A Holistically Engineered Laccase Nanozyme With Enhanced Activity for Phenolic Compound Removal

ABSTRACT

To achieve high catalytic activity in laccase‐mimicking nanozymes, it is essential to accurately replicate the enzyme's active catalytic center and ensure effective transport of substrates and oxygen. To address this challenge, this work presents a holistic biomimetic strategy based on the natural laccase catalytic process, designing a novel nanozyme Cu‐Cys@PCN‐222(Cu/Fe). In this nanozyme, the incorporation of Cys and the FeTCPP into the PCN‐222 framework achieves the biomimicry of the substrate binding pocket and oxygen‐carrying hydrophobic sites, respectively. These two key components enables efficient pollutant enrichment and enhances dissolved oxygen capture and transport. Finally, Cys and TCPP within the PCN‐222 framework through the coordination with copper constructed the CuN₄(OH₂)/Cu‐S unsaturated copper clusters. This structure imitates the asymmetric coordination structure of the natural laccase active center, achieving highly efficient catalysis of substrate molecules. The synergy factor confirms the synergistic interaction among the three biomimetic functions, and this synergistic architecture promotes a highly efficient catalytic cycle, endowing the nanozyme with 2.2‐fold higher activity than natural laccase, as well as excellent structural stability and broad substrate universality. Furthermore, the degradation mechanism and intermediate product evolution were systematically investigated. This work establishes a generalizable paradigm for designing high‐performance biomimetic catalysts via the holistic biomimetic strategy.

To achieve high catalytic activity in laccase‐mimicking nanozymes, a holistic biomimetic strategy is presented. Cu‐Cys@PCN‐222(Cu/Fe) replicates the substrate binding pocket, oxygen‐carrying hydrophobic sites, and asymmetric active center (CuN₄/Cu‐S). The synergy factor confirms the synergistic interaction among the three biomimetic functions, and this synergistic architecture promotes a highly efficient catalytic cycle, endowing the nanozyme with 2.2‐fold higher activity than natural laccase, as well as excellent structural stability and broad substrate universality.

1. Introduction

Accelerated industrialization has led to the widespread contamination of industrial wastewater and municipal sewage systems with phenolic pollutants, posing significant threats to aquatic ecosystems and human health [1, 2]. Compared with traditional methods for wastewater treatment, enzymatic catalysis demonstrates outstanding catalytic efficiency and near‐zero byproduct generation [3, 4, 5, 6]. Laccase, as a representative polyphenol oxidase, achieves efficient degradation of phenolic pollutants through a catalytic cycle centered on its active site [7]. Laccase initially recognizes and captures phenolic compounds within its active pocket. Subsequently, theT1/T2/T3 multi‐copper clusters in the catalytic center of laccase oxidizes the captured phenolics (Equation 1). Simultaneously oxygen molecules from the solution are supplied to the clusters through a dedicated hydrophobic channel and reduced to water at the multi‐copper clusters (Equation 2) [8, 9]. Moreover, the practical implementation of native laccase faces substantial challenges by its instability and sensitivity to environmental fluctuations [10].

$${\mathrm{C}}_6{\mathrm{H}}_5\mathrm{OH}\stackrel{\mathrm{Laccase}}{\to }{\mathrm{C}}_6{\mathrm{H}}_4{\mathrm{O}}_2+2{\mathrm{H}}^{+}+2{\mathrm{e}}^{-}$$ (1)

$${\mathrm{O}}_2+4{\mathrm{H}}^{+}+4{\mathrm{e}}^{-}\stackrel{\mathrm{Laccase}}{\to }2{\mathrm{H}}_2\mathrm{O}$$ (2)

To address this limitation, researchers have engineered laccase biomimetic nanozymes to combine catalytic performance with enhanced stability [11]. For example, Liang et al. synthesized a Cu‐N/O‐coordinated nanozymes, successfully mimic the local coordination geometry of the copper active center, achieving affinity comparable to laccase [12, 13, 14]. Subsequent advancements, exemplified by pore‐confined active sites within covalent organic frameworks (COFs). This bioinspired design exhibited that mimicking the active site and the substrate‐confining pocket can yield activity (1.4 times) surpassing the natural laccase [15]. Despite these advancements, a fundamental challenge remains. Currently, most research on the design of nanozymes focuses on independently mimicking the multi‐copper clusters of natural enzymes, overlooking the reaction integrated of the native enzymatic process [16, 17]. However, the overall catalytic process of laccase is governed by the capture and transfer of both substrate [18] and oxygen [19] to the active center, and then triggers the core redox reactions [8, 20]. This leads to the kinetic mismatch between the high catalytic rate of the multi‐copper clusters active center and the low adsorption/transport rates of both substrate and oxygen, which creates a “short‐board effect,” ultimately limiting the overall catalytic performance.

