Polypeptide self-assembled nickel-selenium artificial hydrogenase as well as preparation method and application thereof

Abstract

The invention discloses a polypeptide self-assembled nickel-selenium artificial hydrogenase as well as a preparation method and application thereof. The artificial hydrogenase is a polypeptide-nickel compound with a binuclear coordination center, wherein the polypeptide-nickel compound is formed by a self-assembly reaction of polypeptide and nickel ions in a buffer solution; a main chain of the polypeptide provides two strongly coordinated main chain amide groups, polypeptide cysteine and selenocysteine residues provide two side chain mercaptan and / or selenol, and the two side chain mercaptan and / or selenol are coordinated with Ni < 2 + > together to form a binuclear coordination center. The stability of the artificial hydrogenase metal coordination structure is improved, a binuclear metal center can be stably constructed, disordered polypeptide aggregation is reduced, the structural repeatability is improved, the electron transfer path of the metal center is more controllable, and an active structure can be kept under conventional solution and air conditions. An artificial biomimetic catalytic system with a clear structure, good repeatability and higher applicability can be obtained.

Description

Technical Field

[0001] This invention relates to the fields of biomimetic catalytic materials and artificial enzyme engineering, and particularly to a polypeptide self-assembled nickel-selenium artificial hydrogenase, its preparation method, and its application. Background Technology

[0002] Hydrogen, as a clean and high-energy-density secondary energy source, is considered to have significant application potential in future energy systems. Natural hydrogenases can efficiently catalyze the generation and oxidation of hydrogen under ambient temperature and pressure conditions, playing a crucial role in bioenergy conversion. Natural hydrogenases mainly include [NiFe] hydrogenase and [FeFe] hydrogenase, whose active centers are composed of various metal clusters and sulfur ligands, exhibiting a highly complex three-dimensional configuration and finely regulated electron transport pathways.

[0003] Despite the excellent catalytic performance of natural hydrogenases, their intricate structures and extreme sensitivity to oxygen lead to rapid inactivation in vitro. Furthermore, extraction is difficult, costly, and scalable. Therefore, recent years have seen a surge in research on artificial hydrogenases and biomimetic metallocatalytic centers, aiming to simplify the structures of natural enzymes through strategies such as small molecule ligands, short peptides, protein fragments, or peptide self-assembly, and to construct artificial systems capable of efficiently catalyzing hydrogen production under mild conditions.

[0004] In existing technologies, peptides are increasingly becoming important templates for constructing artificial metal active sites due to their structural designability, ease of modification, and self-assembly capabilities. For example, some studies have utilized short peptides containing coordination groups such as Cys and His with Ni... 2+ Fe 2+ Metal ions bind to each other, forming metal coordination structures similar to those of natural hydrogenases. These systems can mimic the metal-binding environment of natural enzymes to some extent, but they still suffer from the following common problems: 1. Insufficient structural stability Most short peptide-metal coordination structures are easily oxidized under air conditions, which leads to the destruction of the coordination environment and a rapid decline in catalytic activity, making it difficult to meet the stability requirements of practical applications.

[0005] 2. Difficulty in constructing a stable dual-core metallic structure The active site of natural hydrogenases is a binuclear metal structure with strict geometric constraints. In existing short peptide systems, due to the high flexibility of ligands and loose spatial structure, it is often difficult to achieve a stable construction of the bimetallic center, resulting in insufficient electron transfer efficiency.

[0006] 3. Limited electron transport control capability Existing artificial systems generally lack clear electron transport pathways and the synergistic relationships between metals are unclear, resulting in their overall catalytic performance being lower than that of natural enzymes.

[0007] 4. Lack of effective control over peptide self-assembly behavior Peptides are prone to uncontrolled aggregation or oligomerization under metal induction, resulting in structural heterogeneity and affecting the reproducibility of metal coordination and catalytic reliability.

[0008] 5. Insufficient research on the use of selenium ligands to stabilize metal centers. Although selenium ligands theoretically possess stronger affinity for soft tissues and antioxidant capacity, and may provide greater stability in metal coordination, there are few publicly available studies on the specific construction methods of selenium substitution strategies in short peptide-metal systems and their impact on binuclear structures.

[0009] For the reasons mentioned above, existing technologies have not yet provided an artificial biomimetic hydrogenase system capable of forming a stable and controllable binuclear metal coordination structure under mild conditions and maintaining catalytic activity in an air environment for a long period of time. In particular, existing technologies still have significant shortcomings in areas such as "how to accurately construct metal active centers through the self-assembly of short peptide sequences, how to improve their antioxidant capacity, and how to avoid the disordered aggregation of peptides". Summary of the Invention

[0010] To overcome the shortcomings of existing artificial metal catalytic systems constructed using short peptides or polypeptides in terms of poor structural stability, precise metal coordination, and controllability of electron transfer, the main objective of this invention is to provide a polypeptide self-assembled nickel-selenium artificial hydrogenase, its preparation method, and its application. This invention solves the technical problems existing in current artificial hydrogenase systems, such as unstable metal coordination structures, difficulty in constructing binuclear metal centers, easy disordered aggregation of polypeptides, and unclear electron transfer pathways.

