Assembling a single active pocket by enzyme and metal modules for simultaneously catalyzing oxidation-reduction cascades

Assembling a single active pocket by enzyme and metal modules for simultaneously catalyzing oxidation-reduction cascades | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Assembling a single active pocket by enzyme and metal modules for simultaneously catalyzing oxidation-reduction cascades Jun Ge, Yunkai Fan, Jia Hu, Qilu Wu, Mengyu Zhu, Haozhi Wang, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6659547/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted You are reading this latest preprint version Abstract Chemoenzymatic cascades, which combine the merits of enzymatic catalysis and chemical catalysis, have blossomed into a powerful technology for new-to-nature reactions. The diffusion resistance of reaction intermediates is a major rate-limiting factor in cascade reactions, which can be reduced by integrating metal catalytic modules and enzymes in a single catalyst due to the proximity effect. Here we assemble enzymatic and dual metal-single-atom photocatalytic modules in a single active pocket for “one binding two reactions” catalysis, which can eliminate the diffusion resistance of reaction intermediates. The enzyme-metal hybrid active pocket exhibited excellent activity in simultaneously catalyzing transfer hydrogenation and oxidation reactions under visible light. The diffusion and rebinding of intermediates between the multiple catalytic modules are eliminated in the artificial active pocket, achieving efficient oxidation-reduction cascades for the directed detoxification of low-concentration mycotoxins, which is not reachable through engineered enzymes or photocatalysts alone. This work proposes a new type of cascade process and establishes a powerful tool for editing enzyme active pockets with metal catalytic modules. Physical sciences/Chemistry/Catalysis/Biocatalysis Physical sciences/Chemistry/Catalysis/Photocatalysis Physical sciences/Chemistry/Catalysis/Catalyst synthesis Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction The increasing demands for expanding the spectrum of biocatalysis has accelerated the development of cascade reactions merging enzymatic catalysis with metal/photoredox catalysis. The chemoenzymatic cascades that cannot be achieved either by enzyme or by chemical catalysis alone are receiving significant attentions, unveiling the frontier of new-to-nature biocatalysis 1 – 4 . Learn from multi-enzyme cascade catalysis in nature, where the substrate channeling plays an important role in the enhancement of efficiency 5 – 7 , the metal catalytic module and enzyme has been integrated in a single catalyst to construct the enzyme-metal hybrid catalyst 8 – 10 . As the distance between enzyme and metal catalytic module decreases, the efficiency of chemoenzymatic cascade reaction can be greatly enhanced due to the proximity effect which decreases the diffusion hinderance of reaction intermediates of cascades 11 – 15 . Inspired by the above achievements, we hypothesized that if multiple catalytic modules were assembled properly with a precise spatial structure to form a single active pocket, the reactant could undergo the whole cascade reactions with very low probability that the reaction intermediate diffused out the active pocket. This new type of “one binding two reactions” catalysis could theoretically eliminate the diffusion of intermediates for cascade reactions, and greatly improve the catalytic efficiency of cascade reactions especially that with low substrate concentrations which have the serious obstacle in substrate binding and diffusion 16 . For example, this type of catalysis will highly promote the efficiency of catalytic detoxification of toxins in food and feed industry, which is a big challenge for human health and food safety 17 – 19 . However, the assembly of metal catalytic modules with enzyme to form an active site which can simultaneously catalyze multi-step cascade reactions has not been achieved yet. In this work, we constructed an enzyme-metal hybrid active pocket with high stability by co-coordinating dual metal single-atom photocatalytic modules and enzyme with a zirconium (Zr) cluster in metal-organic frameworks (MOFs) (Fig. 1 ). The tunable structure and assembly process of MOFs allows the self-organization of the spatial position of enzymatic and metallic modules in the hybrid active pocket. The enzyme-metal hybrid active pocket constructed by dual metal single-atom photocatalytic modules and an oxidoreductase, exhibits excellent activity in simultaneously catalyzing oxidation-reduction cascades of low-concentration macromolecular substrates including some toxins difficult to remove. In vitro experiments in liver and kidney cells demonstrated that the chemoenzymatic cascade reactions catalysed by the enzyme-metal hybrid active pocket significantly detoxified mycotoxins. The mechanism of the high efficiency was interpreted as the efficient utilization of intermediates and electron transfer within the artificial active pocket. The “one binding two reactions” catalysis provide tremendous possibilities for new chemoenzymatic cascade reactions applicable to synthesis and environmental remediation, particularly for the low-concentration substrates. Results and Discussion Creation of enzyme-metal hybrid active pocket The enzyme-metal hybrid active pocket was created by co-coordinating metal atoms and enzyme catalytic modules with the assistance of MOFs containing metal clusters. The amino acid residue on the enzyme surface, can theoretically coordinate with metal clusters 20 . The binding sites of laccase with three common metal clusters of MOFs (Zr 6 O 4 (OH) 4 cluster from PCN-224, Cu₂(CO₂)₄ cluster from HKUST-1, and Fe₃O(CO₂)₆ cluster from MIL-100(Fe)) were investigated by molecular docking (Supplementary Figs. 1–6). For Zr and Fe, the metal clusters most likely to coordinate with the active center-adjacent amino acids of laccase, with the binding energies of -5.36 and − 5.14 Kcal/mol, respectively. These results indicated that the enzyme active site can oriented binding to the metal clusters from MOFs with the hydrophobic pocket near T1 Cu active site towards the frameworks. Due to metal atoms can be theoretically anchored on the organic ligand of PCN-224 (tetrakis(4-carboxyphenyl)porphyrin, TCPP) to form the photocatalytic modules 21 , PCN-224 is selected as the template for constructing the enzyme-metal hybrid active pocket (Supplementary Figs. 7 and 8). Pt and Pd atoms were anchored onto the porphyrin linker of Zr cluster by a solvothermal method using H 2 PtCl 6 and Pd(OAc) 2 as the precursors. Subsequently, enzymes were used as “macro ligands” for oriented binding to Zr clusters through the active center-adjacent amino acids, generating the enzyme-metal hybrid active pocket (Fig. 2 a). High-angle annular dark-field scanning transmission electron microscopy (HAADF-STEM) and SEM images showed that PCN(PtPd) were spherical particles with an average size of 160 nm (Supplementary Figs. 9 and 10). No obvious change in the morphology of the nanocomposite was observed after the incorporating of enzymes (Supplementary Figs. 11 and 12). No aggregation of metals was detected (Fig. 2 b). The mass loadings of Pt and Pd were 3.1% and 2.7%, respectively, as measured by inductively coupled plasma–atomic emission spectrometry (Supplementary Table 1). Aberration-corrected scanning transmission electron microscopy (AC-STEM) was performed to gain direct insight into the atomic structure of PCN(PtPd)-Lac. The obvious individual bright dots observed in the images indicated the atomic dispersion of single metal atoms over PCN(PtPd)-Lac (Fig. 2 c and 2 d). Elemental mapping further revealed the uniform distribution of Pt and Pd atoms (Supplementary Fig. 13). The element analysis of PCN(PtPd)-Lac under HAADF-STEM (Supplementary Fig. 14) demonstrated the presence S from enzymes, indicating that Lac was immobilized on PCN(PtPd). The S element was well-distributed in both the inside and outside of the nanocomposite, showing that the enzyme is evenly incorporated on PCN(PtPd). The successful incorporation of the enzyme was further confirmed by cryo-electron tomography (Cryo-ET) (Supplementary Fig. 16), Fourier transform infrared spectroscopy (Supplementary Fig. 17) and confocal laser scanning microscopy (CLSM) (Supplementary Fig. 18). The loading of Lac was 24.5% as determined by the thermal gravity analysis (TGA) under nitrogen (Supplementary Fig. 19). In addition, the loading of Zr, Pt, and Pd of PCN(PtPd)-Lac decreased to 8%, 1.3% and 0.5% compared to PCN(PtPd), indicating that the coordination between enzymes and Zr clusters leads to the release of a portion of metal nodes and organic ligands (Supplementary Table 1). The mesopores generated from the coordination of enzyme and Zr clusters were characterized by Cryo-ET. Compared with PCN(PtPd)-Lac (Fig. 2 e), the electron density scan curve of PCN(PtPd) (Supplementary Fig. 22) showed a more frequent electron density variation, demonstrating that the pore sizes of PCN(PtPd) are generally smaller. The pore size distribution of PCN(PtPd) and PCN(PtPd)-Lac were analyzed by the fast Fourier transformation of the electron density. As shown in Fig. 2 h, mesopores from 7–10 nm were generated in PCN(PtPd)-Lac, whereas 5–7 nm of pores were detected in the PCN(PtPd) (Supplementary Fig. 23). The increase in the pore size of PCN(PtPd)-Lac was further confirmed by Brunauer-Emmett-Teller (BET). The proportion of 2–10 nm mesoporous pores increased significantly in the PCN(PtPd)-Lac sample, which could be beneficial for the molecular transfer in the enzymatic reactions (Supplementary Fig. 24). The distance between the metal single atoms and the original active site of laccase is approximately 15 Å (Supplementary Fig. 25). The coordination environment and chemical state of Pt and Pd atoms of PCN(PtPd)-Lac were investigated by X-ray absorption fine structures (XAFS). The Pt L-edge and Pd K-edge X-ray absorption near-edge structures (XANES) revealed the oxidation state of Pt and Pd in PCN(PtPd)-Lac. In comparison with Pt foil and Pd foil, the white line peaks of PCN(PtPd)-Lac were significant higher and showed significant shifts to higher energy, suggesting that the Pt and Pd atoms were in oxidized state (Fig. 3 a and 3 b). The sole presence of Pt and Pd atoms was confirmed by extended X-ray absorption fine structure spectroscopy (EXAFS). As shown in Fig. 3 c and Fig. 3 d, the R-space EXAFS spectra of PCN(PtPd)-Lac showed major peaks at 1.8 Å and 1.5 Å which were close to the Pt-O and Pd-O peaks, and were attributed to the Pt-N/Cl and Pd-N/O backscattering, respectively. Quantitative EXAFS curve-fitting analysis was then performed to investigate the coordination configuration. For the Pt L-edge, the fitting of PCN(PtPd)-Lac showed two coordination shells of Pt-N and Pt-Cl with coordination numbers of 2.9 and 1.4, respectively, suggesting a porphyrin-based PtN 3 structure with an axial chlorine ligand (Fig. 3 e and Supplementary Table 2). For the Pd K-edge, the fitting of PCN(PtPd)-Lac revealed two coordination shells of Pd-N and Pd-O with coordination numbers of 4.0 and 1.9, respectively, indicating a porphyrin-based PdN 4 structure with two axial oxygen ligands (Fig. 3 f and Supplementary Table 3). From the wavelet transforms (WT) of the Pt L-edge and Pd K-edge EXAFS signals of PCN(PtPd)-Lac, the intensity maximum at 1.6 Å −1 and 5.4 Å −1 are ascribed to Pt-N/Cl and Pd-N/O coordination, respectively, whereas no intensity maximum (11.4 Å −1 and 9.2 Å −1 ) corresponds to the Pt-Pt and Pd-Pd coordination were detected compared with Pt foil and Pd foil (Fig. 3 g and 3 h). Evaluation of enzyme and metal catalytic performances of the hybrid active pocket. The introduction of Pt and Pd single atoms into PCN-224 can theoretically enhance the photocatalytic performance. The photo-response capability of the dual-single-atom catalyst was evaluated by photoelectrochemical experiments. The UV/Vis diffuse reflectance spectrum of PCN(PtPd) demonstrated strong adsorption in the range of 200–800 nm (Supplementary Fig. 30). Based on the UV diffuse reflectance spectra, the reflectance data were converted into optical absorbance via the Kubelka-Munk function. The optical bandgaps were estimated by extrapolating the linear region of the plots ([F(R)hν] 2 against photon energy hν) to intersect with the hν axis (Supplementary Fig. 31). The optical bandgaps of PCN, PCN(Pt), PCN(Pd), and PCN(PtPd) were 2.90 eV, 2.85 eV, 2.84 eV, and 2.69 eV, respectively. The conduction and valence band positions were calculated by Mott-Schottky analysis (Supplementary Fig. 32). These results reveal that the anchoring of Pt and Pd dual-single-atoms can significantly decrease the optical bandgap (Fig. 4 a), potentially enhancing the capability for visible-light response. However, the optical bandgap of PCN(PtPd)-Lac (2.81 eV) was larger than that of PCN(PtPd), possibly due to the defects generated by enzyme incorporation. Photocurrent response measurements have been conducted to unveil the charge-separation