Inspired by the holistic functionality of natural laccase [20], this work fabricated a multi‐functional laccase‐mimetic nanozyme through the holistic biomimetic strategy, named Cu‐Cys@PCN‐222(Cu/Fe). This approach aims to synergistically enhance both mass transfer of both substrate and oxygen and catalytic reaction kinetics. The design of Cu‐Cys@PCN‐222(Cu/Fe) nanozyme achieves the synergistic biomimicry of three key functional components. The porous framework of PCN‐222 mimics the substrate binding pocket of natural laccase, enabling efficient capture of phenolic pollutants. Simultaneously, the incorporated heme‐like FeTCPP unit establishes hydrophobic sites to facilitate dissolved oxygen capture and transport, thereby mimicking the native oxygen supply function [21, 22]. Furthermore, Cys and TCPP within the PCN‐222 framework through the coordination with copper constructed the CuN₄(OH₂)/Cu‐S unsaturated copper clusters, which imitated the asymmetric coordination structure of the natural laccase active center. The synergy of the three key functional components ensures a highly efficient catalytic progress, as well as promoting substrate enrichment, oxygen supply, and efficient electron transfer. The synergistic enhancement among the three key functional components was verified by systematically comparing different functional systems, enabling Cu‐Cys@PCN‐222(Cu/Fe) to achieve 2.2‐fold higher catalytic efficiency than natural laccase. Furthermore, the nanozyme combines broad‐spectrum degradation capability with excellent stability in industrial wastewater, outperforming most previously reported laccase‐mimicking nanozymes. This work present a novel strategy for high‐performance biomimetic catalysts through holistic mimicry of natural enzymatic systems. This approach overcomes the “short‐board effect” to achieve superior nanozyme performance.

2. Experimental

2.1. Characterizations and Reagents

Detailed types of experimental characterizations (Section S1) and reagents (Section S2) could be found in the supporting information.

2.2. Preparation Of Nanozymes

All materials were synthesized based on a reported procedure for PCN‐222(H) [23]. Briefly, PCN‐222(H/Fe) and Cys@PCN‐222(H/Fe) were prepared by adding FeTCPP and L‐cysteine during the synthesis. Finally, Cu‐Cys@PCN‐222(Cu/Fe) was obtained by introducing Cu²⁺ ions into Cys@PCN‐222(H/Fe) via hydrothermal coordination. The preparation process of Cu‐Cys@PCN‐222(Cu/Fe) was depicted in Scheme S1. The detailed preparation procedures of all materials were provided in Section S3.

2.3. Catalytic Experiment and Analysis Methods

In this work, 2,4‐dichlorophenol and 4‐aminoantipyrine were used as reaction substrates and chromogenic reagent, and the laccase and nanozyme catalytic activity was detected by coupling chromogenic reaction. Subsequently, the catalytic process and ability of nanozymes were evaluated through the stability comparison experiments, determination of enzyme kinetic, electrochemical tests, and investigation of the nanozyme catalytic mechanism. The detailed specific process was described in Section S4.

3. Results and Discussion

3.1. Structural Characterizations

Scanning electron microscope (SEM) images of Cu‐Cys@PCN‐222(Cu/Fe) nanozyme and its comparative materials were presented in Figure 1a–d. PCN‐222(H) exhibited elongated rod‐like structures (Figure 1a) with lengths ranging from 5 to 20 µm, consistent with previous reports [24]. After the incorporation of the structurally similar iron porphyrin ligand, PCN‐222(H/Fe) maintained essentially identical morphology to PCN‐222(H) (Figure 1b). The rod‐like structures in Cys@PCN‐222(H/Fe) showed an increased diameter with the further incorporation of Cys (Figure 1c), which could be primarily attributed to the alterations in the crystalline structure induced by Cys [15]. Following copper coordination, the resulting Cu‐Cys@PCN‐222(Cu/Fe) maintained similar morphology to Cys@PCN‐222(H/Fe) (Figure 1d), confirming that copper coordination did not affect the material's morphology. Furthermore, EDS mapping (Figure 1e) revealed a homogeneous distribution of Fe, Cu, and S elements on Cu‐Cys@PCN‐222(Cu/Fe), providing evidence for successful incorporation of FeTCPP, Cu, and Cys within the PCN‐222 framework.

FIGURE 1.

SEM images of PCN‐222(H) (a), PCN‐222(H/Fe) (b), Cys@PCN‐222(H/Fe) (c), Cu‐Cys@PCN‐222(Cu/Fe) (d); EDS images of Cu‐Cys@PCN‐222(Cu/Fe) (e); PXRD patterns (f); N₂ adsorption‐desorption isotherms (g); FT‐IR spectra (h) of PCN‐222(H), PCN‐222(H/Fe), Cys@PCN‐222(H/Fe) and Cu‐Cys@PCN‐222(Cu/Fe).