[0011] To achieve the aforementioned objectives, the technical solution adopted by this invention includes: A polypeptide sequence for self-assembly of nickel-selenium artificial hydrogenase, wherein the polypeptide is selected from any one of NI, Sec-1, Sec-3, Sec-5, and Sec-12, wherein the amino acid sequence of NI is as shown in SEQ ID No. 1, the amino acid sequence of Sec-1 is as shown in SEQ ID No. 2, the amino acid sequence of Sec-3 is as shown in SEQ ID No. 3, the amino acid sequence of Sec-5 is as shown in SEQ ID No. 4, and the amino acid sequence of Sec-12 is as shown in SEQ ID No. 5.

[0012] A peptide-based nickel-selenium artificial hydrogenase, wherein the artificial hydrogenase is a peptide-nickel complex with a binuclear coordination center formed by the self-assembly reaction of the peptide with nickel ions in a buffer solution; the amino acid sequence of the peptide is selected from any one of SEQ ID NO.1 to 5 as described in claim 1; the main chain of the peptide provides two strongly coordinating main chain amide groups; and the peptide cysteine ​​and selenocysteine ​​residues provide two side chain thiols and / or selenools, which coordinate with Ni2+ to form a binuclear coordination center.

[0013] A method for preparing a polypeptide self-assembled nickel-selenium artificial hydrogenase includes the following steps: Step 1: Design an artificial hydrogenase polypeptide sequence, wherein the polypeptide sequence is selected from any one of SEQ ID No. 1-5 as described in claim 1; Step 2: Synthesize artificial hydrogenase peptides. The five peptides described in Step 1 are synthesized using a standard solid-phase peptide synthesis method. Step 3: Prepare self-assembled artificial hydrogenase. The five polypeptides described in Step 2 undergo self-assembly reactions with nickel ions in a buffer solution to form a polypeptide-nickel complex with a binuclear coordination center. The main chain of the polypeptide provides two strongly coordinating main chain amide groups, and the polypeptide cysteine ​​and selenocysteine ​​residues provide two side chain thiols and / or selenools, which coordinate with Ni2+ to form a binuclear coordination center.

[0014] The standard solid-phase polypeptide synthesis method described in step 2 uses Wang-resin as the solid-phase support.

[0015] The buffer solution in step 3 contains 25 mM HEPES, 100 mM NaCl and 3.75 mM TCEP, and the pH value of the buffer solution is 8.5.

[0016] The final concentration of the polypeptide in step 3 is 0.75 mM, and the nickel ions are from the NiCl2 solution with a final concentration of 1.65 mM.

[0017] The self-assembly reaction time described in step 3 is 2 hours at 50°C and 48 hours at room temperature.

[0018] In the application of the peptide-assembled nickel-selenium artificial hydrogenase in hydrogen production, the photosensitizer and the sacrificial electron donor coexist with the peptide-assembled nickel-selenium artificial hydrogenase in the same reaction system, and synergistically achieve the catalytic hydrogen evolution of protons in aqueous solution under visible light irradiation.

[0019] The photosensitizer is Eosin Y, and the sacrificial electron donor is triethanolamine.

[0020] Compared with the prior art, the beneficial effects of the present invention are as follows: 1. Improved stability of metal coordination structure By introducing specific ligands into short peptide sequences and regulating their spatial distribution, a metal coordination environment with a well-defined geometric configuration can be stably formed, effectively reducing the damage to the metal center structure caused by air oxidation, thereby improving the overall structural stability.

[0021] 2. Capable of stably constructing dual-core metal centers The short peptide sequence designed in this application can form suitable spatial constraints through self-assembly, so that metal ions maintain a reasonable distance and angle between them, which is conducive to obtaining a structurally controllable binuclear metal center, which is difficult to achieve with existing peptide systems.

[0022] 3. Reduce disordered aggregation of peptides and improve structural reproducibility. By adjusting the distribution of hydrophobic and hydrophilic amino acids in the sequence, this technique can significantly inhibit the disordered aggregation of peptides under metal-induced conditions, resulting in a more uniform coordination structure and improved reproducibility of the system.

[0023] 4. The electron transport path in the metal center is more controllable. Since the binuclear structure can be formed under a relatively stable geometric configuration, this technical solution can, to a certain extent, regulate the electron transfer path, which is beneficial to improving the catalytic efficiency of the metal center.

[0024] 5. It can maintain its active structure under normal solution and air conditions. By rationally designing the coordinating groups and sequence conformations, the metal coordination centers obtained by this technical solution can still maintain structural integrity under air conditions, making them suitable for a wider range of experimental and application scenarios.