efficiency. The photocurrent density of PCN(PtPd) was 2.5, 3.6, and 5.3 times higher than that of PCN(Pd), PCN(Pt), and PCN-224, respectively, showing that PCN(PtPd) has excellent performance for generating and suppressing the recombination of photogenerated electron-hole pairs (Fig. 4 b). The charge-transfer resistance property was further determined by the electrochemical impedance spectroscopy (EIS). As shown in Fig. 4 c, the arc radius of the Nyquist curves of the photocatalytic materials decreased after doping Pt or Pd single atoms. To investigate the cooperative effect of Pt and Pd atoms on the photoelectrochemical performance, the HOMO-LUMO gaps and partial density of states (pDOS) of PCN(Pt), PCN(Pd), and PCN(PtPd) were calculated via density functional theory (DFT). The molecular orbital diagrams are shown in Fig. 4 d and Supplementary Fig. 32. The HOMO-LOMO gaps of PCN(Pt), PCN(Pd), and PCN(PtPd) were 2.256, 2.106, and 1.993 eV, respectively (Fig. 4 e), showing that the energy required for electron transition in PCN(PtPd) is lower, which facilitates light absorption. As shown in Fig. 4 f, there are multiple orbital overlaps in the pDOS of Pd and Pt over PCN(PdPt). The orbitals overlap of Pb and Pt suggests that the orbital hybridization and electron transfer between Pb and Pt, further indicating that the synergy between the Pt and Pd atoms enhance the surface electronic structure. The electron transfer between Pd to Pt atoms in PCN(PtPd) was detected by X-ray photoelectron spectroscopy (XPS) (Supplementary Fig. 35). Compared with homogeneous metal atoms, there was subtle electron transfer from Pd to Pt through the metal-organic framework due to the electronegativity of Pt (2.28) is greater than that of Pd (2.20), which enhances the utilization efficiency of photoelectrons. The photocatalytic performance of PCN(PtPd) was evaluated in the water-donating transfer hydrogenation of aflatoxin B 1 (AFB 1 ) under visible-light irradiation (420-650nm LED) at 25 o C. AFB 1 is the most potent hepatocarcinogen known in mammals, which is classified as a group I carcinogen by the International Agency of Research on Cancer. The double C 8 = C 9 bond is the teratogenic and toxic site 22 . The C 8 = C 9 bond was converted to C 8 -C 9 bond in the transfer hydrogenation of AFB 1 , as analyzed by ultrahigh-performance liquid chromatography-quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF MS) (Supplementary Figs. 37–39). The conversion reached 100% within 5 hours using PCN(PtPd) as the catalyst (0.01 mg), with a substrate concentration of 6 mM. Calculating from the initial activity, the turnover frequency (TOF) for PCN(PtPd) (104.5 s − 1 ) was 2.9-fold and 4.4-fold that of PCN(Pd) (35.9 s − 1 ) and PCN(Pt) (23.9 s − 1 ), respectively (Fig. 4 g). To verify the direct hydrogenation mechanism with the in situ-generated protons from water splitting under visible-light irradiation, nuclear magnetic resonance (NMR) measurements were conducted. As shown in the 1 H NMR spectra of the AFB 1 transformation products (Supplementary Figs. 40–42), when D 2 O is used as the solvent, the signals from the protons of C 8 and C 9 shifted and the intensities reduced to half that of the product obtained when H 2 O is used as the solvent, unambiguously demonstrating that the hydrogen source for transfer hydrogenation originates from water. To gain insight into the superior performance of PCN(PtPd) and explore the detailed reaction path of AFB 1 hydrogenation, we further conducted DFT calculations. The AFB 1 hydrogenation capabilities of PCN(Pt), PCN(Pd), and PCN(PtPd) were investigated. As shown in Fig. 4 h, AFB 1 was adsorbed within the MOFs channels, with two terminal oxygen atoms coordinating to the dual metal atoms. Due to the synergistic interaction between Pt and Pd atoms, PCN(PtPd) displayed the strongest adsorption energy (-0.57 eV). The reaction pathway analysis identified that the first hydrogenation step (AFB 1 *+H→AFB 1 *-1H) is the rate-determining step. The reaction energy of the first hydrogenation step using PCN(Pt), PCN(Pd), and PCN(PtPd) as the catalysts were 0.70, 0.75, and 0.59 eV, respectively. The low reaction energy on the Pt-Pd dual-single-atoms configuration is considered to be the reason why PCN(PtPd) exhibited superior catalytic activity in AFB 1 hydrogenation. To further investigate the effect of dual-hetero-metal-single atoms on the activation of reactants, the charge density differences and corresponding Bader charge transfers in PCN(Pt), PCN(Pd), and PCN(PtPd) were calculated (Supplementary Fig. 44). The Pd and Pt atoms received Bader charge of -1.24 |e| and − 1.16 |e| from the framework, respectively, indicating strong charge interactions between the Pd/Pt atoms and the PCN-224 framework. Compared with PCN(Pt) and PCN(Pd), the asymmetric electronic configuration of PCN(PtPd) facilitated the charge redistribution within the catalyst, thereby significantly enhancing the activation capacity for reactants in photocatalytic reactions. These results demonstrated that the synergistic interaction between Pt and Pd atoms enables the catalyst to exhibit superior transfer hydrogenation activity under visible-light irradiation. The enzyme activity of PCN(PtPd)-Lac was evaluated in the oxidation reaction of TMB. The relative activity of laccase in PCN(PtPd)-Lac was 33% compared with native laccase at the same protein amount (Supplementary Fig. 45). The kinetics parameters of PCN(PtPd)-Lac were further investigated. As results, the K m of Lac and PCN(PtPd)-Lac were 2.27 and 0.789 mM, respectively, showing that PCN(PtPd)-Lac had a better substrate affinity than that of Lac (Supplementary Fig. 46). Oxidation-reduction cascades catalyzed in the active pocket of PCN(PtPd)-Lac. Since the enzyme-metal hybrid active pocket exhibited excellent activity in both the photocatalytic transfer hydrogenation reaction and enzyme-catalyzed oxidation reaction, it is possible to simultaneously perform oxidation and reduction reactions in a single active pocket. The products (O 2 or H 2 O) of these two reactions serve as substrates for each other. O 2 is generated in the transfer hydrogenation reaction catalyzed by Pt-Pd atoms, which is followed reduced to H 2 O catalyzed by laccase. H 2 O serves as the hydrogen source in the transfer hydrogenation reaction. Theoretically, the simultaneous action of metal and enzyme catalytic modules in single active pocket can mutually drive the reactions and eliminate intermediate diffusion, thus achieving efficient chemoenzymatic cascade reactions for low-concentration macromolecular substrates which is a grand challenge in chemistry. Mycotoxins contamination is an important issue for food safety and human health. However, the removal of low concentrations of mycotoxins in feed and food is a challenging task. Although strategies such as photocatalysis and microbial metabolism can effectively degrade toxins, the catalytic processes are uncontrollable, resulting in multiple transformation products which may increase toxicity. Mycotoxins can be directed transformed through enzymatic catalysis due to the high specificity and clear catalytic path of enzyme. However, the available detoxification enzymes are very limited and suffer from low catalytic activity. Due to the O 2 /H 2 O oxidation-reduction cascade, it is possible to directed transform mycotoxins efficiently by combining the visible-light-driven transfer hydrogenation reaction and enzyme-catalyzed oxidation reaction in a single active pocket (Fig. 5 a). Additionally, it is hypothesized that the electron transfer between metal and enzyme modules can accelerate the catalytic efficiency and broaden the substrate scope of laccase. The efficiency of PCN(PtPd)-Lac for the detoxification of AFB 1 was evaluated. The oxidation of AFB 1 by native laccase is challenging. As shown in Fig. 5 b, the conversion was only 1.2% within 1.5 h when using native laccase as the catalyst with a substrate concentration of 6 mM under visible-light irradiation. The conversion of AFB 1 catalyzed by PCN(PtPd)-Lac, PCN(PtPd) + Lac (physical mixing of PCN(PtPd) and Lac), PCN(PtPd), and Lac + ABTS(mediator) were 100%, 11%, 10%, and 2.3%, respectively. The AFB 1 transformation efficiency of PCN(PtPd)-Lac was 9 and 44 times higher than that of PCN(PtPd) + Lac and Lac + ABTS, respectively, indicating that the enzyme-metal hybrid active pocket greatly enhanced the degradation efficiency of AFB 1 . To investigate the mechanism for the high catalytic activity of the hybrid active pocket, the reaction solutions catalyzed by PCN(PtPd) + Lac and PCN(PtPd)-Lac were analyzed by HPLC. The chromatographic peak with retention time of 3.0 − 3.3 min that attributed to the intermediate of cascade reaction (AFB 1 -2H) was observed when using the combination of PCN(PtPd) and Lac as the catalysts. Whereas, no intermediate was detected when using PCN(PtPd)-Lac as the catalyst, indicating that no intermediate diffused into the bulk solution due to the in-situ transformation in the enzyme-metal hybrid active pocket (Fig. 5 c). The AFB 1 transformation product (AFB 1 -P) was purified via preparative HPLC (Supplementary Fig. 47), and the molecular structure was analyzed by UPLC-Q-TOF MS (Supplementary Figs. 48–49). Based on the structure of AFB 1 -P, it is observed that the C 8 = C 9 double bond of AFB 1 (the main site of toxicity and carcinogenicity, which forms adducts with proteins, DNA, and RNA, leading to physiological disorders) is converted into a C-C bond in the photocatalytic hydrogenation reaction, while the carbon-hydrogen bond of the cyclopenteneone ring (a toxicity site) of AFB 1 is oxidized in the laccase-catalyzed reaction (Fig. 5 a). The influence of light intensity on the catalytic efficiency of PCN(PtPd)-Lac was further investigated. As shown in Supplementary Fig. 50, PCN(PtPd)-Lac exhibited significant catalytic activity even under low light intensity (< 4 mW/cm²), potentially due to its excellent visible-light responsiveness. Subsequently, we tested the photo-enzyme catalytic efficiency of PCN(PtPd)-Lac under sunlight. Notably, PCN(PtPd)-Lac exhibited excellent catalytic activity even on a cloudy or raining day. After exposing to sunlight for 1.5 hours on a sunny, cloudy, and rainy day, the conversions of AFB 1 were 72.5%, 69.4%, and 66.7%, respectively (Fig. 5 d). To gain insight the superior performance of the Lac-PtPd hybrid active site, characterizations for the electron transfer between the dual single atoms and laccase were performed. The reduction peak at 0.5 V which corresponding to the electron transfer in redox reaction in the cyclic voltammetry (CV) curve of PCN(PtPd)-Lac is significantly higher than that of Lac and PCN(PtPd), indicating an increase in electron mobility and electrochemical activity (Supplementary Fig. 55). The major enhancement in the electron mobility of PCN(PtPd)-Lac is possibly due to the directional electron transfer from PCN(PtPd) to laccase, which facilitates charge separation and prevent the formation of the photogenerated electron-hole complex. Laccase is a multicopper oxidase, in which the type 1 cooper (T1 Cu) center has been shown to play a key role in electron transfer processes and substrate oxidation. As shown in Supplementary Fig. 2, laccase orientedly binds to PCN(PtPd) with the hydrophobic pocket near T1 Cu active site towards the photocatalyst. According to Marcus theory, the distance between the active site of oxidoreductase and the semiconductor should be less than 20 Å for direct electron transfer 23 . The distance between T1 Cu and the binding position is 10.4 Å, which is beneficial to realize effective direct electron transfer (Supplementary Fig. 25). The ESR spectra of T1 Cu (g = 2.0250) of laccase and PCN(PtPd)-Lac were used to assess the magnetic properties. As shown in Supplementary Fig. 56, the signal intensity for T1 Cu (as the active center) of PCN(PtPd)-Lac exhibited a significant increase compared with that of the free laccase, indicating the enhanced oxidation ability of laccase in PCN(PtPd)-Lac. The O 2 /H 2 O oxidation-reduction cascade in the enzyme-metal hybrid active pocket was further investigated (Fig. 5 e). To verify the in-situ generation of oxygen in the active pocket, the catalytic activities of Lac and PCN(PtPd)-Lac under oxygen-depleted environment (N 2 atmosphere) were determined. As shown in Fig. 5 f, Lac exhibited no activity under N 2 , demonstrating that the enzyme-catalyzed reaction was oxygen-dependent. PCN(PtPd)-Lac exhibited a significant activity under N 2 with a comparable turnover number (TON = 57053) to the activity in air. These results suggest that the in-situ generation of O 2 by the Pt-Pd atoms can be effectively utilized by laccase in the enzyme-metal hybrid active pocket, achieving efficient chemoenzymatic cascade catalysis. Artificial metalloenzymes has emerged as powerful tool to incorporate metal complexes into protein scaffolds for creating new active sites. Yet, artificial metalloenzymes can catalyze only one type of reactions 8 , 9 . To our knowledge, this work is the first to report two-step cascade reactions simultaneously