The crystal structures of the materials were characterized by powder X‐ray diffraction (PXRD) analysis (Figure 1f). The characteristic diffraction peaks of all materials matched well with the simulated pattern of PCN‐222(H) reported in the literature [24, 25], indicating that the PCN‐222 framework is maintained during the modification process [26, 27]. In the final Cu‐Cys@PCN‐222(Cu/Fe), the intensities of all major diffraction peaks decreased [28]. Detailed analysis and XRD images of Cys@PCN‐222(H), PCN‐222(Cu), Cu‐Cys@PCN‐222(Cu), and PCN‐222(Cu/Fe) were provided in Figure S1 to confirm the successful synthesis of materials. N₂ adsorption‐desorption isotherms were employed to investigate the specific surface areas of the four nanozymes, with results shown in Figure 1g and Table S1. All materials exhibited typical Type IV adsorption‐desorption curves, indicating the coexistence of abundant mesopores and micropores in their pore structures [29]. Detailed analysis and the NLDFT pore size distribution was provided in the Figure S2. Subsequent Fourier transform infrared spectroscopy (FT‐IR) analysis (Figure 1h, Figure S3a) showed that the characteristic peaks corresponding to the porphyrin ligand structure and Zr─O bond were presented in all four materials. This verified the successful synthesis of PCN‐222 framework [30]. Detailed analysis and the thermogravimetric analysis (TGA) stability tests was provided in the Figure S3.

High‐resolution X‐ray photoelectron spectroscopy (XPS) confirmed the presence of expected elements in PCN‐222(H), PCN‐222(H/Fe), Cys@PCN‐222(H/Fe) and Cu‐Cys@PCN‐222(Cu/Fe) (Figure S4a, Table S2), indicating the successful incorporation of FeTCPP, Cys, and Cu. Detailed analysis of the N 1s region (Figure 2a) confirmed the presence of characteristic pyrrolic N and C═N─C species from the porphyrin structure. After incorporating of FeTCPP, a new peak appeared at 398.8 eV, corresponding to Fe─N coordination [31]. The N 1s spectrum remained nearly identical after Cys introduction, indicating that the Cys modification did not affect the coordination environment of nitrogen in the porphyrin framework. In contrast, subsequent coordination with copper ions produced a distinct Cu─N peak at 396.8 eV, which unambiguously confirms the metalation of the porphyrin center. Notably, the coordination of Cu ions induced a binding energy shift (−0.1 eV) in both Fe─N and C═N─C environments, indicating that the formation of Cu─N bond promoted an increase in electron density [32]. This electronic perturbation was further reflected in the Fe 2p energy level (Figure S4b). After the incorporation of Cys, the binding energy of Fe 2p_1/2 and Fe 2p_3/2 peaks decreased by 0.1 eV, followed by further shifts of 0.2 and 0.3 eV, respectively upon copper coordination. The S 2p spectra (Figure S4c) provided direct evidence for the successful anchoring and subsequent metal coordination of Cys. The S‐H signal in Cys@PCN‐222(H/Fe) confirmed successful loading of Cys. The appearance of a Cu─S peak at 162.3 eV in Cu‐Cys@PCN‐222(Cu/Fe) directly demonstrated coordination between copper and Cys, forming the Cu‐S site [33]. In the Cu 2p spectra (Figure 2b), Cu‐Cys@PCN‐222(Cu/Fe) exhibited characteristic peaks at 935.0 and 933.1 eV, assigned to Cu²⁺ and Cu⁺/Cu⁰ species [34], respectively. The Cu LM2 spectra of Cu‐Cys@PCN‐222(Cu/Fe) (Figure 2c) further confirmed that copper species exist in the form of mixed‐valence states of Cu⁺ (28.9%) and Cu²⁺ (72.1%). The above results collectively demonstrated the coexistence of both Cu─N and Cu─S sites in Cu‐Cys@PCN‐222(Cu/Fe), along with the formation of a mixed Cu⁺/Cu²⁺ valence state.

FIGURE 2.

N 1s XPS (a); Cu 2p (b) and Cu LM2 XPS (c) of PCN‐222(H), PCN‐222(H/Fe), Cys@PCN‐222(H/Fe) and Cu‐Cys@PCN‐222(Cu/Fe); Cu K edge XANES spectra (d), FT‐XANES (e), WT for the FT k‐weighted χ(k) function for Cu‐Cys@PCN‐222(Cu/Fe) and standard samples (f).