[0025] In summary, the technical solution of this application has significant improvements over the prior art in terms of metal coordination structure stability, binuclear metal center construction, controllability of peptide self-assembly, and electron transfer regulation. It can obtain an artificial biomimetic catalytic system with a well-defined structure, good reproducibility, and greater applicability. Attached Figure Description

[0026] Figure 1 The sequences and structures of five polypeptides are shown below: (a) NI; (b) Sec-1; (c) Sec-3; (d) Sec-5; (e) Sec-12. Figure 2 The following are HPLC chromatograms of five peptides: (a) Apo-NI; (b) Apo-Sec-1; (c) Apo-Sec-3; (d) Apo-Sec-5; (e) Apo-Sec-12; Figure 3Mass spectra of five polypeptides: (a) Apo-NI; (b) Apo-Sec-1; (c) Apo-Sec-3; (d) Apo-Sec-5; (e) Apo-Sec-12; Figure 4 (A) UV-Vis absorption spectra of the artificial hydrogenase of the present invention after preparation and after 7 days of aerobic storage; (a) NI; (b) Sec-1; (c) Sec-3; (d) Sec-5; (e) Sec-12; (B) Circular dichroism chromatograms of the artificial hydrogenase of the present invention after preparation and after 7 days of aerobic storage: (a) NI; (f) Sec-1; (g) Sec-3; (h) Sec-5; (i) Sec-12; Figure 5 The following is a comparison of the particle size results of the artificial hydrogenase of the present invention after preparation and after 7 days of aerobic storage; (a) NI; (b) Sec-1; (c) Sec-3; (d) Sec-5; (e) Sec-12; Figure 6 The following are electron paramagnetic resonance (EPR) results of the artificial hydrogenase of this invention: (a) NI; (b) Sec-1; (c) Sec-3; (d) Sec-5; (e) Sec-12; Figure 7 The artificial hydrogenase and Ni of this invention 2+ The structure diagram of the coordination-formed binuclear complex is shown, with Se atoms in red, S atoms in pink, Ni atoms in green, N atoms in blue, and O atoms in blue-violet. Figure 8 (a) is a graph showing the catalytic results of different artificial hydrogenases of the present invention at pH=8 and in an anaerobic environment; (b) is a graph showing the TON results of different artificial enzymes at pH=8 and in an anaerobic environment. P <0.001, P <0.001, P <0.01, P <0.05, ns: no significant difference; one-way ANOVA, multiple comparisons; data are expressed as mean ± standard deviation; Figure 9 The following are the results of photocatalytic hydrogen evolution of the artificial hydrogenase of the present invention under aerobic conditions at different pH values: (a) Photocatalytic hydrogen evolution of the artificial hydrogenase at pH=5 under aerobic conditions; (b) Photocatalytic hydrogen evolution of the artificial hydrogenase at pH=7 under aerobic conditions; (c) Photocatalytic hydrogen evolution of the artificial hydrogenase at pH=9 under aerobic conditions; (d) Photocatalytic hydrogen evolution of the artificial enzyme at different pH values ​​under aerobic conditions. P <0.001, P <0.001, P <0.01, P <0.05, ns: no significant difference; one-way ANOVA, multiple comparisons; data are expressed as mean ± standard deviation; Figure 10 The following are the results of photocatalytic hydrogen evolution of the artificial hydrogenase of the present invention after 7 days of storage under different conditions: (a) Photocatalytic hydrogen evolution of the artificial enzyme after 7 days of storage under nitrogen; (b) Photocatalytic hydrogen evolution of the artificial enzyme after 7 days of storage under oxygen and carbon dioxide; (c) Comparison of photocatalytic hydrogen evolution of the artificial enzyme after 7 days of storage under different conditions. P <0.001, P <0.001, P <0.01, P <0.05, ns: no significant difference; one-way ANOVA, multiple comparisons; data are expressed as mean ± standard deviation; Figure 11 This is a protein membrane voltammetric curve of the artificial hydrogenase of the present invention. Detailed Implementation

[0027] The embodiments described below are exemplary descriptions of key experimental evidence and are not intended to limit the core content and application scope of this invention due to the amount of evidence. It should be noted that all the accompanying drawings and corresponding descriptions merely illustrate the concept, principles, and representative experimental evidence of the disclosed embodiments of this invention. Where the chain of evidence is complete, it is unnecessary to show all the specific details and extended details of the various embodiments listed in this invention.

[0028] Unless otherwise defined, the technical terms used in the following embodiments have the same meaning as commonly understood by those skilled in the art to which this invention pertains.

[0029] The present invention will now be described in detail with reference to the accompanying drawings and embodiments.

[0030] This invention designs short peptide sequences with specific coordinating groups and spatial conformations, enabling them to form stable bonds with metal ions under solution conditions and construct well-defined binuclear metal centers through self-assembly. This technical solution allows control over the metal coordination geometry and assembly method by adjusting the coordination sites, the ratio of hydrophobic amino acids, and their distribution within the short peptide sequence, thereby obtaining metal-active structures with well-defined conformations and good reproducibility. Simultaneously, by introducing specific sulfur- or selenium-containing ligand groups, the structural stability of the metal center under air conditions is improved, reducing coordination disruption caused by oxidation.