perform in a single active pocket. The enhancement of the electron transfer from the T1 Cu center to the type 2/type 3 trinuclear Cu center in the enzyme, theoretically expands the substrate scope of laccase. To investigate whether PCN(PtPd)-Lac can oxidize a wide spectrum of substrates, several mycotoxins including AFB 1 ( 1a ), zearalenone (ZEN, 2a ), trichothecenes (T-2, 3a ), deoxynivalenol (DON, 4a ), and ochratoxin A (OTA, 5a ) that typically unreactive with laccase alone were used as the substrates. These mycotoxins frequently co-exist in contaminated grains, food, and feed. The possible binding sites of the mycotoxins with laccase and the corresponding binding energies were calculated by molecular docking (Supplementary Figs. 58 and 57). The binding energies for AFB 1 , DON, OTA, T-2, and ZEN were − 5.21, -4.53, -5.47, -3.16, and − 5.61 Kcal/mol, respectively, showing good affinities (Fig. 6 b). However, in the directed degradation reactions of mycotoxins, except for ZEN (0.7% conversion), the conversion of other mycotoxins catalyzed by laccase was 0 (Fig. 6 c). Even with the addition of mediators (ABTS), the conversions of DON, OTA, T-2, and ZEN catalyzed by laccase were only 0.4%, 0.3%, 0.4%, 1.2%, respectively. Notably, the catalytic activity of PCN(PtPd)-Lac for the degradation of DON, OTA, T-2, and ZEN were 31, 49, 33, 17 times higher than that of the combination of laccase and mediator (Lac + ABTS) under visible-light irradiation. The enhancement in the catalytic activity of PCN(PtPd)-Lac may due to the efficient cooperative catalysis of Pt-Pd atoms and enzyme catalytic module in a single active pocket. The non-directed degradation of mycotoxins by photocatalysis is attributed to the non-specific oxidation of oxygen radicals. When PCN(PtPd)-Lac was exposed to high intensity of light (Xe-lamp, 100 mW/cm 2 ), hydroxyl radical (·OH) and superoxide radical (·O 2− ) were detected in the ESR spectra (Supplementary Fig. 59). To investigate whether oxygen radicals are generated in the reaction under the irradiation of weak visible light (LED, 4 mW/cm 2 ), radical quenching experiments were further carried out using isopropyl alcohol (IPA) and tryptophan (TRP) as the ·OH and ·O 2− quenching agents, respectively. As shown in Fig. 6 d, 97% and 93% of the activity retained after the addition of IPA and TRP when the reaction conducted under the LED irradiation, showing that almost no free radicals are produced. Thus, the transformation of AFB 1 in the photocatalysis was possibly due to the transfer hydrogenation reaction catalyzed by the Pt-Pd dual single atoms. Therefore, mycotoxins can be directed transformed by photo-enzyme coupled catalysis. Additionally, the decline in enzyme activity caused by free radicals can be avoided. PCN(PtPd)-Lac can be easily recovered by centrifugation, and exhibited more than 96% residual activity for seven batches of reuse, showing an excellent stability (Fig. 6 e). The transformation products (DON-P, OTA-P, T-2-P, and ZEN-P) were purified via preparative HPLC (Supplementary Figs. 60–63), and the molecular structures were analyzed by UPLC-Q-TOF MS (Supplementary Figs. 64–71). It is found that the laccase in PCN(PtPd)-Lac displayed the capability to catalyze hydroxylation of mycotoxins (Fig. 6 a). Additionally, the C = C or C = O bonds of these mycotoxins were converted to C-C or C-OH bonds in the photocatalytic water-donating transfer hydrogenation reactions catalyzed by the Pt-Pd dual single atoms. The performance of PCN(PtPd)-Lac in simultaneously catalyzing the detoxification of multiple mycotoxins was further characterized. As shown in Fig. 6 f, the conversion of mycotoxins reached 100% within 180 minutes using 0.05 mg of PCN(PtPd)-Lac as the catalyst, with the initial concentration of each mycotoxin is 6 mM. The toxicity of the transformation products on liver and kidney cells were assessed using a crystal violet staining assay (Supplementary Figs. 72–73). The cytotoxicity of transformation products on normal Alpha mouse liver 12 (AML 12) hepatocyte is presented in Fig. 6 g, demonstrating that the hepatotoxicity of the transformation products was considerably reduced compared to the mycotoxins. Similarly, the transformation products exhibited significantly lower renal toxicity than the mycotoxins. After a 36-hour exposure to AFB 1 -P, AFQ 1 (transformation product catalyzed by Lac + ABTS), and AFB 1 with concentration of 1 mg/mL, the viabilities of AML 12 cells were determined as 85%, 55%, and 11%, respectively. The significant decrease in the hepatotoxicity and renal toxicity of AFB 1 -P compared to AFQ 1 , may due to the simultaneous conversion of two toxic sites of AFB 1 by efficiently coupling the photocatalysis and enzyme catalysis when using PCN(PtPd)-Lac as the catalyst. Conclusion We have presented a design of an enzyme-metal hybrid active pocket, created by co-coordinating Pt-Pd single-atom photocatalytic modules and laccase with a Zr cluster in MOFs. Due to the efficient utilization of intermediates and electron transfer within the artificial active pocket, PCN(PtPd)-Lac is highly active in the simultaneously catalyzing oxidation-reduction cascades for directed conversion of low-concentration mycotoxins to low-toxicity products, which is not reachable through engineered enzymes or photocatalysis alone. The construction of enzyme-metal hybrid active pocket provides an attractive way forward to achieving chemoenzymatic catalysis of new-to-nature reactions. Methods Preparation of PCN-224 PCN-224 was synthesized using the method reported previously with few modifications. Briefly, 150 mg of ZrOCl 2 ·8H 2 O, 1500 mg of benzoic acid, and 50 mg of tetrakis(4-carboxyphenyl) porphyrin (TCPP) were dissolved in DMF (50 mL) in a 100-mL round-bottom flask. The mixture was sonicated for 10 min, and then was heated at 90°C for 5 h. After cooling down to room temperature, PCN-224 was collected by centrifugation. The precipitated PCN-224 was then washed by ethanol for three times. The powder of PCN-224 was obtained by vacuum drying. Preparation of PCN(PtPd) The dual-single-atom catalyst PCN(PtPd) was fabricated by anchoring Pt and Pd atoms on PCN-224 using a hydrothermal method. PCN-224 (10 mg) was dispersed in DMF (4 mL). 200 µL of H 2 PtCl 6 aqueous solution (100 mg/mL) and 200 µL of Pd(OAc) 2 aqueous solution (20 mg/mL) were added simultaneously under magnetic stirring. The mixture was sonicated at 25 o C for 10 min, and then transferred to a 20 mL stainless-steel autoclave and heated at 80 o C for 4 h. Then, PCN(PtPd) was collected by centrifugation at 10000 rpm for 5 minutes and washed by water for three times. Preparation of PCN(PtPd)-Lac PCN(PtPd)-Lac hybrid catalyst was constructed by a dynamic exchange strategy. PCN(PtPd) (10 mg) was dispersed in phosphate buffer (1 mL, 50 mM, pH 7.0). Laccase (4 mg) was added under magnetic stirring at 25 o C. The reaction solution was stirred for 40 minutes. PCN(PtPd)-Lac was collected by centrifugation and washed by water for three times. The powder of PCN(PtPd)-Lac was obtained by lyophilization and stored at 4 o C in dark. Enzyme activity assay The enzyme activities of Lac and PCN(PtPd)-Lac were assayed by a standard method using ABTS as the substrate. The reaction was conducted by adding 10 µL of laccase solution or catalyst suspension to 990 µL of 0.5 mM ABTS solution (prepared in 10 mM acetate buffer, pH 5.0), and the absorbance change at 420 nm was monitored using a UV-Vis spectrophotometer. Detoxification of mycotoxins by PCN(PtPd)-Lac : First, mycotoxins (6 nmol) was dissolved in deionized water (1 mL). Then, 10 µL of the catalyst suspension (1 mg/mL) involving PCN(PtPd)-Lac was added with vigorously stirring. The mixture was subsequently placed in a PCX50C photoreactor equipped with a white LED light with a wavelength of 420–650 nm. After the reaction, the supernatant was collected by centrifugation. The conversions were analyzed by HPLC. HPLC conditions: The concentration of mycotoxins in the solution was determined by an HPLC system (Agilent 1260). HPLC was performed on an Agilent C18 column (250 mm × 4.6 mm; 5 µm) at a flow rate of 0.6 mL min − 1 . The mobile phase consisted of water and acetonitrile (45:55 v/v for AFB 1, 80:20 v/v for DON, 51:49 v/v for OTA, 70:30 v/v for ZEN, 25:75 v/v forT-2).The column temperature was 40°C. The injection volume was 20 µL. AFB1, DON, and ZEN were analysed using a UV detector at wavelengths of 365 nm, 218 nm, and 236 nm, respectively. In contrast, OTA and T-2 were detected using a fluorescence detector. The excitation and emission wavelengths for OTA were set at 333 nm and 460 nm, respectively, while those for T-2 were 381 nm and 470 nm. DFT calculations DFT calculations are performed using the Vienna Ab initio Software Package (VASP 5.4.4) within the Perdew-Burke-Ernzerhof (PBE) generalized gradient approximation and projected enhancement wave (PAW) method 24 – 26 . The cutoff energy of the plane-wave basis set is set to 400 eV. Monkhorst–Pack special k-point meshes of 3 × 3 × 1 were proposed to carry out geometry optimization and electronic structure calculation. In the process of geometric optimization, all atoms can relax without any restriction until the convergence thresholds of the maximum force and energy are less than 0.01 eV/Å and 1.0 × 10 − 5 eV/atom, respectively. A 15 Å vacuum layer was introduced to avoid interaction between periodic images. All DFT calculations were carried out using the CP2K code 27 . All calculations employed a mixed Gaussian and planewave basis sets. Core electrons were represented with norm-conserving Goedecker-Teter-Hutter pseudopotentials 28 – 30 , and the valence electron wavefunction was expanded in a double-zeta basis set with polarization functions 31 along with an auxiliary plane wave basis set with an energy cutoff of 400 Ry. The generalized gradient approximation exchange-correlation functional of Perdew, Burke, and Enzerhof (PBE) 32 was used. Each configuration was optimized with the Broyden-Fletcher-Goldfarb-Shanno (BGFS) algorithm with SCF convergence criteria of 1.0×10 − 5 au. To compensate the long-range van der Waals dispersion interaction, the DFT-D3 scheme 33 with an empirical damped potential term was added into the energies obtained from exchange-correlation functional in all calculations. Molecular Docking simulation Molecular docking simulations were performed to investigate the binding interactions between the toxins AFB 1 , OTA, ZEN, DON, and T-2 with laccase. The docking calculations were carried out using AutoDock with 50 docking runs. The structures of the ligands were drawn using ChemDraw and subsequently energy-minimized using Chem3D. The receptor protein, laccase, was obtained from the PDB database. The binding affinity of each ligand was evaluated, and potential hydrogen bonding interactions between the ligands and amino acids near the enzyme’s active pocket were analyzed based on the docking results. Declarations Competing interests The authors declare no competing interests. Author contributions X. L. and J. G. supervised the project. X. L. and J. G. conceived of the idea. Y. F. performed the experiments with technical help from J. H. H. W. performed the calculations. Y. F., J. H., Q. W. and M. Z. participated in analyzing the results. X. L., Y. F., and J. G. wrote the paper. Acknowledgements This research was supported by the National Key Research and Development Program of China (2022YFF1102800), the National Natural Science Foundation of China (Grant No. 22308143, 22168024, 22425803), the Jiangxi Provincial Natural Science Foundation (Grant No. 20232ACB215008), the Beijing Natural Science Foundation (Grant No. Z240030), the Shenzhen Science and Technology Program (Grant No. KCXFZ20240903093102004). The authors thank beamline BL14W1 (Shanghai Synchrotron Radiation Facility) for providing the beam time. Data availability The data that support the plots within this paper and other findings of this study are available from the corresponding author upon request. References Miller DC, Athavale SV, Arnold FH (2022) Combining chemistry and protein engineering for new-to-nature biocatalysis. Nat Synth 1:18–23 Raps FC, Rivas-Souchet A, Jones CM, Hyster TK (2025) Emergence of a distinct mechanism of C–N bond formation in photoenzymes. Nature 637:362–368 Ward TR, Copéret C (2023) Introduction: bridging the gaps: learning from catalysis across boundaries. Chem Rev 123:5221–5224 Huang X et al (2022) Photoinduced chemomimetic biocatalysis for enantioselective intermolecular radical conjugate addition. Nat Catal 5:586–593 Wheeldon I, Minteer S, Banta S, Atannassov P, Sigman M (2016) Substrate channelling as an approach to cascade reactions. Nat Chem 8:299–309 Sweetlove LJ, Fernie AR (2018) The role of dynamic enzyme assemblies and substrate channelling in metabolic regulation. Nat Commun 9:2136 Abdallah W, Hong X, Banta S, Wheeldon I (2022) Microenvironmental effects can masquerade as substrate channelling in cascade biocatalysis. Curr Opin Biotechnol 73:233–239 Alonso S et al (2020) Genetically engineered proteins with two active sites for enhanced biocatalysis and synergistic