To precisely elucidate the local coordination structure and electronic state of the copper centers in the catalyst, this work employed X‐ray absorption spectroscopy (XAS) to perform a detailed analysis of the active site structure of Cu‐Cys@PCN‐222(Cu/Fe) (Figure 2d–f, Tables S3 and S4). The Cu K‐edge X‐ray absorption near‐edge structure (XANES) spectrum (Figure 2d) revealed the absorption edge position of the Cu‐Cys@PCN‐222(Cu/Fe). This position lies between those of the Cu₂O (Cu⁺) and CuO/CuPc (Cu²⁺) reference samples, indicating an intermediate valence state between Cu⁺ and Cu²⁺ [35]. Subsequent linear combination fitting (LCF) quantitatively confirmed the mixed‐valence nature of the copper centers, with Cu²⁺ accounting for approximately 84.3% and Cu⁺ for about 15.7% (Table S3). The presence of Cu⁺ was attributed to the electron‐donating effect of the negatively charged porphyrin ligand, while being stabilized within the framework of the porphyrin ring. Fourier‐transform extended X‐ray absorption fine structure (FT‐EXAFS) analysis in R‐space (Figure 2e, Figure S5) further resolved and fitted the local coordination environment. The fitting results (Table S4) showed a fitted Cu‐N bond length of 1.97 Å, which agreed with literature values reported for metal‐porphyrin ring center coordination structures [36, 37]. This result indicated that copper ions were incorporated into the porphyrin center of the PCN‐222 ligand. Meanwhile, the average coordination number of 5.4 confirmed that, in addition to the four equatorial nitrogen atoms of the porphyrin ring, the copper ion was most likely axially coordinated by water molecules [38]. This inference was supported by the fitting data of Cu⁺ species in Table S3 and the aqueous environment during the nanozyme synthesis. This axial coordination created a CuN₄(OH₂) copper clusters structure with the characteristic of an unsaturated laccase active center. The presence of the axially coordinated water molecule created a weak ligand field and a labile proton transfer site, which effectively reduced the energy barrier for catalytic reactions [39]. Furthermore, the fitting results identified another key scattering path corresponding to Cu─S (R = 2.19 Å). This Cu‐S site was connected to the CuN₄(OH₂) unit via Zr─O clusters, forming the multi‐type copper clusters structure analogous to the active center of laccase. Wavelet transform (WT) analysis (Figure 2f) further verified the mixed coordination structure. This was evidenced by a significant intensity maximum at approximately 5.47 Å⁻¹ in the k‐space spectrum of Cu‐Cys@PCN‑222(Cu/Fe). This maximum was positioned between the CuPc (5.50 Å⁻¹, Cu─N) and CuS (5.39 Å⁻¹, Cu─S) standards. This intermediate maximum was attributed to the combined contribution of scattering signals from two distinct bond types in the material, confirming the coexistence of Cu─N and Cu─S coordination environments from a spatially resolved perspective [36].

Building upon the all the above results confirmed that the key multi‐copper clusters active structure of the Cu‐Cys@PCN‑222(Cu/Fe), composed of CuN₄(OH₂) sites from copper‐porphyrin coordination and Cu‐S sites from copper‐Cys coordination. The axially unsaturated coordination in the porphyrin center not only better biomimicked the unsaturated active site of laccase but also provided a flexible coordination field that was essential for stabilizing the mixed valence state (Cu⁺/Cu²⁺). This unique structure effectively promotes substrate accessibility and activation, thereby significantly reducing the energy barrier of the catalytic reaction. Furthermore, the mixed valence (Cu⁺/Cu²⁺) and mixed coordination (CuN₄(OH₂)/Cu─S) environments work synergistically to facilitate efficient electron transfer during catalytic cycles. To evaluate the electrochemical properties arising from this unique electronic structure, electrochemical impedance spectroscopy (EIS, Figure S6a) and cyclic voltammetry (CV, Figure S6b) were employed. Cu‐Cys@PCN‐222(Cu/Fe) exhibited excellent electrical conductivity and redox properties, which was evidenced by its lowest R_et value (46.3 Ω), the highest peak current difference and the smallest peak pressure difference (Figure S6c) [40].

3.2. Evaluation of Catalytic Performance

To evaluate and compare the superior catalytic performance of nanozymes, this work tested the catalytic performances of Cu‐Cys@PCN‐222(Cu/Fe) and six comparative materials. The activity was investigated through the coupling chromogenic reaction of 2,4‐DP and 4‐AP reported in the literature (Figure 3a) [14]. As shown in the spectral data, the strongest absorption peak of Cu‐Cys@PCN‐222(Cu/Fe) at 510 nm reflected its exceptional catalytic performance. In contrast, PCN‐222(H), PCN‐222(H/Fe), Cys@PCN‐222(H/Fe), and Cys@PCN‐222(H/Fe) showed negligible catalytic activity due to the lack of active copper ions. The activity of Cu‐Cys@PCN‐222(Cu/Fe) was 2.2‐fold higher than that of natural laccase. This result confirmed excellent biomimetic enzymatic activity of Cu‐Cys@PCN‐222(Cu/Fe). Additionally, Cu‐Cys and PCN‐222(Cu) were served as contrast nanozymes with single‐type copper cluster active sites. Cu‐Cys@PCN‐222(Cu/Fe) exhibited a 3.1‐fold enhancement over Cu‐Cys and a 1.7‐fold increase relative to PCN‐222(Cu). The remarkable enhancement could be attributed to the synergistic effect between the Cu‐Cys and Cu‐porphyrin sites. Subsequently, we systematically compared the final material Cu‐Cys@PCN‐222(Cu/Fe) with three bifunctional control materials: PCN‐222(Cu/Fe) (without Cys), Cu‐Cys@PCN‐222(Cu) (without FeTCPP), and Cys@PCN‐222(H/Fe) (without Cu). The results showed that the catalytic activity of Cu‐Cys@PCN‐222(Cu/Fe) was significantly superior to that of the other bifunctional control materials (Figure S7). Furthermore, to quantitatively evaluate the synergistic effect, we introduced the synergy factor α (defined in Section S4.8). The three bifunctional combinations showed synergy factors of 1.07 (Cu/FeTCPP), 1.13 (Cu/Cys), and 1.06 (Cys/FeTCPP). All bifunctional combinations exhibited only weak synergistic enhancement. In contrast, the final material Cu‐Cys@PCN‐222(Cu/Fe) showed a significantly higher synergy factor (α = 1.34). This indicated that the combination of any two functional components yields only limited synergy, while the combination of all three components significantly enhanced the overall catalytic performance (Figure S8). The four aforementioned most active materials were subsequently evaluated by degrading the model pollutant 2,4‐dichlorophenol (2,4‐DP) (Figure 3b). After 10 h reaction, the final degradation efficiency of Cu‐Cys@PCN‐222(Cu/Fe) reached 68.9%. This value substantially exceeded those of laccase (50.9%), Cu‐Cys (22.4%), and PCN‐222(Cu) (58.8%). Kinetic fitting (Figure 3c,d) of the degradation profiles revealed that the rate constant of Cu‐Cys@PCN‐222(Cu/Fe) (0.18 min⁻¹) was 2.0‐fold, 3.6‐fold, and 1.4‐fold greater than those of laccase, Cu‐Cys, and PCN‐222(Cu), respectively.