[0031] This technical solution can obtain an artificial biomimetic catalytic system that can form a stable binuclear metal structure under conventional solution conditions and is not prone to disordered aggregation. By rationally designing the sequence and coordination environment, it can be made to have controllable electron transfer characteristics, providing a reproducible, tunable and structurally well-defined path for the construction of artificial hydrogenases.

[0032] Example 1 Step 1: Design of artificial hydrogenase polypeptide sequences Both natural [NiFe] hydrogenase and acetyl-CoA synthase (ACS) possess a metal coordination structure centered on cysteine ​​(Cys) at their catalytic center, commonly in Cys–Ni–S or Cys–Ni–Fe bridging configurations. Based on structural analysis of the active sites of these metalloenzymes, researchers abstracted the functional motif CXCGCXXXXXXCG from their conserved sequences and constructed a short peptide model system capable of coordinating with divalent nickel ions. This motif was further specified as CNCGCGNNNDRCG, whose Cys residues can coordinate with Ni… 2+ Coordination with a metal center allows short peptides to self-assemble into catalytically active artificial enzyme structures in solution. However, in such short peptide systems with Cys as the sole coordinating group, the metal center is sensitive to oxygen and easily oxidized in air, leading to disruption of the coordination environment, which in turn reduces catalytic efficiency and reduces stability. Since artificial hydrogenase systems with pure sulfur coordination are generally limited in their oxygen tolerance, their ability to maintain structure and activity in air environments still needs improvement. Studies of natural [NiFeSe] hydrogenase show that replacing the key coordination site with selenium enhances the metal center's affinity for softness, electron transferability, and antioxidant capacity, enabling it to maintain high catalytic activity under oxygen-containing conditions. Therefore, to improve the poor oxygen tolerance and limited catalytic performance of short peptide artificial enzymes constructed with Cys motifs, it is necessary to introduce selenocysteine ​​(Sec) into the short peptide sequence to establish a more stable metal coordination environment, thereby obtaining artificial enzyme systems that can maintain structure and catalytic activity under air conditions.

[0033] Therefore, we constructed five artificial enzyme peptide sequences from the N-terminus to the C-terminus, as follows: Figure 1 As shown, NI (SEQ ID No. 1), Sec-1 (SEQ ID No. 2), Sec-3 (SEQ ID No. 3), Sec-5 (SEQ ID No. 4), and Sec-12 (SEQ ID No. 5) were synthesized by the Fmoc solid-phase synthesis method. The first polypeptide NI does not contain selenocysteine ​​(Sec), while the other polypeptides replace Cys with Sec at different positions.

[0034] (To distinguish between the peptides before and after self-assembly, Apo is added before the sequence of the unassembled peptide to indicate the difference.)

[0035] Step 2: Synthesis of artificial hydrogenase polypeptide 2.1 Five artificial enzyme peptides were synthesized using Wang-resin as a solid-phase carrier and the standard solid-phase peptide synthesis (SPPS) method.