chemo- and biocatalysis. Nat Catal 3:319–328 Zhou Z, Roelfes G (2020) Synergistic catalysis in an artificial enzyme by simultaneous action of two abiological catalytic sites. Nat Catal 3:289–294 Vornholt T et al (2024) Artificial metalloenzymes. Nat Rev Methods Primers 4:78 Gröger H, Gallou F, Lipshutz BH (2022) Where chemocatalysis meets biocatalysis: in water. Chem Rev 123:5262–5296 Tian D et al (2023) Multi-compartmental MOF microreactors derived from Pickering double emulsions for chemo-enzymatic cascade catalysis. Nat Commun 14:3226 González-Granda S, Albarrán-Velo J, Lavandera I (2023) Gotor-Fernández, V. Expanding the synthetic toolbox through metal–enzyme cascade reactions. Chem Rev 123:5297–5346 Guðmundsson A, Manna S, Bäckvall JE (2021) Iron (II)-Catalyzed Aerobic Biomimetic Oxidation of Amines using a Hybrid Hydroquinone/Cobalt Catalyst as Electron Transfer Mediator. Angew Chem Int Ed 60:11819–11823 Görbe T et al (2017) Design of a Pd (0)-CalB CLEA biohybrid catalyst and its application in a one-pot cascade reaction. ACS Catal 7:1601–1605 Gopich IV, Szabo A (2013) Diffusion modifies the connectivity of kinetic schemes for multisite binding and catalysis. Proc. Natl. Acad. Sci. 110, 19784–19789 Magnoli AP, Poloni VL, Cavaglieri L (2019) Impact of mycotoxin contamination in the animal feed industry. Curr Opin Food Sci 29:99–108 Dey DK et al (2023) Mycotoxins in food and feed: Toxicity, preventive challenges, and advanced detection techniques for associated diseases. Crit Rev Food Sci Nutr 63:8489–8510 Santos Pereira C, Cunha C, S., Fernandes JO (2019) Prevalent mycotoxins in animal feed: Occurrence and analytical methods. Toxins 11:290 Feng Y et al (2023) A Dynamic Defect Generation Strategy for Efficient Enzyme Immobilization in Robust Metal-Organic Frameworks for Catalytic Hydrolysis and Chiral Resolution. Angew Chem Int Ed 62:e202302436 Sui J et al (2022) A general strategy to immobilize single-atom catalysts in metal–organic frameworks for enhanced photocatalysis. Adv Mater 34:2109203 Martínez J (2023) Computational studies of aflatoxin B1 (AFB1): A review. Toxins 15:135 Lou X et al (2024) Enhanced Interfacial Electron Transfer in Photocatalyst-Natural Enzyme Coupled Artificial Photosynthesis System: Tuning Strategies and Molecular Simulations. Small 20:2404055 Perdew JP, Burke K, Ernzerhof M (1996) Generalized gradient approximation made simple. Phys Rev Lett 77:3865 Hammer BHLB, Hansen LB, Nørskov JK (1999) Improved adsorption energetics within density-functional theory using revised Perdew-Burke-Ernzerhof functionals. Phys Rev B 59:7413 Blöchl PE (1994) Projector augmented-wave method. Phys Rev B 50:17953 VandeVondele J, Quickstep (2005) Fast and accurate density functional calculations using a mixed Gaussian and plane waves approach. Comput Phys Commun 167:103–128 Goedecker S, Teter M, Hutter J (1996) Separable dual-space Gaussian pseudopotentials. Phys Rev B 54:1703 Hartwigsen C, Gœdecker S, Hutter J (1998) Relativistic separable dual-space Gaussian pseudopotentials from H to Rn. Phys Rev B 58:3641 Krack M, Parrinello M (2000) All-electron ab-initio molecular dynamics. Phys Chem Chem Phys 2:2105–2112 VandeVondele J, Hutter J (2007) Gaussian basis sets for accurate calculations on molecular systems in gas and condensed phases. J Phys Chem 127 Perdew JP, Burke K, Ernzerhof M (1996) Generalized gradient approximation made simple. Phys Rev Lett 77:3865 Grimme S, Antony J, Ehrlich S, Krieg H (2010) A consistent and accurate ab initio parametrization of density functional dispersion correction (DFT-D) for the 94 elements H-Pu. J Phys Chem 132 Additional Declarations There is NO Competing Interest. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6659547","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":463630958,"identity":"06426026-7c2f-4807-a0cd-765312a6aafd","order_by":0,"name":"Jun Ge","email":"data:image/png;base64,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","orcid":"https://orcid.org/0000-0001-5503-8899","institution":"Tsinghua University","correspondingAuthor":true,"prefix":"","firstName":"Jun","middleName":"","lastName":"Ge","suffix":""},{"id":463630959,"identity":"85ad0c75-1d02-4321-9a4a-d5344b5c1e7f","order_by":1,"name":"Yunkai Fan","email":"","orcid":"","institution":"Nanchang University","correspondingAuthor":false,"prefix":"","firstName":"Yunkai","middleName":"","lastName":"Fan","suffix":""},{"id":463630960,"identity":"4ab6db93-6cdc-4a9a-b8b5-14f570e6ebb8","order_by":2,"name":"Jia Hu","email":"","orcid":"","institution":"Nanchang University","correspondingAuthor":false,"prefix":"","firstName":"Jia","middleName":"","lastName":"Hu","suffix":""},{"id":463630961,"identity":"2cd69688-1f7e-4e1a-bbd5-49cbab859a1e","order_by":3,"name":"Qilu Wu","email":"","orcid":"","institution":"Tsinghua University","correspondingAuthor":false,"prefix":"","firstName":"Qilu","middleName":"","lastName":"Wu","suffix":""},{"id":463630962,"identity":"2e714bcf-5444-4ffb-884c-81f32c1d44d1","order_by":4,"name":"Mengyu Zhu","email":"","orcid":"","institution":"Tsinghua University","correspondingAuthor":false,"prefix":"","firstName":"Mengyu","middleName":"","lastName":"Zhu","suffix":""},{"id":463630963,"identity":"8855eceb-e7df-472a-a984-a4e1dd542d68","order_by":5,"name":"Haozhi Wang","email":"","orcid":"","institution":"Hainan University","correspondingAuthor":false,"prefix":"","firstName":"Haozhi","middleName":"","lastName":"Wang","suffix":""},{"id":463630964,"identity":"c8709e8c-5308-4d96-9c59-ef4234bf7a4e","order_by":6,"name":"Xiaoyang Li","email":"","orcid":"https://orcid.org/0000-0002-4762-7105","institution":"Nanchang University","correspondingAuthor":false,"prefix":"","firstName":"Xiaoyang","middleName":"","lastName":"Li","suffix":""}],"badges":[],"createdAt":"2025-05-14 02:35:08","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6659547/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6659547/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":85179977,"identity":"57442837-9d6f-411c-ada4-c11ec84ba5df","added_by":"auto","created_at":"2025-06-23 07:09:29","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":583372,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eChemoenzymatic cascade reactions on enzyme-metal hybrid catalysts. a, \u003c/strong\u003eSeparate enzyme and metal active sites for multiple-step chemoenzymatic cascade reactions.\u003cstrong\u003e b,\u003c/strong\u003e The enzyme-metal hybrid active pocket for simultaneously catalyzing two step chemoenzymatic cascade reactions. The diffusion of intermediates is eliminated in the “one binding two reactions” catalysis. \u003cstrong\u003ec,\u003c/strong\u003eOverview of the structure of enzyme-metal hybrid active pocket and the proposed mechanism for cascade reactions.\u003cstrong\u003e \u003c/strong\u003eThe intermediate is generated on the metal catalytic module and bound in situ by the enzyme module of the active pocket. Green and pink: metal atoms, orange: enzyme catalytic module, yellow: substrate, blue: intermediate. Asp is the key amino acid residues responsible for binding with substrate of enzyme.\u003c/p\u003e","description":"","filename":"image1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6659547/v1/6e2c8aa955d262e5b52bcb41.jpg"},{"id":85180698,"identity":"9e34553c-72f1-48a7-976f-c188ac896b8b","added_by":"auto","created_at":"2025-06-23 07:17:29","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1619791,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFabrication and characterization of enzyme-metal hybrid active pocket. a\u003c/strong\u003e, Schematic of the preparation process of PCN(PtPd)-Lac. The PtPd-Lac hybrid active site is constructed by co-coordinating dual-metal hetero-single atoms and enzyme with a Zr cluster in PCN-224. \u003cstrong\u003eb\u003c/strong\u003e, HAADF-STEM image of PCN(PtPd)-Lac. \u003cstrong\u003ec\u003c/strong\u003e, Magnified AC-STEM image of PCN(PtPd)-Lac in which the bright dots represent Pt and Pd single atoms. \u003cstrong\u003ed\u003c/strong\u003e, AC-STEM image of PCN(PtPd)-Lac. \u003cstrong\u003ee\u003c/strong\u003e, Cryo-ET reconstruction and \u003cstrong\u003ef\u003c/strong\u003e, its zoomed image of a single PCN(PtPd)-Lac nanocomposite. \u003cstrong\u003eg,\u003c/strong\u003e Linear scan of electron density along the dashed line. Four characteristic pore sizes are shown on the plot, tiny peaks possibly representing the encapsulated enzymes are marked by arrowheads.\u003cstrong\u003e h, \u003c/strong\u003eFast Fourier transformation of the electron density linear scan is converted to pore size distribution of the PCN(PtPd)-Lac in the left panel. Normalized PDI: normalized pore distribution intensity.\u003c/p\u003e","description":"","filename":"image2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6659547/v1/b75cff7b1a453386b32ef85c.jpg"},{"id":85179979,"identity":"62a4e41c-d374-43ed-a9e0-a87da83291b7","added_by":"auto","created_at":"2025-06-23 07:09:29","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":619970,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eStructural characterizations of the Pt and Pd single atoms by XAFS. \u003c/strong\u003eNormalized XANES spectra: \u003cstrong\u003ea\u003c/strong\u003e, Pt L-edge. \u003cstrong\u003eb\u003c/strong\u003e, Pd K-edge. R-space EXAFS spectra: \u003cstrong\u003ec\u003c/strong\u003e, Pt samples. \u003cstrong\u003ed\u003c/strong\u003e, Pd samples. EXAFS fitting: \u003cstrong\u003ee\u003c/strong\u003e, Pt, \u003cstrong\u003ef\u003c/strong\u003e, Pd of PCN(PtPd). 3D contour maps of WT-XAFS spectra: \u003cstrong\u003eg,\u003c/strong\u003ePt of Pt foil and PCN(PtPd). \u003cstrong\u003eh, \u003c/strong\u003ePd of Pd foil and PCN(PtPd).\u003c/p\u003e","description":"","filename":"image3.png","url":"https://assets-eu.researchsquare.com/files/rs-6659547/v1/5d4d6880f994e80bdb211941.png"},{"id":85179981,"identity":"3c91d36d-f7a1-48b7-89b0-8329eb0296c6","added_by":"auto","created_at":"2025-06-23 07:09:29","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":6761302,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePhotocatalytic performance of PCN(PtPd)-Lac. a\u003c/strong\u003e, Schematic illustration of the band positions of PCN, PCN(Pt), PCN(Pd), PCN(PtPd), and PCN(PtPd)-Lac. \u003cstrong\u003eb\u003c/strong\u003e, Photocurrent response measurements. \u003cstrong\u003ec\u003c/strong\u003e, Electrochemical impedance spectroscopy (EIS) Nyquist plots. \u003cstrong\u003ed\u003c/strong\u003e, The molecular orbital diagrams of PCN(PdPt). Green: Pt, Pink: Pd. \u003cstrong\u003ee\u003c/strong\u003e, DFT calculation of the LUMO and HOMO of PCN(Pt), PCN(Pd), and PCN(PdPt). \u003cstrong\u003ef\u003c/strong\u003e, Partial density of state (pDOS) of Pd over PCN(Pd), pDOS of Pt over PCN(Pt), and pDOS of Pd and Pt over PCN(PdPt). The multiple orbital overlaps of the pDOS of Pd and Pt are shown in the red frame. \u003cstrong\u003eg\u003c/strong\u003e, Catalytic performance of PCN(Pt), PCN(Pd), and PCN(PdPt) in the photocatalytic transfer hydrogenation reaction of AFB\u003csub\u003e1\u003c/sub\u003e using H\u003csub\u003e2\u003c/sub\u003eO as proton source under visible-light irradiation. \u003cstrong\u003eh\u003c/strong\u003e, Free energy diagrams of AFB\u003csub\u003e1\u003c/sub\u003e hydrogenation over PCN(Pt), PCN(Pd), and PCN(PdPt).\u003c/p\u003e","description":"","filename":"image4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6659547/v1/e822f3907921ad3aadc32e01.jpg"},{"id":85179985,"identity":"51ce5d41-b003-419e-95c1-cc5d83bc5436","added_by":"auto","created_at":"2025-06-23 07:09:29","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":7636780,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDirected degradation of mycotoxins by PCN(PtPd)-Lac.\u003c/strong\u003e \u003cstrong\u003ea, \u003c/strong\u003eReaction scheme for coupling photocatalytic transfer hydrogenation and enzyme-catalyzed hydroxylation for degradation of AFB\u003csub\u003e1\u003c/sub\u003e. \u003cstrong\u003eb, \u003c/strong\u003eThe conversion of AFB\u003csub\u003e1 \u003c/sub\u003ein the degradation reaction. Conditions: 6 nmol AFB\u003csub\u003e1\u003c/sub\u003e, 0.01 mg PCN(PtPd)-Lac (1.3 wt% Pt, 0.5 wt% Pd, and 24.5 wt% laccase), 1 mL H\u003csub\u003e2\u003c/sub\u003eO, 25 \u003csup\u003eo\u003c/sup\u003eC under visible-light irradiation for 1.5 hours. All catalysts have the same amounts of enzyme or/and metal as PCN(PtPd)-Lac. \u003cstrong\u003ec,\u003c/strong\u003e HPLC chromatograms of reaction solutions catalyzed by PCN(PtPd)+Lac and PCN(PtPd)-Lac. \u003cstrong\u003ed, \u003c/strong\u003eComparison of AFB\u003csub\u003e1\u003c/sub\u003e conversion catalyzed by Lac, PCN(PtPd), and PCN(PtPd)-Lac under sunlight radiation on different weather days. The sunlight irradiance of raining, cloudy, and sunny are 1.8, 3.4, and 7.8 mW/cm\u003csup\u003e2\u003c/sup\u003e, respectively. \u003cstrong\u003ee,\u003c/strong\u003e Scheme for the simultaneous action of reduction reaction and oxidation reaction in the laccase-PtPd hybrid active site. \u003cstrong\u003ef,\u003c/strong\u003e Turnover number (TON) of Lac and PCN(PtPd)-Lac in the directed degradation of AFB\u003csub\u003e1\u003c/sub\u003e, ZEN, T-2, DON, and OTA at air or N\u003csub\u003e2\u003c/sub\u003e atmosphere under visible-light irradiation.