FIGURE 3.

2,4‐DP and 4‐AP coupled chromogenic reactions catalyzed by laccase or laccase nanozymes and their corresponding UV‐Vis spectra (a); Degradation curves (b), degradation kinetic curves (c), and degradation ability comparison (d) of 2,4‐DP by laccase, PCN‐222(Cu), Cu‐Cys, and Cu‐Cys@PCN‐222(Cu/Fe); Lineweaver‐Burk curve (e) and Kinetic parameters of laccase and Cu‐Cys@PCN‐222(Cu/Fe) (f).

To gain deeper insight into the catalytic capabilities toward substrates, the Michaelis‐Menten kinetics of laccase and Cu‐Cys@PCN‐222(Cu/Fe) were investigated via Lineweaver‐Burk analysis (Figure 3e,f; Table S5) [18, 41]. The Michaelis constant (K _m) of the Cu‐Cys@PCN‐222(Cu/Fe) (0.51 mM) was lower than that of laccase (0.72 mM). Meanwhile, the maximum reaction rate (V _max) of the Cu‐Cys@PCN‐222(Cu/Fe) (0.66 mM·min⁻¹) exceeded that of the natural enzyme (0.54 mM·min⁻¹). The lower K _m value and higher V _max value indicated that Cu‐Cys@PCN‐222(Cu/Fe) exhibited a stronger substrate affinity and a higher catalytic rate compared to laccase [42, 43, 44]. More significantly, the catalytic rate constant (k _cat) of Cu‐Cys@PCN‐222(Cu/Fe) was 2.3‐fold higher than that of laccase, revealing its superior intrinsic activity. Consequently, the calculated catalytic efficiency (k _cat/K _m) of Cu‐Cys@PCN‐222(Cu/Fe) was 3.63 × 10⁴ mM⁻¹·min⁻¹, which represented a 3.2‐fold higher than that of the natural laccase (1.13 × 10⁴ mM⁻¹·min⁻¹). This value confirmed its exceptional overall catalytic proficiency. To further evaluate the substrate universality of the final material, Cu‐Cys@PCN‐222(Cu/Fe) were used to mediate the chromogenic coupling reaction between a series of phenolic compounds (molecular structures shown in Table S6) and 4‐AP (as shown in Figure S9) [45, 46, 47]. Under identical experimental conditions, the measured absorbance values ranged from 0.503 to 1.268. The results demonstrate that the Cu‐Cys@PCN‐222(Cu/Fe) nanozyme exhibits broad‐spectrum substrate degradation performance, showing high catalytic activity toward various phenolic pollutants.

The catalytic stability of Cu‐Cys@PCN‐222(Cu/Fe) nanozyme was investigated by adjusting different environmental conditions (Figure S10a–e). The results showed that Cu‐Cys@PCN‐222(Cu/Fe) maintained significantly higher reactivity than natural laccase under all tested conditions. It exhibited superior tolerance under conditions of pH 3–9, temperature 30°C–90°C, storage within 9 days, high concentration of NaCl (500 mm) and pure ethanol. This excellent stability originated from the stabilizing effect of the PCN‐222 framework. Furthermore, reusability tests showed,84% of the catalytic activity was still retained after seven cycles (Figure S10f). The stability of the nanozyme structure before and after use was fully demonstrated by PXRD analysis (Figure S11). Collectively, Cu‐Cys@PCN‐222(Cu/Fe) nanozyme demonstrated excellent stability. For a more comprehensive assessment, we compared Cu‐Cys@PCN‐222(Cu/Fe) nanozyme with previously reported laccase‐mimicking nanozymes in terms of pH tolerance, thermal stability, storage stability, reusability, and catalytic activity (Table S7) [48, 49, 50, 51, 52]. This comparison demonstrated that Cu‐Cys@PCN‐222(Cu/Fe) nanozyme exhibited a well‐balanced and competitive overall performance among the reported systems. To evaluate the practical application potential of the nanozyme, we extended the stability tests to synthetic industrial wastewater based on compositions reported in the literature (detailed composition shown in Table S8) [53, 54]. Under these conditions, Cu‐Cys@PCN‐222(Cu/Fe) maintained higher catalytic activity than natural laccase (Figure S12). The results showed that the degradation activity of natural laccase gradually decreased over time with a flattening trend, while Cu‐Cys@PCN‐222(Cu/Fe) consistently maintained higher catalytic activity. After 10 h of reaction, the remaining degradation ability of the nanozyme was 1.65‐fold than that of natural laccase, demonstrating its promising practical application potential in complex wastewater environments.