[0036] Preparation Example 1 This preparation example illustrates the preparation method of NI. The specific operation method is as follows: (1) Weigh 0.1 mmol Fmoc-cysteine ​​(Trt)-Wang resin, put it into a polypeptide solid phase synthesis tube, add 4 mL N,N-dimethylformamide (DMF), tighten and fix the synthesis tube cap, place the synthesis tube on a vertical rotary mixer and mix for at least 30 minutes to allow the resin to fully expand in the DMF solution. (2) Filter to remove DMF liquid, and rinse the resin thoroughly with DMF / DCM (dichloromethane) / DMF in sequence; (3) Add 2-3 mL of 20% piperidine / DMF (v / v) solution to the synthesis tube, mix well for 1 minute and then remove the solution to achieve the purpose of pre-deprotection; (4) Transfer 2-3 mL of 20% piperidine / DMF (v / v) solution to the synthesis tube, seal the synthesis tube tightly, and place the synthesis tube on a vertical rotary mixer to mix for 20 minutes. (5) Filter to remove piperidine / DMF solution, and thoroughly wash the resin three times each with DMF / DCM / DMF to complete the removal of the Fmoc protecting group of the first amino acid residue on Wang-resin—glycine (Gly); (6) Weigh 8 equivalents (0.8 mmol) of Fmoc-cysteine ​​(Cys) containing a protecting group, add 1.6 mL of 2-(7-azobenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (HATU) solution (0.5 mol / L), and then add 8 equivalents (0.8 mmol) of N,N-diisopropylethylamine (DIPEA). At this time, the amino acid solution should be yellow. (7) Add the prepared solution to the solid phase synthesis tube, tighten the synthesis tube cap, place the synthesis tube on a vertical rotary mixer and mix for 25 minutes. The carboxyl group of cysteine ​​undergoes a condensation reaction with the amino group of glycine on the resin, and the second amino acid Fmoc-cysteine ​​is fixed onto the above resin. (8) Filter to remove excess reaction solution, and thoroughly wash the resin three times each with DMF / DCM / DMF to complete the coupling of one amino acid residue; (9) Repeat steps 3-8 and carry out condensation reaction according to the order of amino acid sequence (CNCGCGNNNDRCG see SEQ ID NO. 1). The carboxyl group of each subsequent amino acid condenses with the amino group of the previous amino acid to obtain a polypeptide with the desired amino acid sequence on the resin. (10) It is worth noting that, in order to prevent the amino acid residues from being deselenic during the coupling of selenocysteine, the resin needs to be rinsed with 20% DCM / N-methylpyrrolidone (v / v), DCM, and N-methylpyrrolidone for 2 minutes each, and then the Fmoc group of selenocysteine ​​is removed with 20% piperidine / N-methylpyrrolidone (v / v) (3 minutes). (11) Dry the resin and continue to filter the resin under negative pressure for at least 2 hours; (12) Prepare at least 10 mL of fresh trifluoroacetic acid (TFA):phenol:water:thioanisole:1,2-ethanedithiol (87.5:2.5:5:5:2.5) (v / v) lysis buffer; (13) Transfer the lysis solution to the synthesis tube, tighten the synthesis tube cap, and place it on a vertical rotary mixer to mix for 3 hours. This step can remove all side chain protecting groups except for the Mob protecting group. (14) Filter under negative pressure, collect the lysis solution in a flask, wash the resin twice with 3 mL of lysis solution and collect it in the flask. (15) Rotary evaporate the pyrolysis solution until 500 μL or less of the pyrolysis solution remains; (16) Transfer the lysis buffer to a centrifuge tube, add 20 mL of cold ether (-20°C), and gently shake to allow a white precipitate to form; (17) Pre-cool the centrifuge to 4°C, centrifuge at 6000 rpm for 20 minutes, and carefully discard the supernatant; (18) Add 20 mL of cold diethyl ether to the precipitate again; repeat step 17 three times in total; (19) Use a nitrogen blower and inert gas to remove residual ether; (20) The crude product was dissolved in a 30% acetonitrile / water (v / v) solution and freeze-dried to obtain a fluffy white powder; (21) The lyophilized peptide was transferred to a flask and a lysis buffer of trimethylbromosilane: m-cresol: benzyl sulfide: TFA (12.9:4.7:11.3:70.9) (v / v) was added. The reaction was carried out at 37°C under inert gas protection for 1.5 hours in the dark. This step can remove the Mob protecting group of Sec. (22) Repeat steps 15-20 to obtain a white, fluffy polypeptide.

[0037] Preparation Example 2: Preparation of Sec-1 The method of Preparation Example 1 was followed, except that the condensation reaction was carried out in the order of the amino acid sequence (UNCGCGNNNDRCG, see SEQ ID No. 2).

[0038] Preparation Example 3: Preparation of Sec-3 The method of Preparation Example 1 was followed, except that the condensation reaction was carried out in the order of the amino acid sequence (CNUGCGNNNDRCG, see SEQ ID No. 3).

[0039] Preparation Example 4: Preparation of Sec-5 The method of Preparation Example 1 was followed, except that the condensation reaction was carried out in the order of the amino acid sequence (CNCGUGNNNDRCG, see SEQ ID No. 4).

[0040] Preparation Example 5: Preparation of Sec-12 The method of Preparation Example 1 was followed, except that the condensation reaction was carried out in the order of the amino acid sequence (CNCGCGNNNDRUG, see SEQ ID No. 5).

[0041] 2.2 Purification and Identification of Artificial Hydrogenase Peptides This article describes the purification of artificial hydrogenase peptides using preparative HPLC. A high-efficiency column (Gemini® 5 µm C18 110 Å) was selected to separate impurities from the crude product. The specific experimental procedure is as follows: The lyophilized peptides were dissolved in 30% acetonitrile / water (v / v) and then filtered using a 0.45 µm filter. Gradient elution was performed using two mobile phases: mobile phase A: 100% methanol + 0.1% TFA, and mobile phase B: 95% water + 5% methanol + 0.1% TFA. Two wavelengths were set for the UV detector: 220 nm and 254 nm. The mobile phase gradient settings are shown in Table 1.

[0042] Table 1 Gradient elution conditions for reversed-phase high-performance liquid chromatography

[0043] The HPLC chromatograms of the five artificial enzyme peptides are shown below. Figure 2 As shown, the chromatograms of the five peptides all showed only one obvious peak, indicating that the purity of the peptides was higher than 95%. Furthermore, the peak positions of the five peptides were similar, indicating that the five peptides had similar hydrophilicity and hydrophobicity, which is beneficial for subsequent use of similar self-assembly environments to promote peptide folding and the formation of artificial enzymes with secondary structures.