\u003c/p\u003e","description":"","filename":"image5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6659547/v1/f4c6a7b3e15537f638825f63.jpg"},{"id":85179983,"identity":"153bbfdd-4a88-44ba-8475-d6993c4f76e7","added_by":"auto","created_at":"2025-06-23 07:09:29","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":5910145,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCatalytic performance of PCN(PtPd)-Lac in the directed detoxification of mycotoxins. a, \u003c/strong\u003edirected degradation of AFB\u003csub\u003e1\u003c/sub\u003e, ZEN, T-2, DON, and OTA by PCN(PtPd)-Lac under visible light irradiation.\u003cstrong\u003e b, \u003c/strong\u003eConversions of mycotoxins catalyzed by Lac, Lac+ABTS, and PCN(PtPd)-Lac with the same amount of enzyme. Conditions: 6 nmol mycotoxins, PCN(PtPd)-Lac (0.01 mg), 1 mL H\u003csub\u003e2\u003c/sub\u003eO, 25 \u003csup\u003eo\u003c/sup\u003eC under visible-light irradiation for 30 minutes. \u003cstrong\u003ec, \u003c/strong\u003eThe binding energies for AFB\u003csub\u003e1\u003c/sub\u003e, ZEN, T-2, DON, and OTA with laccase. \u003cstrong\u003ed,\u003c/strong\u003e Effects of radical scavengers on the activity of PCN(PtPd)-Lac in the degradation of AFB\u003csub\u003e1\u003c/sub\u003e under irradiation of LEDs (4 mW/cm\u003csup\u003e2\u003c/sup\u003e) and Xe-lamp (100 mW/cm\u003csup\u003e2\u003c/sup\u003e). IPA and TRP are the quenching agents for ·OH and ·O\u003csup\u003e2−\u003c/sup\u003e, respectively.\u003cstrong\u003e e,\u003c/strong\u003e Relative enzyme activity of PCN(PtPd)-Lac in seven cycles of the reaction. \u003cstrong\u003ef,\u003c/strong\u003e Catalytic performance of PCN(PtPd)-Lac (0.05 mg) in the degradation of the mixture of AFB\u003csub\u003e1\u003c/sub\u003e, ZEN, T-2, DON, and OTA. The initial concentration of each mycotoxin is 2 μg/mL. \u003cstrong\u003eg,\u003c/strong\u003e Cell viability assays of AML 12 liver cells treated with mycotoxins and the corresponding transformation products at a concentration of 1 mg/mL.\u003c/p\u003e","description":"","filename":"image6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6659547/v1/d31cd2dbb7097e33dcc82b0d.jpg"},{"id":85181797,"identity":"caf6429c-b90f-496e-a56e-79874be407b6","added_by":"auto","created_at":"2025-06-23 07:25:37","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":23906455,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6659547/v1/bf330b63-c263-4855-aaff-8239f9edfb4a.pdf"},{"id":85179982,"identity":"4ac52161-9bf7-4984-8ba9-352e58a060a3","added_by":"auto","created_at":"2025-06-23 07:09:29","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":5830662,"visible":true,"origin":"","legend":"Supplementary information","description":"","filename":"SupplementaryInformation7.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6659547/v1/004c1819266564dbb6618e7a.pdf"}],"financialInterests":"There is \u003cb\u003eNO\u003c/b\u003e Competing Interest.","formattedTitle":"Assembling a single active pocket by enzyme and metal modules for simultaneously catalyzing oxidation-reduction cascades","fulltext":[{"header":"Introduction","content":"\u003cp\u003eThe increasing demands for expanding the spectrum of biocatalysis has accelerated the development of cascade reactions merging enzymatic catalysis with metal/photoredox catalysis. The chemoenzymatic cascades that cannot be achieved either by enzyme or by chemical catalysis alone are receiving significant attentions, unveiling the frontier of new-to-nature biocatalysis\u003csup\u003e\u003cspan additionalcitationids=\"CR2 CR3\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eLearn from multi-enzyme cascade catalysis in nature, where the substrate channeling plays an important role in the enhancement of efficiency\u003csup\u003e\u003cspan additionalcitationids=\"CR6\" citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e, the metal catalytic module and enzyme has been integrated in a single catalyst to construct the enzyme-metal hybrid catalyst\u003csup\u003e\u003cspan additionalcitationids=\"CR9\" citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e. As the distance between enzyme and metal catalytic module decreases, the efficiency of chemoenzymatic cascade reaction can be greatly enhanced due to the proximity effect which decreases the diffusion hinderance of reaction intermediates of cascades\u003csup\u003e\u003cspan additionalcitationids=\"CR12 CR13 CR14\" citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eInspired by the above achievements, we hypothesized that if multiple catalytic modules were assembled properly with a precise spatial structure to form a single active pocket, the reactant could undergo the whole cascade reactions with very low probability that the reaction intermediate diffused out the active pocket. This new type of \u0026ldquo;one binding two reactions\u0026rdquo; catalysis could theoretically eliminate the diffusion of intermediates for cascade reactions, and greatly improve the catalytic efficiency of cascade reactions especially that with low substrate concentrations which have the serious obstacle in substrate binding and diffusion\u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e. For example, this type of catalysis will highly promote the efficiency of catalytic detoxification of toxins in food and feed industry, which is a big challenge for human health and food safety\u003csup\u003e\u003cspan additionalcitationids=\"CR18\" citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e. However, the assembly of metal catalytic modules with enzyme to form an active site which can simultaneously catalyze multi-step cascade reactions has not been achieved yet.\u003c/p\u003e \u003cp\u003eIn this work, we constructed an enzyme-metal hybrid active pocket with high stability by co-coordinating dual metal single-atom photocatalytic modules and enzyme with a zirconium (Zr) cluster in metal-organic frameworks (MOFs) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). The tunable structure and assembly process of MOFs allows the self-organization of the spatial position of enzymatic and metallic modules in the hybrid active pocket. The enzyme-metal hybrid active pocket constructed by dual metal single-atom photocatalytic modules and an oxidoreductase, exhibits excellent activity in simultaneously catalyzing oxidation-reduction cascades of low-concentration macromolecular substrates including some toxins difficult to remove. In vitro experiments in liver and kidney cells demonstrated that the chemoenzymatic cascade reactions catalysed by the enzyme-metal hybrid active pocket significantly detoxified mycotoxins. The mechanism of the high efficiency was interpreted as the efficient utilization of intermediates and electron transfer within the artificial active pocket. The \u0026ldquo;one binding two reactions\u0026rdquo; catalysis provide tremendous possibilities for new chemoenzymatic cascade reactions applicable to synthesis and environmental remediation, particularly for the low-concentration substrates.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"Results and Discussion","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eCreation of enzyme-metal hybrid active pocket\u003c/h2\u003e \u003cp\u003eThe enzyme-metal hybrid active pocket was created by co-coordinating metal atoms and enzyme catalytic modules with the assistance of MOFs containing metal clusters. The amino acid residue on the enzyme surface, can theoretically coordinate with metal clusters\u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e. The binding sites of laccase with three common metal clusters of MOFs (Zr\u003csub\u003e6\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e(OH)\u003csub\u003e4\u003c/sub\u003e cluster from PCN-224, Cu₂(CO₂)₄ cluster from HKUST-1, and Fe₃O(CO₂)₆ cluster from MIL-100(Fe)) were investigated by molecular docking (Supplementary Figs.\u0026nbsp;1\u0026ndash;6). For Zr and Fe, the metal clusters most likely to coordinate with the active center-adjacent amino acids of laccase, with the binding energies of -5.36 and \u0026minus;\u0026thinsp;5.14 Kcal/mol, respectively. These results indicated that the enzyme active site can oriented binding to the metal clusters from MOFs with the hydrophobic pocket near T1 Cu active site towards the frameworks.\u003c/p\u003e \u003cp\u003eDue to metal atoms can be theoretically anchored on the organic ligand of PCN-224 (tetrakis(4-carboxyphenyl)porphyrin, TCPP) to form the photocatalytic modules\u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e, PCN-224 is selected as the template for constructing the enzyme-metal hybrid active pocket (Supplementary Figs.\u0026nbsp;7 and 8). Pt and Pd atoms were anchored onto the porphyrin linker of Zr cluster by a solvothermal method using H\u003csub\u003e2\u003c/sub\u003ePtCl\u003csub\u003e6\u003c/sub\u003e and Pd(OAc)\u003csub\u003e2\u003c/sub\u003e as the precursors. Subsequently, enzymes were used as \u0026ldquo;macro ligands\u0026rdquo; for oriented binding to Zr clusters through the active center-adjacent amino acids, generating the enzyme-metal hybrid active pocket (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea).\u003c/p\u003e \u003cp\u003eHigh-angle annular dark-field scanning transmission electron microscopy (HAADF-STEM) and SEM images showed that PCN(PtPd) were spherical particles with an average size of 160 nm (Supplementary Figs.\u0026nbsp;9 and 10). No obvious change in the morphology of the nanocomposite was observed after the incorporating of enzymes (Supplementary Figs.\u0026nbsp;11 and 12). No aggregation of metals was detected (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb). The mass loadings of Pt and Pd were 3.1% and 2.7%, respectively, as measured by inductively coupled plasma\u0026ndash;atomic emission spectrometry (Supplementary Table\u0026nbsp;1). Aberration-corrected scanning transmission electron microscopy (AC-STEM) was performed to gain direct insight into the atomic structure of PCN(PtPd)-Lac. The obvious individual bright dots observed in the images indicated the atomic dispersion of single metal atoms over PCN(PtPd)-Lac (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ec and \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ed). Elemental mapping further revealed the uniform distribution of Pt and Pd atoms (Supplementary Fig.\u0026nbsp;13). The element analysis of PCN(PtPd)-Lac under HAADF-STEM (Supplementary Fig.\u0026nbsp;14) demonstrated the presence S from enzymes, indicating that Lac was immobilized on PCN(PtPd). The S element was well-distributed in both the inside and outside of the nanocomposite, showing that the enzyme is evenly incorporated on PCN(PtPd). The successful incorporation of the enzyme was further confirmed by cryo-electron tomography (Cryo-ET) (Supplementary Fig.\u0026nbsp;16), Fourier transform infrared spectroscopy (Supplementary Fig.\u0026nbsp;17) and confocal laser scanning microscopy (CLSM) (Supplementary Fig.\u0026nbsp;18). The loading of Lac was 24.5% as determined by the thermal gravity analysis (TGA) under nitrogen (Supplementary Fig.\u0026nbsp;19). In addition, the loading of Zr, Pt, and Pd of PCN(PtPd)-Lac decreased to 8%, 1.3% and 0.5% compared to PCN(PtPd), indicating that the coordination between enzymes and Zr clusters leads to the release of a portion of metal nodes and organic ligands (Supplementary Table\u0026nbsp;1).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe mesopores generated from the coordination of enzyme and Zr clusters were characterized by Cryo-ET. Compared with PCN(PtPd)-Lac (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ee), the electron density scan curve of PCN(PtPd) (Supplementary Fig.\u0026nbsp;22) showed a more frequent electron density variation, demonstrating that the pore sizes of PCN(PtPd) are generally smaller. The pore size distribution of PCN(PtPd) and PCN(PtPd)-Lac were analyzed by the fast Fourier transformation of the electron density. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eh, mesopores from 7\u0026ndash;10 nm were generated in PCN(PtPd)-Lac, whereas 5\u0026ndash;7 nm of pores were detected in the PCN(PtPd) (Supplementary Fig.\u0026nbsp;23). The increase in the pore size of PCN(PtPd)-Lac was further confirmed by Brunauer-Emmett-Teller (BET). The proportion of 2\u0026ndash;10 nm mesoporous pores increased significantly in the PCN(PtPd)-Lac sample, which could be beneficial for the molecular transfer in the enzymatic reactions (Supplementary Fig.\u0026nbsp;24). The distance between the metal single atoms and the original active site of laccase is approximately 15 \u0026Aring; (Supplementary Fig.\u0026nbsp;25).