3.3. Catalytic Reaction Mechanism of Cu‐Cys@PCN‐222(Cu/Fe)

The above research results clearly revealed the multi‐copper clusters active structure composed of the nanozyme. The mixed valence (Cu⁺/Cu²⁺) and mixed coordination (CuN₄(OH₂)/Cu‐S) environments enhanced the catalytic performance of Cu‐Cys@PCN‐222(Cu/Fe) in the degradation of 2,4‐DP. In the following section, we provide a detailed analysis of the adsorption behavior of the nanozyme toward substrate and oxygen molecules, as well as its degradation mechanism.

The substrate adsorption capability of the Cu‐Cys@PCN‐222(Cu/Fe) nanozyme was first evaluated. Given the prominent catalytic activity of Cu‐modification nanozymes, PCN‐222(H/Fe) and Cys@PCN‐222(H/Fe) without Cu modification were selected as adsorbents. The adsorption isotherms of both materials were measured across a range of 2,4‐DP concentrations. The data were fitted with both Langmuir and Freundlich models (Figure S13) [55, 56], showing a better alignment with the Langmuir model. The fitting results (Figure 4a) indicated that PCN‐222(H/Fe) had a maximum adsorption capacity (Q _max) of 263.79 mg/g and a Langmuir constant (K _L) of 0.10 L/g, while Cys@PCN‐222(H/Fe) exhibited a Q _max of 233.26 mg/g and a K _L of 0.13 L/g. Since the specific surface area of Cys@PCN‐222(H/Fe) after cysteine modification was approximately 0.27‐fold that of PCN‐222(H/Fe), we compared the fitted parameters normalized by specific surface area. As shown in Figure 4b, the specific surface area‐normalized Q _max and K _L of Cys@PCN‐222(H/Fe) were 3.25‐fold and 4.21‐fold higher than those of PCN‐222(H/Fe), respectively. The Cys‐modified Cys@PCN‐222(H/Fe) framework exhibited superior adsorption capacity and substrate binding affinity, primarily attributable to the enhanced specific interactions between the incorporated Cys and the substrate molecules. The adsorption kinetics of Cys@PCN‐222(H/Fe) toward 2,4‐DP were subsequently analyzed (Figure 4c) [57, 58]. After 20 min of adsorption, Cys@PCN‐222(H/Fe) reached near‐equilibrium adsorption for 2,4‐DP (Figure S14). Kinetic analysis using both pseudo first‐order and pseudo second‐order models revealed that the adsorption process followed pseudo‐first‐order kinetics, with the rate constant (k) of 0.12 min⁻¹. The calculated k value was compared with those of other recently reported adsorbents under similar conditions (Figure 4d). Notably, the adsorption rate constant of Cys@PCN‐222(H/Fe) was approximately 1.1 to 60.0 times higher than those of other adsorbents, underscoring the superior kinetics of Cys@PCN‐222(H/Fe) [59, 60, 61, 62, 63, 64, 65]. These results demonstrated that the biomimetic active pocket structure within the Cys‐modified porous framework effectively enhanced the nanozyme's substrate capture capability.

FIGURE 4.

Langmuir fitting for 2,4‐DP using PCN‐222(H/Fe) and Cys@PCN‐222(H/Fe) (a); Comparison of the Langmuir fitted parameters normalized by specific surface area (b); Pseudo‐first‐order kinetic simulations for 2,4‐DP using Cys@PCN‐222(H/Fe) (c); Comparison of adsorption ability of 2,4‐DP by different adsorbents reported in the literatures (d); Raman spectra of PCN‐222(Cu/Fe) and Cu‐Cys@PCN‐222(Cu/Fe) before and after puming O₂ (e,f); The comparison of current response under different initial oxygen concentration in 1 mm 2,4‐DP solution between Cu‐Cys@PCN‐222(Cu/Fe) and Cu‐Cys@PCN‐222(Cu) (g).