[0044] After purification, a mixed solution containing the target peptide was obtained. Methanol was removed from the solution using a rotary evaporator. The solution was then transferred to centrifuge tubes and lyophilized to obtain a white powdered peptide. The peptide powder was weighed, dissolved in water to obtain a peptide solution with a concentration of 1 mg / mL, aliquoted into 1.5 mL EP tubes, lyophilized to remove water, and the peptide powder was stored at -20 °C to avoid repeated freeze-thaw cycles.

[0045] Molecular weight was determined using matrix-assisted laser desorption / ionization time-of-flight mass spectrometry (MALDI-TOF-MS).

[0046] (1) α-cyano-4-hydroxycinnamic acid (CHCA) was dissolved in acetonitrile water at a ratio of 1:2 to form a saturated CHCA solution (with precipitate), and 0.1% TFA was added; (2) Dissolve the peptide in ultrapure water to prepare a solution with a concentration of 10 fmol / μl to 1 pmol / μl; (3) Mix the polypeptide solution and CHCA matrix solution at a ratio of 1:1, and take 1 μl of the mixture for spotting.

[0047] The MALDI-TOF-MS analysis results of the 5 peptides are as follows: Figure 3 As shown, the theoretical molecular weight of Apo-NI is 1329.45, while the molecular weight in the mass spectrum is 333.25, corresponding to the theoretical molecular weight plus 4H. + The theoretical molecular weight of the selenocysteine-containing polypeptide is 1379.45, such as... Figure 3As shown, the mass spectrometry results for Apo-Sec-1, Apo-Sec-3, Apo-Sec-5, and Apo-Sec-12 are 467.3, 474.6, 474.33, and 230.3, respectively, corresponding to theoretical molecular weights plus 2H. + + Na + 2Na + + H + 2Na + + H + 6H + This indicates that five high-purity target peptides were successfully synthesized, which will facilitate further experiments.

[0048] Step 3: Preparation of self-assembled artificial hydrogenase After obtaining the target peptide, a self-assembly environment needs to be constructed so that the peptide can interact with Ni. 2+ A special spatial structure is constructed and folded to obtain catalytic activity. The preparation method is as follows: (1) Take 1 mL of freshly prepared 4-hydroxyethylpiperazine ethanesulfonic acid (HEPES)-tris(2-chloroethyl) phosphate (TCEP)-NaCl solution, add 9 mL of ultrapure water to obtain a buffer solution with pH=8.5 containing 25 mM HEPES, 100 mM NaCl and 3.75 mM TCEP. (2) Add 1 mL of buffer solution to the polypeptide powder that has been dispensed in the previous step, mix thoroughly to ensure that the polypeptide is completely dissolved in the buffer solution, and the final polypeptide concentration is 0.75 mM. (3) Transfer the polypeptide solution to a vial, tighten the stopper to seal it completely, and purge the vial with 100% nitrogen gas for at least 20 minutes to deoxygenate the polypeptide solution. (4) The temperature-controlled magnetic stirrer was set to 50 ℃ and 400 rpm. The vial was heated in a water bath to maintain a constant temperature of 50 ℃. 10 μL of NiCl2 solution was continuously added dropwise to the vial using a microsyringe. The solution contained Ni 2+ The final concentration was 1.65 mM, and the reaction was carried out at 50°C in the absence of oxygen for 2 hours. Alternatively, the same conditions could be used, with the reaction proceeding at room temperature (25°C) for 48 hours.

[0049] (5) After the reaction is complete, the solution is a light yellow liquid and should be stored at 4 ℃ in an oxygen-free environment.

[0050] Example 2: Conformation and Performance Testing of Artificial Enzymes To investigate the relationship between peptides and Ni 2+The secondary structures of each assembly were investigated using ultraviolet-visible absorption spectroscopy (UV-VIS), circular dichroism spectroscopy (CD), and electron paramagnetic resonance (EPR) to determine the conformations formed after complexation. To further explore whether peptides will oligomerize to form polymers during self-assembly, and whether the artificial enzyme is stable under oxygen and carbon dioxide conditions, this study used a nanoparticle size analyzer, ultraviolet spectrophotometer, and circular dichroism chromatograph to measure the particle size, UV-VIS absorption spectrum, and circular dichroism spectrum of the artificial enzyme immediately after preparation and after 7 days of exposure to air.

[0051] 2.1 Characterization of the structure and stability of artificial enzymes Leveraging the advantages of UV-Vis absorption spectroscopy and circular dichroism spectroscopy, we used UV-Vis absorption spectroscopy and circular dichroism spectroscopy to investigate the secondary structure of peptides after folding into artificial enzymes.

[0052] (1) Determination of ultraviolet-visible absorption spectrum: The ultraviolet spectrophotometer was a Hitachi U-3900, the optical path of the quartz sample cell was 1 cm, the scanning range was 200-380 nm, and the measurement was performed at room temperature.