\u003c/p\u003e \u003cp\u003eThe coordination environment and chemical state of Pt and Pd atoms of PCN(PtPd)-Lac were investigated by X-ray absorption fine structures (XAFS). The Pt L-edge and Pd K-edge X-ray absorption near-edge structures (XANES) revealed the oxidation state of Pt and Pd in PCN(PtPd)-Lac. In comparison with Pt foil and Pd foil, the white line peaks of PCN(PtPd)-Lac were significant higher and showed significant shifts to higher energy, suggesting that the Pt and Pd atoms were in oxidized state (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea and \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb). The sole presence of Pt and Pd atoms was confirmed by extended X-ray absorption fine structure spectroscopy (EXAFS). As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ec and Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ed, the R-space EXAFS spectra of PCN(PtPd)-Lac showed major peaks at 1.8 \u0026Aring; and 1.5 \u0026Aring; which were close to the Pt-O and Pd-O peaks, and were attributed to the Pt-N/Cl and Pd-N/O backscattering, respectively. Quantitative EXAFS curve-fitting analysis was then performed to investigate the coordination configuration. For the Pt L-edge, the fitting of PCN(PtPd)-Lac showed two coordination shells of Pt-N and Pt-Cl with coordination numbers of 2.9 and 1.4, respectively, suggesting a porphyrin-based PtN\u003csub\u003e3\u003c/sub\u003e structure with an axial chlorine ligand (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ee and Supplementary Table\u0026nbsp;2). For the Pd K-edge, the fitting of PCN(PtPd)-Lac revealed two coordination shells of Pd-N and Pd-O with coordination numbers of 4.0 and 1.9, respectively, indicating a porphyrin-based PdN\u003csub\u003e4\u003c/sub\u003e structure with two axial oxygen ligands (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ef and Supplementary Table\u0026nbsp;3). From the wavelet transforms (WT) of the Pt L-edge and Pd K-edge EXAFS signals of PCN(PtPd)-Lac, the intensity maximum at 1.6 \u0026Aring;\u003csup\u003e\u0026minus;1\u003c/sup\u003e and 5.4 \u0026Aring;\u003csup\u003e\u0026minus;1\u003c/sup\u003e are ascribed to Pt-N/Cl and Pd-N/O coordination, respectively, whereas no intensity maximum (11.4 \u0026Aring;\u003csup\u003e\u0026minus;1\u003c/sup\u003e and 9.2 \u0026Aring;\u003csup\u003e\u0026minus;1\u003c/sup\u003e) corresponds to the Pt-Pt and Pd-Pd coordination were detected compared with Pt foil and Pd foil (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eg and \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eh).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eEvaluation of enzyme and metal catalytic performances of the hybrid active pocket.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eThe introduction of Pt and Pd single atoms into PCN-224 can theoretically enhance the photocatalytic performance. The photo-response capability of the dual-single-atom catalyst was evaluated by photoelectrochemical experiments. The UV/Vis diffuse reflectance spectrum of PCN(PtPd) demonstrated strong adsorption in the range of 200\u0026ndash;800 nm (Supplementary Fig.\u0026nbsp;30). Based on the UV diffuse reflectance spectra, the reflectance data were converted into optical absorbance via the Kubelka-Munk function. The optical bandgaps were estimated by extrapolating the linear region of the plots ([F(R)hν]\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e against photon energy hν) to intersect with the hν axis (Supplementary Fig.\u0026nbsp;31). The optical bandgaps of PCN, PCN(Pt), PCN(Pd), and PCN(PtPd) were 2.90 eV, 2.85 eV, 2.84 eV, and 2.69 eV, respectively. The conduction and valence band positions were calculated by Mott-Schottky analysis (Supplementary Fig.\u0026nbsp;32). These results reveal that the anchoring of Pt and Pd dual-single-atoms can significantly decrease the optical bandgap (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea), potentially enhancing the capability for visible-light response. However, the optical bandgap of PCN(PtPd)-Lac (2.81 eV) was larger than that of PCN(PtPd), possibly due to the defects generated by enzyme incorporation. Photocurrent response measurements have been conducted to unveil the charge-separation efficiency. The photocurrent density of PCN(PtPd) was 2.5, 3.6, and 5.3 times higher than that of PCN(Pd), PCN(Pt), and PCN-224, respectively, showing that PCN(PtPd) has excellent performance for generating and suppressing the recombination of photogenerated electron-hole pairs (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eb). The charge-transfer resistance property was further determined by the electrochemical impedance spectroscopy (EIS). As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ec, the arc radius of the Nyquist curves of the photocatalytic materials decreased after doping Pt or Pd single atoms.\u003c/p\u003e \u003cp\u003eTo investigate the cooperative effect of Pt and Pd atoms on the photoelectrochemical performance, the HOMO-LUMO gaps and partial density of states (pDOS) of PCN(Pt), PCN(Pd), and PCN(PtPd) were calculated via density functional theory (DFT). The molecular orbital diagrams are shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ed and Supplementary Fig.\u0026nbsp;32. The HOMO-LOMO gaps of PCN(Pt), PCN(Pd), and PCN(PtPd) were 2.256, 2.106, and 1.993 eV, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ee), showing that the energy required for electron transition in PCN(PtPd) is lower, which facilitates light absorption. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ef, there are multiple orbital overlaps in the pDOS of Pd and Pt over PCN(PdPt). The orbitals overlap of Pb and Pt suggests that the orbital hybridization and electron transfer between Pb and Pt, further indicating that the synergy between the Pt and Pd atoms enhance the surface electronic structure. The electron transfer between Pd to Pt atoms in PCN(PtPd) was detected by X-ray photoelectron spectroscopy (XPS) (Supplementary Fig.\u0026nbsp;35). Compared with homogeneous metal atoms, there was subtle electron transfer from Pd to Pt through the metal-organic framework due to the electronegativity of Pt (2.28) is greater than that of Pd (2.20), which enhances the utilization efficiency of photoelectrons.\u003c/p\u003e \u003cp\u003eThe photocatalytic performance of PCN(PtPd) was evaluated in the water-donating transfer hydrogenation of aflatoxin B\u003csub\u003e1\u003c/sub\u003e(AFB\u003csub\u003e1\u003c/sub\u003e) under visible-light irradiation (420-650nm LED) at 25 \u003csup\u003eo\u003c/sup\u003eC. AFB\u003csub\u003e1\u003c/sub\u003e is the most potent hepatocarcinogen known in mammals, which is classified as a group I carcinogen by the International Agency of Research on Cancer. The double C\u003csub\u003e8\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;C\u003csub\u003e9\u003c/sub\u003e bond is the teratogenic and toxic site\u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e. The C\u003csub\u003e8\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;C\u003csub\u003e9\u003c/sub\u003e bond was converted to C\u003csub\u003e8\u003c/sub\u003e-C\u003csub\u003e9\u003c/sub\u003e bond in the transfer hydrogenation of AFB\u003csub\u003e1\u003c/sub\u003e, as analyzed by ultrahigh-performance liquid chromatography-quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF MS) (Supplementary Figs.\u0026nbsp;37\u0026ndash;39). The conversion reached 100% within 5 hours using PCN(PtPd) as the catalyst (0.01 mg), with a substrate concentration of 6 mM. Calculating from the initial activity, the turnover frequency (TOF) for PCN(PtPd) (104.5 s\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) was 2.9-fold and 4.4-fold that of PCN(Pd) (35.9 s\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) and PCN(Pt) (23.9 s\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e), respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eg). To verify the direct hydrogenation mechanism with the in situ-generated protons from water splitting under visible-light irradiation, nuclear magnetic resonance (NMR) measurements were conducted. As shown in the \u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003eH NMR spectra of the AFB\u003csub\u003e1\u003c/sub\u003e transformation products (Supplementary Figs.\u0026nbsp;40\u0026ndash;42), when D\u003csub\u003e2\u003c/sub\u003eO is used as the solvent, the signals from the protons of C\u003csub\u003e8\u003c/sub\u003e and C\u003csub\u003e9\u003c/sub\u003e shifted and the intensities reduced to half that of the product obtained when H\u003csub\u003e2\u003c/sub\u003eO is used as the solvent, unambiguously demonstrating that the hydrogen source for transfer hydrogenation originates from water.\u003c/p\u003e \u003cp\u003eTo gain insight into the superior performance of PCN(PtPd) and explore the detailed reaction path of AFB\u003csub\u003e1\u003c/sub\u003e hydrogenation, we further conducted DFT calculations. The AFB\u003csub\u003e1\u003c/sub\u003e hydrogenation capabilities of PCN(Pt), PCN(Pd), and PCN(PtPd) were investigated. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eh, AFB\u003csub\u003e1\u003c/sub\u003e was adsorbed within the MOFs channels, with two terminal oxygen atoms coordinating to the dual metal atoms. Due to the synergistic interaction between Pt and Pd atoms, PCN(PtPd) displayed the strongest adsorption energy (-0.57 eV). The reaction pathway analysis identified that the first hydrogenation step (AFB\u003csub\u003e1\u003c/sub\u003e*+H\u0026rarr;AFB\u003csub\u003e1\u003c/sub\u003e*-1H) is the rate-determining step. The reaction energy of the first hydrogenation step using PCN(Pt), PCN(Pd), and PCN(PtPd) as the catalysts were 0.70, 0.75, and 0.59 eV, respectively. The low reaction energy on the Pt-Pd dual-single-atoms configuration is considered to be the reason why PCN(PtPd) exhibited superior catalytic activity in AFB\u003csub\u003e1\u003c/sub\u003e hydrogenation. To further investigate the effect of dual-hetero-metal-single atoms on the activation of reactants, the charge density differences and corresponding Bader charge transfers in PCN(Pt), PCN(Pd), and PCN(PtPd) were calculated (Supplementary Fig.\u0026nbsp;44). The Pd and Pt atoms received Bader charge of -1.24 |e| and \u0026minus;\u0026thinsp;1.16 |e| from the framework, respectively, indicating strong charge interactions between the Pd/Pt atoms and the PCN-224 framework. Compared with PCN(Pt) and PCN(Pd), the asymmetric electronic configuration of PCN(PtPd) facilitated the charge redistribution within the catalyst, thereby significantly enhancing the activation capacity for reactants in photocatalytic reactions. These results demonstrated that the synergistic interaction between Pt and Pd atoms enables the catalyst to exhibit superior transfer hydrogenation activity under visible-light irradiation.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe enzyme activity of PCN(PtPd)-Lac was evaluated in the oxidation reaction of TMB. The relative activity of laccase in PCN(PtPd)-Lac was 33% compared with native laccase at the same protein amount (Supplementary Fig.\u0026nbsp;45). The kinetics parameters of PCN(PtPd)-Lac were further investigated. As results, the K\u003csub\u003em\u003c/sub\u003e of Lac and PCN(PtPd)-Lac were 2.27 and 0.789 mM, respectively, showing that PCN(PtPd)-Lac had a better substrate affinity than that of Lac (Supplementary Fig.\u0026nbsp;46).\u003c/p\u003e \u003cp\u003e \u003cb\u003eOxidation-reduction cascades catalyzed in the active pocket of PCN(PtPd)-Lac.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eSince the enzyme-metal hybrid active pocket exhibited excellent activity in both the photocatalytic transfer hydrogenation reaction and enzyme-catalyzed oxidation reaction, it is possible to simultaneously perform oxidation and reduction reactions in a single active pocket. The products (O\u003csub\u003e2\u003c/sub\u003e or H\u003csub\u003e2\u003c/sub\u003eO) of these two reactions serve as substrates for each other. O\u003csub\u003e2\u003c/sub\u003e is generated in the transfer hydrogenation reaction catalyzed by Pt-Pd atoms, which is followed reduced to H\u003csub\u003e2\u003c/sub\u003eO catalyzed by laccase. H\u003csub\u003e2\u003c/sub\u003eO serves as the hydrogen source in the transfer hydrogenation reaction. Theoretically, the simultaneous action of metal and enzyme catalytic modules in single active pocket can mutually drive the reactions and eliminate intermediate diffusion, thus achieving efficient chemoenzymatic cascade reactions for low-concentration macromolecular substrates which is a grand challenge in chemistry.\u003c/p\u003e \u003cp\u003eMycotoxins contamination is an important issue for food safety and human health. However, the removal of low concentrations of mycotoxins in feed and food is a challenging task. Although strategies such as photocatalysis and microbial metabolism can effectively degrade toxins, the catalytic processes are uncontrollable, resulting in multiple transformation products which may increase toxicity. Mycotoxins can be directed transformed through enzymatic catalysis due to the high specificity and clear catalytic path of enzyme. However, the available detoxification enzymes are very limited and suffer from low catalytic activity. Due to the O\u003csub\u003e2\u003c/sub\u003e/H\u003csub\u003e2\u003c/sub\u003eO oxidation-reduction cascade, it is possible to directed transform mycotoxins efficiently by combining the visible-light-driven transfer hydrogenation reaction and enzyme-catalyzed oxidation reaction in a single active pocket (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea). Additionally, it is hypothesized that the electron transfer between metal and enzyme modules can accelerate the catalytic efficiency and broaden the substrate scope of laccase.