Building upon the excellent 2,4‐DP adsorption of the framework, we further investigated the promoting effect of incorporating FeTCPP biomimetic hydrophobic sites with oxygen‐carrying ability to capture dissolved oxygen. The catalytic activities of PCN‐222(Cu), PCN‐222(Cu/Fe), Cu‐Cys@PCN‐222(Cu), and Cu‐Cys@PCN‐222(Cu/Fe) were determined by coupling color reaction (Figure S15) [14]. The results demonstrated that after doping FeTCPP with the oxygen‐carrying ability, the catalytic performance of PCN‐222 (Cu/Fe) and Cu‐Cys@PCN‐222 (Cu/Fe) was significantly better than that of the corresponding materials without FeTCPP doping. Subsequently, Raman spectroscopy was employed to analyze the spectral changes of FeTCPP‐doped PCN‐222(Cu/Fe) and Cu‐Cys@PCN‐222(Cu/Fe) under oxygen and nitrogen atmospheres (Figure 4e,f, Figure S16). Under nitrogen atmosphere, the Raman spectra of both nanozymes showed negligible changes. After introducing oxygen, characteristic peaks corresponding to the pyrrole ring (1325 and 1450 cm⁻¹) and the ferrous ion (1560 cm⁻¹) in both PCN‐222(Cu/Fe) and Cu‐Cys@PCN‐222(Cu/Fe) shifted to lower wavenumbers. This indicated that the porphyrin rings and ferrous ions on the nanozyme surface successfully adsorbed oxygen molecules via coordination after FeTCPP doping [66, 67]. The electrochemical current‐time (I‐t) curves were further employed to compare the electrochemical responses of Cu‐Cys@PCN‐222(Cu/Fe) and Cu‐Cys@PCN‐222(Cu) during their reaction with 2,4‐DP under different oxygen concentrations (Figure S17, Section S4.4). Analysis revealed that under different oxygen concentration conditions, the difference in I‐t signal peak values (ΔI) of FeTCPP‐doped Cu‐Cys@PCN‐222(Cu/Fe) was significantly higher than that of the undoped Cu‐Cys@PCN‐222(Cu). Moreover, as the dissolved oxygen concentration in the solution increased, the ΔI ratio of the two nanozymes increased from 1.12 to 3.72 (Figure 4g). A larger peak current difference (ΔI) indicated a stronger ability of the nanozyme to utilize dissolved oxygen [68]. These electrochemical results fully demonstrated that the FeTCPP biomimetic hydrophobic sites with oxygen‐carrying ability enhanced the ability of nanozyme to capture and reduce dissolved oxygen.

The reaction pathway of Cu‐Cys@PCN‐222(Cu/Fe) was further investigated using various in situ characterization techniques. First, in situ Raman spectroscopy was applied to monitor the dynamic evolution of the catalyst's active centers during the reaction (Figure 5a). The characteristic peaks observed at 227, 385, and 891 cm⁻¹ corresponded to the signals of Cu(I) and Cu(II) species [69, 70], which reflected the adsorption of trace water around the copper active centers [71]. The synchronous enhancement of the signal intensities of the Cu(I) and Cu(II) was attributed to the stretching vibrations of Cu⁺─N/O bonds and Cu²⁺‐N/O bonds, which were due to the coordination between the active centers and reaction intermediates. The changes in the intensity of these peaks directly reflected the evolution of the coordination relationship between the reaction intermediates and the active centers during the catalytic process. To gain deeper understanding of the dynamic behaviors of these species, the corresponding peak areas were further analyzed (Figure 5e, Table S9). The growth trend of the peak areas stabilized after 15 min of reaction, indicating that the catalytic cycle reached a steady‐state operation phase due to the dynamic equilibrium in the accumulation of reaction intermediates. Subsequently, in situ attenuated total reflectance FTIR (ATR‐FTIR) spectroscopy was utilized to track the formation of reaction intermediates during the Cu‐Cys@PCN‐222(Cu/Fe)‐catalyzed degradation of 2,4‐DP. As shown in Figure 5b, two characteristic absorption peaks at 1241 cm⁻¹ (C─O intermediate) and 1650 cm⁻¹ (benzoquinone C─O) were observed to continuously increase in intensity [72, 73]. Meanwhile, a broad characteristic peak attributed to H₂O appeared in the 3000–4000 cm⁻¹ region. These results confirmed the reaction pathway in which 2,4‐DP was converted into quinone products through oxidative intermediates. Further analysis of the peak areas for these characteristic bands (Figure 5e, Table S10) revealed that their increasing trend likewise stabilized after 15 min, which corroborates the observations from the in situ Raman spectroscopy. Next, the catalytic process was monitored and analyzed using electron paramagnetic resonance (EPR) spectroscopy. Using 5,5‐Dimethyl‐1‐pyrroline N‐oxide (DMPO) as a spin‐trapping agent, distinct characteristic signals of quinone‐type radicals and DMPO‐H• adducts were detected during the reaction (Figure S18) [71]. The intensities of both signals gradually increased as the reaction proceeded (Figure 5c). The presence of the DMPO‐H• adduct confirmed that the reaction proceeds via a radical‐based dehydrogenation pathway. The resulting intermediate was a quinone‐type radical, which was consistent with the in situ infrared spectroscopy. Finally, the oxygen reduction pathway of Cu‐Cys@PCN‐222 leading to water formation was verified using a rotating ring‐disk electrode (RDE) (Figure 5d, Section S4.6) [74, 75]. The experimental results showed that as the electrode rotation rate increased (Figure S19), the disk current exhibited an upward trend, confirming the occurrence of oxygen reduction [76]. The average electron transfer number (Figure 5d Illustration) of the oxygen reduction reaction was calculated to be 3.30 using the Koutecky‐Levich (Equation S1, Section S4.7), which was consistent with the four‐electron process described in Equation (2).

