the first coordination sphere of the T1 center copper ion are highlighted in pink. ThL—Trametes hirsuta; TvL—Trametes versicolor; ToL—Trametes ochracea; CcL—Coriolopsis caperata; CtL—Coriolopsis trogii; CgL—Coriolopsis gallica; SmL—Steccherinum murashkinskyi; SoL—Steccherinum ochraceum; AfL—Antrodiella faginea; AcL—Antrodiella citronella.
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Figure 5. Solvent accessibility of variable loops S1, S4, S5, and S6 near the T1 center of (a) medium- (SmL, AfL) and (b) high- (CcL, ThL)-redox-potential laccases. Blue—the least accessible regions, red—the most accessible regions.
Figure 5. Solvent accessibility of variable loops S1, S4, S5, and S6 near the T1 center of (a) medium- (SmL, AfL) and (b) high- (CcL, ThL)-redox-potential laccases. Blue—the least accessible regions, red—the most accessible regions.
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Table 1. Some biochemical properties of laccases.
Table 1. Some biochemical properties of laccases.
LaccaseMw, kDapICarbohydrate Content, %E0T1, mV *Copper ContentRef.
CcL633.516780 ± 104[40]
ThL664.012780 ± 104[40]
SmL633.09650 ± 204[41]
AfL654.517620 ± 504[41]
* vs. NHE.
Table 2. Some characteristics of MWCNT-electrodes with adsorbed laccases.
Table 2. Some characteristics of MWCNT-electrodes with adsorbed laccases.
LaccaseΓ, pmol cm−2Ess, * mV Eonset, * mV E½, * mV Δi/ΔE, μA mV−1
CcL1.86 ± 0.31856 ± 16 a843 ± 11 a818 ± 9 a1.65
ThL1.67 ± 0.23830 ± 18 ab818 ± 14 ab789 ± 11 a1.43
SmL1.58 ± 0.25809 ± 19 ab787 ± 13 bc742 ± 10 b0.54
AfL1.81 ± 0.20792 ± 18 b776 ± 10 c747 ± 7 b0.66
* Measurements were performed in 0.1 M Na–citrate–phosphate buffer, pH 5.5, at 23 °C. All the potentials are given vs. NHE. Means within the same column with different superscripts are significantly different (p < 0.05).
Table 3. Kinetic parameters of laccase-catalyzed oxidation of electron donor substrates *.
Table 3. Kinetic parameters of laccase-catalyzed oxidation of electron donor substrates *.
LaccaseK4Mo(CN)8
E0 = 782 mV [46]		ABTS
E0 = 682 mV [47]		K4Fe(CN)6
E0 = 412 mV [48]
kcat, s−1KM, μMkcat/KM, s−1 μM−1kcat, s−1KM, μMkcat/KM, s−1 μM−1kcat, s−1KM, μMkcat/KM, s−1 μM−1
CcL74.6 ± 3.3334.7 ± 19.40.22373.2 ± 3.911.8 ± 0.56.2081.8 ± 3.350.4 ± 2.81.62
ThL23.7 ± 2.41685.0 ± 56.90.01474.7 ± 3.116.7 ± 0.24.4765.6 ± 3.2245.1 ± 7.20.27
SmL9.3 ± 0.31270.3 ± 48.60.00733.3 ± 1.471.7 ± 2.90.4662.7 ± 1.990.0 ± 2.70.70
AfL20.6 ± 0.4480.0 ± 22.10.04332.7 ± 1.14.9 ± 0.46.6757.2 ± 1.688.1 ± 6.40.65
* Measurements were performed in 0.1 M Na–citrate–phosphate buffer, pH 5.5, at 23 °C. The substrate potentials are given vs. NHE.
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Morozova, O.; Khlupova, M.; Vasil’eva, I.; Yaropolov, A.; Fedorova, T. Fungal Laccases with High and Medium Redox Potential: Is the T1 Center Potential a Key Characteristic of Catalytic Efficiency in Heterogeneous and Homogeneous Reactions? Int. J. Mol. Sci. 2025, 26, 7488. https://doi.org/10.3390/ijms26157488

AMA Style

Morozova O, Khlupova M, Vasil’eva I, Yaropolov A, Fedorova T. Fungal Laccases with High and Medium Redox Potential: Is the T1 Center Potential a Key Characteristic of Catalytic Efficiency in Heterogeneous and Homogeneous Reactions? International Journal of Molecular Sciences. 2025; 26(15):7488. https://doi.org/10.3390/ijms26157488

Chicago/Turabian Style

Morozova, Olga, Maria Khlupova, Irina Vasil’eva, Alexander Yaropolov, and Tatyana Fedorova. 2025. "Fungal Laccases with High and Medium Redox Potential: Is the T1 Center Potential a Key Characteristic of Catalytic Efficiency in Heterogeneous and Homogeneous Reactions?" International Journal of Molecular Sciences 26, no. 15: 7488. https://doi.org/10.3390/ijms26157488

APA Style

Morozova, O., Khlupova, M., Vasil’eva, I., Yaropolov, A., & Fedorova, T. (2025). Fungal Laccases with High and Medium Redox Potential: Is the T1 Center Potential a Key Characteristic of Catalytic Efficiency in Heterogeneous and Homogeneous Reactions? International Journal of Molecular Sciences, 26(15), 7488. https://doi.org/10.3390/ijms26157488

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