\u003c/p\u003e \u003cp\u003eThe efficiency of PCN(PtPd)-Lac for the detoxification of AFB\u003csub\u003e1\u003c/sub\u003e was evaluated. The oxidation of AFB\u003csub\u003e1\u003c/sub\u003e by native laccase is challenging. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eb, the conversion was only 1.2% within 1.5 h when using native laccase as the catalyst with a substrate concentration of 6 mM under visible-light irradiation. The conversion of AFB\u003csub\u003e1\u003c/sub\u003e catalyzed by PCN(PtPd)-Lac, PCN(PtPd)\u0026thinsp;+\u0026thinsp;Lac (physical mixing of PCN(PtPd) and Lac), PCN(PtPd), and Lac\u0026thinsp;+\u0026thinsp;ABTS(mediator) were 100%, 11%, 10%, and 2.3%, respectively. The AFB\u003csub\u003e1\u003c/sub\u003e transformation efficiency of PCN(PtPd)-Lac was 9 and 44 times higher than that of PCN(PtPd)\u0026thinsp;+\u0026thinsp;Lac and Lac\u0026thinsp;+\u0026thinsp;ABTS, respectively, indicating that the enzyme-metal hybrid active pocket greatly enhanced the degradation efficiency of AFB\u003csub\u003e1\u003c/sub\u003e. To investigate the mechanism for the high catalytic activity of the hybrid active pocket, the reaction solutions catalyzed by PCN(PtPd)\u0026thinsp;+\u0026thinsp;Lac and PCN(PtPd)-Lac were analyzed by HPLC. The chromatographic peak with retention time of 3.0\u0026thinsp;\u0026minus;\u0026thinsp;3.3 min that attributed to the intermediate of cascade reaction (AFB\u003csub\u003e1\u003c/sub\u003e-2H) was observed when using the combination of PCN(PtPd) and Lac as the catalysts. Whereas, no intermediate was detected when using PCN(PtPd)-Lac as the catalyst, indicating that no intermediate diffused into the bulk solution due to the in-situ transformation in the enzyme-metal hybrid active pocket (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ec).\u003c/p\u003e \u003cp\u003eThe AFB\u003csub\u003e1\u003c/sub\u003e transformation product (AFB\u003csub\u003e1\u003c/sub\u003e-P) was purified via preparative HPLC (Supplementary Fig.\u0026nbsp;47), and the molecular structure was analyzed by UPLC-Q-TOF MS (Supplementary Figs.\u0026nbsp;48\u0026ndash;49). Based on the structure of AFB\u003csub\u003e1\u003c/sub\u003e-P, it is observed that the C\u003csub\u003e8\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;C\u003csub\u003e9\u003c/sub\u003e double bond of AFB\u003csub\u003e1\u003c/sub\u003e (the main site of toxicity and carcinogenicity, which forms adducts with proteins, DNA, and RNA, leading to physiological disorders) is converted into a C-C bond in the photocatalytic hydrogenation reaction, while the carbon-hydrogen bond of the cyclopenteneone ring (a toxicity site) of AFB\u003csub\u003e1\u003c/sub\u003e is oxidized in the laccase-catalyzed reaction (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea). The influence of light intensity on the catalytic efficiency of PCN(PtPd)-Lac was further investigated. As shown in Supplementary Fig.\u0026nbsp;50, PCN(PtPd)-Lac exhibited significant catalytic activity even under low light intensity (\u0026lt;\u0026thinsp;4 mW/cm\u0026sup2;), potentially due to its excellent visible-light responsiveness. Subsequently, we tested the photo-enzyme catalytic efficiency of PCN(PtPd)-Lac under sunlight. Notably, PCN(PtPd)-Lac exhibited excellent catalytic activity even on a cloudy or raining day. After exposing to sunlight for 1.5 hours on a sunny, cloudy, and rainy day, the conversions of AFB\u003csub\u003e1\u003c/sub\u003e were 72.5%, 69.4%, and 66.7%, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ed).\u003c/p\u003e \u003cp\u003eTo gain insight the superior performance of the Lac-PtPd hybrid active site, characterizations for the electron transfer between the dual single atoms and laccase were performed. The reduction peak at 0.5 V which corresponding to the electron transfer in redox reaction in the cyclic voltammetry (CV) curve of PCN(PtPd)-Lac is significantly higher than that of Lac and PCN(PtPd), indicating an increase in electron mobility and electrochemical activity (Supplementary Fig.\u0026nbsp;55). The major enhancement in the electron mobility of PCN(PtPd)-Lac is possibly due to the directional electron transfer from PCN(PtPd) to laccase, which facilitates charge separation and prevent the formation of the photogenerated electron-hole complex. Laccase is a multicopper oxidase, in which the type 1 cooper (T1 Cu) center has been shown to play a key role in electron transfer processes and substrate oxidation. As shown in Supplementary Fig.\u0026nbsp;2, laccase orientedly binds to PCN(PtPd) with the hydrophobic pocket near T1 Cu active site towards the photocatalyst. According to Marcus theory, the distance between the active site of oxidoreductase and the semiconductor should be less than 20 \u0026Aring; for direct electron transfer\u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e. The distance between T1 Cu and the binding position is 10.4 \u0026Aring;, which is beneficial to realize effective direct electron transfer (Supplementary Fig.\u0026nbsp;25). The ESR spectra of T1 Cu (g\u0026thinsp;=\u0026thinsp;2.0250) of laccase and PCN(PtPd)-Lac were used to assess the magnetic properties. As shown in Supplementary Fig.\u0026nbsp;56, the signal intensity for T1 Cu (as the active center) of PCN(PtPd)-Lac exhibited a significant increase compared with that of the free laccase, indicating the enhanced oxidation ability of laccase in PCN(PtPd)-Lac.\u003c/p\u003e \u003cp\u003eThe O\u003csub\u003e2\u003c/sub\u003e/H\u003csub\u003e2\u003c/sub\u003eO oxidation-reduction cascade in the enzyme-metal hybrid active pocket was further investigated (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ee). To verify the in-situ generation of oxygen in the active pocket, the catalytic activities of Lac and PCN(PtPd)-Lac under oxygen-depleted environment (N\u003csub\u003e2\u003c/sub\u003e atmosphere) were determined. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ef, Lac exhibited no activity under N\u003csub\u003e2\u003c/sub\u003e, demonstrating that the enzyme-catalyzed reaction was oxygen-dependent. PCN(PtPd)-Lac exhibited a significant activity under N\u003csub\u003e2\u003c/sub\u003e with a comparable turnover number (TON\u0026thinsp;=\u0026thinsp;57053) to the activity in air. These results suggest that the in-situ generation of O\u003csub\u003e2\u003c/sub\u003e by the Pt-Pd atoms can be effectively utilized by laccase in the enzyme-metal hybrid active pocket, achieving efficient chemoenzymatic cascade catalysis. Artificial metalloenzymes has emerged as powerful tool to incorporate metal complexes into protein scaffolds for creating new active sites. Yet, artificial metalloenzymes can catalyze only one type of reactions\u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e,\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e. To our knowledge, this work is the first to report two-step cascade reactions simultaneously perform in a single active pocket.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe enhancement of the electron transfer from the T1 Cu center to the type 2/type 3 trinuclear Cu center in the enzyme, theoretically expands the substrate scope of laccase. To investigate whether PCN(PtPd)-Lac can oxidize a wide spectrum of substrates, several mycotoxins including AFB\u003csub\u003e1\u003c/sub\u003e(\u003cb\u003e1a\u003c/b\u003e), zearalenone (ZEN, \u003cb\u003e2a\u003c/b\u003e), trichothecenes (T-2, \u003cb\u003e3a\u003c/b\u003e), deoxynivalenol (DON, \u003cb\u003e4a\u003c/b\u003e), and ochratoxin A (OTA, \u003cb\u003e5a\u003c/b\u003e) that typically unreactive with laccase alone were used as the substrates. These mycotoxins frequently co-exist in contaminated grains, food, and feed. The possible binding sites of the mycotoxins with laccase and the corresponding binding energies were calculated by molecular docking (Supplementary Figs.\u0026nbsp;58 and 57). The binding energies for AFB\u003csub\u003e1\u003c/sub\u003e, DON, OTA, T-2, and ZEN were \u0026minus;\u0026thinsp;5.21, -4.53, -5.47, -3.16, and \u0026minus;\u0026thinsp;5.61 Kcal/mol, respectively, showing good affinities (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eb). However, in the directed degradation reactions of mycotoxins, except for ZEN (0.7% conversion), the conversion of other mycotoxins catalyzed by laccase was 0 (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ec). Even with the addition of mediators (ABTS), the conversions of DON, OTA, T-2, and ZEN catalyzed by laccase were only 0.4%, 0.3%, 0.4%, 1.2%, respectively. Notably, the catalytic activity of PCN(PtPd)-Lac for the degradation of DON, OTA, T-2, and ZEN were 31, 49, 33, 17 times higher than that of the combination of laccase and mediator (Lac\u0026thinsp;+\u0026thinsp;ABTS) under visible-light irradiation. The enhancement in the catalytic activity of PCN(PtPd)-Lac may due to the efficient cooperative catalysis of Pt-Pd atoms and enzyme catalytic module in a single active pocket.\u003c/p\u003e \u003cp\u003eThe non-directed degradation of mycotoxins by photocatalysis is attributed to the non-specific oxidation of oxygen radicals. When PCN(PtPd)-Lac was exposed to high intensity of light (Xe-lamp, 100 mW/cm\u003csup\u003e2\u003c/sup\u003e), hydroxyl radical (\u0026middot;OH) and superoxide radical (\u0026middot;O\u003csup\u003e2\u0026minus;\u003c/sup\u003e) were detected in the ESR spectra (Supplementary Fig.\u0026nbsp;59). To investigate whether oxygen radicals are generated in the reaction under the irradiation of weak visible light (LED, 4 mW/cm\u003csup\u003e2\u003c/sup\u003e), radical quenching experiments were further carried out using isopropyl alcohol (IPA) and tryptophan (TRP) as the \u0026middot;OH and \u0026middot;O\u003csup\u003e2\u0026minus;\u003c/sup\u003e quenching agents, respectively. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ed, 97% and 93% of the activity retained after the addition of IPA and TRP when the reaction conducted under the LED irradiation, showing that almost no free radicals are produced. Thus, the transformation of AFB\u003csub\u003e1\u003c/sub\u003e in the photocatalysis was possibly due to the transfer hydrogenation reaction catalyzed by the Pt-Pd dual single atoms. Therefore, mycotoxins can be directed transformed by photo-enzyme coupled catalysis. Additionally, the decline in enzyme activity caused by free radicals can be avoided. PCN(PtPd)-Lac can be easily recovered by centrifugation, and exhibited more than 96% residual activity for seven batches of reuse, showing an excellent stability (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ee).\u003c/p\u003e \u003cp\u003eThe transformation products (DON-P, OTA-P, T-2-P, and ZEN-P) were purified via preparative HPLC (Supplementary Figs.\u0026nbsp;60\u0026ndash;63), and the molecular structures were analyzed by UPLC-Q-TOF MS (Supplementary Figs.\u0026nbsp;64\u0026ndash;71). It is found that the laccase in PCN(PtPd)-Lac displayed the capability to catalyze hydroxylation of mycotoxins (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ea). Additionally, the C\u0026thinsp;=\u0026thinsp;C or C\u0026thinsp;=\u0026thinsp;O bonds of these mycotoxins were converted to C-C or C-OH bonds in the photocatalytic water-donating transfer hydrogenation reactions catalyzed by the Pt-Pd dual single atoms.\u003c/p\u003e \u003cp\u003eThe performance of PCN(PtPd)-Lac in simultaneously catalyzing the detoxification of multiple mycotoxins was further characterized. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ef, the conversion of mycotoxins reached 100% within 180 minutes using 0.05 mg of PCN(PtPd)-Lac as the catalyst, with the initial concentration of each mycotoxin is 6 mM. The toxicity of the transformation products on liver and kidney cells were assessed using a crystal violet staining assay (Supplementary Figs.\u0026nbsp;72\u0026ndash;73). The cytotoxicity of transformation products on normal Alpha mouse liver 12 (AML 12) hepatocyte is presented in Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eg, demonstrating that the hepatotoxicity of the transformation products was considerably reduced compared to the mycotoxins. Similarly, the transformation products exhibited significantly lower renal toxicity than the mycotoxins. After a 36-hour exposure to AFB\u003csub\u003e1\u003c/sub\u003e-P, AFQ\u003csub\u003e1\u003c/sub\u003e (transformation product catalyzed by Lac\u0026thinsp;+\u0026thinsp;ABTS), and AFB\u003csub\u003e1\u003c/sub\u003e with concentration of 1 mg/mL, the viabilities of AML 12 cells were determined as 85%, 55%, and 11%, respectively. The significant decrease in the hepatotoxicity and renal toxicity of AFB\u003csub\u003e1\u003c/sub\u003e-P compared to AFQ\u003csub\u003e1\u003c/sub\u003e, may due to the simultaneous conversion of two toxic sites of AFB\u003csub\u003e1\u003c/sub\u003e by efficiently coupling the photocatalysis and enzyme catalysis when using PCN(PtPd)-Lac as the catalyst.