FIGURE 5.

In situ Raman spectra (a) and In situ ATR‐FTIR spectra (b) of Cu‐Cys@PCN‐222(Cu/Fe) catalyzed 2,4‐DP oxidation reaction; DMPO spin‐trapping EPR spectra of 2,4‐DP and 2,4‐DP+Cu‐Cys@PCN‐222(Cu/Fe) system (c); The RDE test of the O₂ reduction (d); The characteristic peak area value of In situ Raman and In situ ATR‐FTIR spectra (e); The schematic mechanistic cycle for Cu‐Cys@PCN‐222(Cu/Fe) (f).

Based on the above mechanistic analysis, the catalytic mechanism of Cu‐Cy@PCN‐222(Cu/Fe) toward 2,4‐DP degradation is illustrated in Figure 5f. The catalytic cycle illustrated in Figure 5f consists of five steps. Step I is substrate capture by the Cys‐modified porous framework, which is supported by Langmuir adsorption fitting (Figure 4a,b) and adsorption kinetics (Figure 4c,d), showing that Cys modification enhances both adsorption capacity and binding affinity. Step II is the dehydrogenation of the captured substrate to form quinone products. In situ ATR‐FTIR (Figure 5b) detects the C─O intermediate at 1241 cm⁻¹ and benzoquinone at 1650 cm⁻¹, while EPR (Figure 5c) detects the DMPO‐H• adduct, confirming a radical‐based dehydrogenation pathway. Step III is electron transfer from the substrate to the copper clusters. In situ Raman (Figure 5a) shows that the characteristic peaks of Cu⁺ and Cu²⁺ increase synchronously during the reaction, directly reflecting the redox cycling of the copper active centers. Step IV is oxygen capture by the FeTCPP sites. Raman spectroscopy (Figure 4e,f) reveals that the pyrrole ring and ferrous ion peaks shift upon O₂ introduction, indicating that FeTCPP successfully captures molecular oxygen. The I‐t curves (Figure 4g) further show that the FeTCPP‐containing nanozyme exhibits a much higher current response under oxygen‐rich conditions, demonstrating its enhanced oxygen utilization capability. Step V is the reduction of captured oxygen to water. The RDE experiment (Figure 5d) gives an electron transfer number of 3.30, close to the theoretical four‐electron value, directly demonstrating that molecular oxygen undergoes a four‐electron reduction pathway (O₂ + 4e⁻ + 4H⁺ → 2H₂O). Together, these five lines of evidence provide a complete and self‐consistent mechanistic picture.

4. Conclusion

This study successfully constructed a high performance laccase‐mimicking nanozyme using a holistic biomimetic strategy. The nanozyme exhibits excellent structural stability and broad substrate universality, with a catalytic performance 2.2‐fold higher than that of natural laccase. This performance results from the holistic biomimicry of three key functions of laccase. Specifically, the incorporation of Cys and the FeTCPP into the PCN‐222 framework achieves the biomimicry of the substrate binding pocket and oxygen‐carrying hydrophobic sites, respectively. Furthermore, the coordination of Cys and TCPP with copper constructs the unsaturated CuN₄(OH₂)/Cu‐S clusters mimics the asymmetric coordination structure of the natural laccase active center. By mimicking the multi‐functional structure of laccase, the ternary synergistic effect is quantitatively validated by a synergy factor α of 1.34. Mechanism study indicates the catalytic cycle involves substrate capture, dehydrogenation, electron‐proton transfer, and the regeneration of the active site, which achieves an overall enhancement of the substrate catalytic ability of the nanozyme. This strategy is achieved by holistically imitating the multi‐functional architecture of natural enzymes, which effectively overcomes the “short‐board effect” inherent in conventional nanozyme designs.

Author Contributions

C.X. and Z.Z. conceived the research idea and designed the experiments. C.X., M.T., J.H., and Z.C. performed the experiments and analyzed the data. X.W., Z.L., Z.W., P.G., L.Z., and Z.Z. provided constructive suggestions for the discussion. C.X. and Z.Z. wrote and revised the paper. Z.Z. supervised the project.

Conflicts of Interest

The authors declare no conflicts of interest.

Supporting information

Supporting File: smll73924‐sup‐0001‐SuppMat.docx.

Data Availability Statement

The data that support the findings of this study are available from the corresponding author upon reasonable request.