[0053] (2) Circular dichroism spectroscopy determination: The circular dichroism spectrometer was a JASCO J-810 instrument from Japan. The test was conducted under nitrogen atmosphere. The optical path length of the quartz sample cell was 1 cm, the scanning sensitivity was 2 mdeg / cm, the wavelength range was 300-600 nm, and the scanning speed was 200 nm / min. The circular dichroism spectrum of 25 mM HEPES-TCEP buffer was used as the baseline at room temperature. The data are expressed as molar ellipticity, and the unit is deg·cm. -2 ·dmol -2 All CD data were scanned four times and the average value was taken.

[0054] In this study, Ni 2+ The concentration of the peptide is far excessive, therefore, at pH 8.5, the peptide readily self-assembles into a binuclear complex. The UV-Vis absorption spectrum of the artificial enzyme is as follows: Figure 4 As shown in Figure A, compared to the absence of Ni 2+ The polypeptide, when Ni 2+ When coexisting with the peptide in a self-assembly environment, a new strong absorption band appears at 260 nm, which is related to S. - → Ni 2+ The charge transfer results indicate that the polypeptide coordinated with the metal ions.

[0055] In addition, the circular dichroism chromatogram of artificial enzymes ( Figure 4 B) A metal ion (dd transition) appears near 550 nm, and a transition belonging to Ni appears at 320 nm. 2+The shoulder peak of the coordination complex formed with S and the negative Cotton effect at 440 nm further prove that Ni 2+ It can bind to deprotonated thiols / selenols, which is a result of the change in the properties of nickel complexes from paramagnetic to diamagnetic.

[0056] In addition, this study further used a nanoparticle size analyzer to determine the particle size of the artificial enzyme at a sample concentration of 1 mg / mL.

[0057] To further analyze the structural stability of the artificial enzyme, after the sample preparation was completed, the sample was removed from the anaerobic environment and exposed to oxygen conditions for 7 days. Then, its UV-Vis absorption spectrum, circular dichroism spectrum, and particle size were measured again.

[0058] A comparison of particle size results of artificial enzymes after preparation and after 7 days of aerobic storage is shown below. Figure 5 As shown, after self-assembly, the particle size of the peptides is concentrated around 1.2 nm, which is consistent with that of compact monomeric peptides. This indicates that the peptides do not oxidize each other during self-assembly to form disulfide bonds or selenium-sulfur bonds, thus avoiding the formation of high molecular weight polymers. After storage for seven days under conditions of simultaneous oxygen and carbon dioxide, the particle size did not change significantly and remained around 1.2 nm, as shown in the UV-Vis absorption spectrum. Figure 4 A) and circular dichroism spectrum ( Figure 4 B) also showed that its structure did not change significantly, indicating that the structure of the artificial enzyme not only has high stability, but also exhibits a certain degree of oxygen resistance, which was further verified in subsequent photocatalysis experiments.

[0059] 2.2 EPR characterization of artificial enzymes Electron paramagnetic resonance (EPR) was used to characterize the coordination environment, electronic structure, and oxidation state of the metal center in order to further confirm the formation of the bimetallic center and the characteristics of its electron distribution.

[0060] The experimental procedure is as follows: (1) All sample preparations were carried out in an anaerobic chamber containing a nitrogen (95%) / hydrogen (5%) gas mixture.

[0061] (2) 100 μL of enzyme solution and 1 μL of 500mM sodium bicarbonate were incubated together at room temperature for 20 minutes; (3) Add 100 μL of Hepes / glycerol buffer; (4) Then add 2 μL of 15 mM EuCl2 and 2 μL of 15 mM diethylenetriaminepentaacetic acid (DTPA) in sequence. After this step is completed, mix quickly and transfer the solution to an EPR tube and freeze it in liquid nitrogen for about 30 seconds between mixing and freezing. (5) After sealing with a rubber cap, add the contents and remove them from the glove box. Store them in liquid nitrogen until the experiment is completed.

[0062] (6) The test was conducted using a Bruker-A300 electron paramagnetic resonance spectrometer with a microwave frequency of 9.496 GHz, a microwave power of 200 μW, a modulation amplitude of 1 mT, and a temperature of 77 K.

[0063] EPR results further reflect the presence of Ni in artificial enzymes. 2+ The position of Ni in the artificial enzyme structure. 2+ Both are in an oxidized state and do not possess EPR activity. Therefore, this study added the strong reducing agent europium diethylenetriaminepentaacetic acid and the small ligand bicarbonate to adjust the pH to 8 in order to capture the Ni reduced by a single electron. 1+ Reduced Ni 1+ Presenting d 9 Electronic configuration, unpaired electrons preferentially located in d x2-y2 Both nickel sites in the artificial enzyme exhibit this coordination property on the orbital plane. For example... Figure 6 As shown, all artificial enzymes Ni 1+ The g-factors were similar, around 2.27, 2.08, and 2.05, indicating that Ni in the artificial enzyme was relatively stable. 1+ The coordination is in a distorted octahedral or square planar configuration, similar to the g values ​​(2.26 to 2.3, 2.11 to 2.12, 2.05) detected by [NiFe] hydrogenase in the single-electron reduction of the "Ni-L" state, and Ni 1+ The EPR characteristic ACS proximal sites also share certain similarities, indicating that the artificial enzyme constructed in this study has a certain structural homology with these two enzymes, further demonstrating the similarity between the peptide and Ni. 2+ Coordination was completed, forming a special secondary structure.