\u003c/p\u003e \u003c/div\u003e"},{"header":"Conclusion","content":"\u003cp\u003eWe have presented a design of an enzyme-metal hybrid active pocket, created by co-coordinating Pt-Pd single-atom photocatalytic modules and laccase with a Zr cluster in MOFs. Due to the efficient utilization of intermediates and electron transfer within the artificial active pocket, PCN(PtPd)-Lac is highly active in the simultaneously catalyzing oxidation-reduction cascades for directed conversion of low-concentration mycotoxins to low-toxicity products, which is not reachable through engineered enzymes or photocatalysis alone. The construction of enzyme-metal hybrid active pocket provides an attractive way forward to achieving chemoenzymatic catalysis of new-to-nature reactions.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"Methods","content":"\u003cp\u003e \u003cstrong\u003ePreparation of PCN-224\u003c/strong\u003e \u003cp\u003ePCN-224 was synthesized using the method reported previously with few modifications. Briefly, 150 mg of ZrOCl\u003csub\u003e2\u003c/sub\u003e\u0026middot;8H\u003csub\u003e2\u003c/sub\u003eO, 1500 mg of benzoic acid, and 50 mg of tetrakis(4-carboxyphenyl) porphyrin (TCPP) were dissolved in DMF (50 mL) in a 100-mL round-bottom flask. The mixture was sonicated for 10 min, and then was heated at 90\u0026deg;C for 5 h. After cooling down to room temperature, PCN-224 was collected by centrifugation. The precipitated PCN-224 was then washed by ethanol for three times. The powder of PCN-224 was obtained by vacuum drying.\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003ePreparation of PCN(PtPd)\u003c/strong\u003e \u003cp\u003eThe dual-single-atom catalyst PCN(PtPd) was fabricated by anchoring Pt and Pd atoms on PCN-224 using a hydrothermal method. PCN-224 (10 mg) was dispersed in DMF (4 mL). 200 \u0026micro;L of H\u003csub\u003e2\u003c/sub\u003ePtCl\u003csub\u003e6\u003c/sub\u003e aqueous solution (100 mg/mL) and 200 \u0026micro;L of Pd(OAc)\u003csub\u003e2\u003c/sub\u003e aqueous solution (20 mg/mL) were added simultaneously under magnetic stirring. The mixture was sonicated at 25 \u003csup\u003eo\u003c/sup\u003eC for 10 min, and then transferred to a 20 mL stainless-steel autoclave and heated at 80 \u003csup\u003eo\u003c/sup\u003eC for 4 h. Then, PCN(PtPd) was collected by centrifugation at 10000 rpm for 5 minutes and washed by water for three times.\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003ePreparation of PCN(PtPd)-Lac\u003c/strong\u003e \u003cp\u003ePCN(PtPd)-Lac hybrid catalyst was constructed by a dynamic exchange strategy. PCN(PtPd) (10 mg) was dispersed in phosphate buffer (1 mL, 50 mM, pH 7.0). Laccase (4 mg) was added under magnetic stirring at 25 \u003csup\u003eo\u003c/sup\u003eC. The reaction solution was stirred for 40 minutes. PCN(PtPd)-Lac was collected by centrifugation and washed by water for three times. The powder of PCN(PtPd)-Lac was obtained by lyophilization and stored at 4 \u003csup\u003eo\u003c/sup\u003eC in dark.\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eEnzyme activity assay\u003c/strong\u003e \u003cp\u003eThe enzyme activities of Lac and PCN(PtPd)-Lac were assayed by a standard method using ABTS as the substrate. The reaction was conducted by adding 10 \u0026micro;L of laccase solution or catalyst suspension to 990 \u0026micro;L of 0.5 mM ABTS solution (prepared in 10 mM acetate buffer, pH 5.0), and the absorbance change at 420 nm was monitored using a UV-Vis spectrophotometer.\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eDetoxification of mycotoxins by PCN(PtPd)-Lac\u003c/b\u003e: First, mycotoxins (6 nmol) was dissolved in deionized water (1 mL). Then, 10 \u0026micro;L of the catalyst suspension (1 mg/mL) involving PCN(PtPd)-Lac was added with vigorously stirring. The mixture was subsequently placed in a PCX50C photoreactor equipped with a white LED light with a wavelength of 420\u0026ndash;650 nm. After the reaction, the supernatant was collected by centrifugation. The conversions were analyzed by HPLC. HPLC conditions: The concentration of mycotoxins in the solution was determined by an HPLC system (Agilent 1260). HPLC was performed on an Agilent C18 column (250 mm \u0026times; 4.6 mm; 5 \u0026micro;m) at a flow rate of 0.6 mL min\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e. The mobile phase consisted of water and acetonitrile (45:55 v/v for AFB\u003csub\u003e1,\u003c/sub\u003e 80:20 v/v for DON, 51:49 v/v for OTA, 70:30 v/v for ZEN, 25:75 v/v forT-2).The column temperature was 40\u0026deg;C. The injection volume was 20 \u0026micro;L. AFB1, DON, and ZEN were analysed using a UV detector at wavelengths of 365 nm, 218 nm, and 236 nm, respectively. In contrast, OTA and T-2 were detected using a fluorescence detector. The excitation and emission wavelengths for OTA were set at 333 nm and 460 nm, respectively, while those for T-2 were 381 nm and 470 nm.\u003c/p\u003e \u003cp\u003e \u003cstrong\u003eDFT calculations\u003c/strong\u003e \u003cp\u003eDFT calculations are performed using the Vienna Ab initio Software Package (VASP 5.4.4) within the Perdew-Burke-Ernzerhof (PBE) generalized gradient approximation and projected enhancement wave (PAW) method\u003csup\u003e\u003cspan additionalcitationids=\"CR25\" citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e. The cutoff energy of the plane-wave basis set is set to 400 eV. Monkhorst\u0026ndash;Pack special k-point meshes of 3 \u0026times; 3 \u0026times; 1 were proposed to carry out geometry optimization and electronic structure calculation. In the process of geometric optimization, all atoms can relax without any restriction until the convergence thresholds of the maximum force and energy are less than 0.01 eV/\u0026Aring; and 1.0 \u0026times; 10\u003csup\u003e\u0026minus;\u0026thinsp;5\u003c/sup\u003e\u0026nbsp;eV/atom, respectively. A 15 \u0026Aring; vacuum layer was introduced to avoid interaction between periodic images. All DFT calculations were carried out using the CP2K code\u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e. All calculations employed a mixed Gaussian and planewave basis sets. Core electrons were represented with norm-conserving Goedecker-Teter-Hutter pseudopotentials\u003csup\u003e\u003cspan additionalcitationids=\"CR29\" citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e, and the valence electron wavefunction was expanded in a double-zeta basis set with polarization functions\u003csup\u003e\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e along with an auxiliary plane wave basis set with an energy cutoff of 400 Ry. The generalized gradient approximation exchange-correlation functional of Perdew, Burke, and Enzerhof (PBE)\u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e was used. Each configuration was optimized with the Broyden-Fletcher-Goldfarb-Shanno (BGFS) algorithm with SCF convergence criteria of 1.0\u0026times;10\u003csup\u003e\u0026minus;\u0026thinsp;5\u003c/sup\u003e au. To compensate the long-range van der Waals dispersion interaction, the DFT-D3 scheme\u003csup\u003e\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e with an empirical damped potential term was added into the energies obtained from exchange-correlation functional in all calculations.\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eMolecular Docking simulation\u003c/strong\u003e \u003cp\u003eMolecular docking simulations were performed to investigate the binding interactions between the toxins AFB\u003csub\u003e1\u003c/sub\u003e, OTA, ZEN, DON, and T-2 with laccase. The docking calculations were carried out using AutoDock with 50 docking runs. The structures of the ligands were drawn using ChemDraw and subsequently energy-minimized using Chem3D. The receptor protein, laccase, was obtained from the PDB database. The binding affinity of each ligand was evaluated, and potential hydrogen bonding interactions between the ligands and amino acids near the enzyme\u0026rsquo;s active pocket were analyzed based on the docking results.\u003c/p\u003e \u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e \u003ch2\u003eCompeting interests\u003c/h2\u003e \u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eAuthor contributions\u003c/h2\u003e \u003cp\u003eX. L. and J. G. supervised the project. X. L. and J. G. conceived of the idea. Y. F. performed the experiments with technical help from J. H. H. W. performed the calculations. Y. F., J. H., Q. W. and M. Z. participated in analyzing the results. X. L., Y. F., and J. G. wrote the paper.\u003c/p\u003e\u003ch2\u003eAcknowledgements\u003c/h2\u003e \u003cp\u003eThis research was supported by the National Key Research and Development Program of China (2022YFF1102800), the National Natural Science Foundation of China (Grant No. 22308143, 22168024, 22425803), the Jiangxi Provincial Natural Science Foundation (Grant No. 20232ACB215008), the Beijing Natural Science Foundation (Grant No. Z240030), the Shenzhen Science and Technology Program (Grant No. KCXFZ20240903093102004). The authors thank beamline BL14W1 (Shanghai Synchrotron Radiation Facility) for providing the beam time.\u003c/p\u003e\u003ch2\u003eData availability\u003c/h2\u003e \u003cp\u003eThe data that support the plots within this paper and other findings of this study are available from the corresponding author upon request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eMiller DC, Athavale SV, Arnold FH (2022) Combining chemistry and protein engineering for new-to-nature biocatalysis. Nat Synth 1:18\u0026ndash;23\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRaps FC, Rivas-Souchet A, Jones CM, Hyster TK (2025) Emergence of a distinct mechanism of C\u0026ndash;N bond formation in photoenzymes. Nature 637:362\u0026ndash;368\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWard TR, Cop\u0026eacute;ret C (2023) Introduction: bridging the gaps: learning from catalysis across boundaries. 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J Phys Chem 132\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"nature-portfolio","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"","title":"Nature Portfolio","twitterHandle":"","acdcEnabled":false,"dfaEnabled":false,"editorialSystem":"ejp","reportingPortfolio":"","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-6659547/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6659547/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eChemoenzymatic cascades, which combine the merits of enzymatic catalysis and chemical catalysis, have blossomed into a powerful technology for new-to-nature reactions. The diffusion resistance of reaction intermediates is a major rate-limiting factor in cascade reactions, which can be reduced by integrating metal catalytic modules and enzymes in a single catalyst due to the proximity effect. Here we assemble enzymatic and dual metal-single-atom photocatalytic modules in a single active pocket for \u0026ldquo;one binding two reactions\u0026rdquo; catalysis, which can eliminate the diffusion resistance of reaction intermediates. The enzyme-metal hybrid active pocket exhibited excellent activity in simultaneously catalyzing transfer hydrogenation and oxidation reactions under visible light. The diffusion and rebinding of intermediates between the multiple catalytic modules are eliminated in the artificial active pocket, achieving efficient oxidation-reduction cascades for the directed detoxification of low-concentration mycotoxins, which is not reachable through engineered enzymes or photocatalysts alone. This work proposes a new type of cascade process and establishes a powerful tool for editing enzyme active pockets with metal catalytic modules.\u003c/p\u003e","manuscriptTitle":"Assembling a single active pocket by enzyme and metal modules for simultaneously catalyzing oxidation-reduction cascades","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-06-23 07:09:24","doi":"10.21203/rs.3.rs-6659547/v1","editorialEvents":[],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"nature-communications","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"NCOMMS","sideBox":"Learn more about [Nature Communications](http://www.nature.com/ncomms/)","snPcode":"","submissionUrl":"https://mts-ncomms.nature.com/","title":"Nature Communications","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature Communications","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"6786a3c1-5f22-40a0-ac9e-5dc9d9bb4641","owner":[],"postedDate":"June 23rd, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[{"id":49230586,"name":"Physical sciences/Chemistry/Catalysis/Biocatalysis"},{"id":49230587,"name":"Physical sciences/Chemistry/Catalysis/Photocatalysis"},{"id":49230588,"name":"Physical sciences/Chemistry/Catalysis/Catalyst synthesis"}],"tags":[],"updatedAt":"2025-06-23T07:09:24+00:00","versionOfRecord":[],"versionCreatedAt":"2025-06-23 07:09:24","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6659547","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6659547","identity":"rs-6659547","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}