[0064] Based on the above results and the properties of the peptides in this study, the five peptides we synthesized formed similar secondary structures in a self-assembly environment. Their polypeptide backbones could provide two strongly coordinating main-chain amide groups and two side-chain thiols or selenols with Ni. 2+ Coordination occurs. For example... Figure 7 As shown, taking peptide Sec-1 as an example, Ni 2+ Induces deprotonation of the amide groups at positions 4 (Gly-4) and 5 (Cys-5), the latter acting as Ni. 2+ The anchoring site binds to it, and the thiol groups of Cys residues at positions 3 (Cys-3) and 5 (Cys-5) act as bridging ligands with Ni. 2+The selenool at position 1 (Sec-1), the carbonyl group at position 11 (Arg-11), and the thiol group at position 12 (Cys-12) further bind with another Ni. 2+ The coordination forms a polypeptide-nickel complex with a square geometry and a binuclear coordination center.

[0065] 2.3 Characterization of photocatalytic hydrogen evolution of artificial enzymes To further test the photocatalytic hydrogen evolution characteristics of the artificial enzyme, we further placed the photosensitizer EosinY and the sacrificial electron donor triethanolamine in the same system with the above-mentioned polypeptide-nickel complex to form a photocatalytic solution.

[0066] Take 1 mL of photocatalytic solution at different pH values ​​and add it to a 4 mL vial. Seal the vial with a 10 mm thick rubber stopper and sealing tape. Before illumination, if it is an oxygen-free test, purge the sample with nitrogen for at least 20 minutes to completely remove oxygen; if it is an aerobic test, deoxygenation is not necessary. During illumination, a 300 W xenon lamp was selected to simulate sunlight. The sample was placed 10 cm away from the light source, and the illumination time was 120 minutes. Eosin Y was used as the photosensitizer, and triethanolamine was used as the sacrificial reagent for the photocatalytic reaction. High-purity helium was used as the carrier gas. The generated hydrogen was periodically analyzed by gas chromatography. The entire process was carried out at a constant temperature of 25°C.

[0067] The results are as follows Figure 8 As shown, Ni 2+ As an inorganic catalyst, it possesses certain catalytic performance, capable of catalyzing the formation of 0.2 μmol of H2 within 2 hours, with a turnover number (TON) of 20. However, as a metal ion, its catalytic activity is limited, showing a significant difference in catalytic efficiency compared to artificial enzymes. The unassembled polypeptide does not exhibit enzymatic catalysis; after 2 hours of reaction, its hydrogen production is similar to the blank group, with only the polypeptide reacting with Ni. 2+After coordination to form a special structure, the self-assembled peptide can reduce the activation energy of the reaction. Furthermore, introducing Se into the peptide sequence not only does not affect the peptide folding and formation of secondary structures, but also exhibits superior enzyme catalytic performance. Sec-12 has a TON of 593.6, while NI has a TON of 442.9, showing a significant difference. Sec-12's catalytic efficiency is increased by 34%, indicating that introducing Se atoms into the peptide's secondary structure is beneficial for improving enzymatic efficiency. Although the constructed artificial enzyme is small in size, it has a higher TON than similar Ni-mimicry enzymes. Ashlee E et al.'s nickel-substituted erydorepinephrine has a TON of only 0.4, while Chakraborty et al.'s Ni-based artificial hydrogenase designed based on Cu storage protein (Csp1) has a TON of 115. This indicates that the [NiFeSe] hydrogenase-based peptide artificial enzyme designed in this study has superior photocatalytic hydrogen evolution activity.

[0068] To obtain the optimal enzyme catalytic efficiency, the hydrogen evolution efficiency of the artificial enzyme was first tested under aerobic conditions at pH 4, 7, and 9, corresponding to acidic, neutral, and alkaline catalytic environments, respectively. The results are as follows: Figure 9 As shown, the artificial enzymes were able to complete photocatalytic hydrogen evolution under aerobic conditions in different pH environments, demonstrating their broad adaptability. The catalytic efficiency increased with increasing pH. At pH=9, artificial enzymes with different sequences exhibited significantly increased hydrogen evolution activity, indicating that the alkaline environment corresponding to pH=9 is conducive to photocatalytic hydrogen evolution. At pH=9, the catalytic properties of different artificial enzymes showed differences. NI without selenocysteine ​​showed the lowest catalytic efficiency, with a TON of only 31, while Sec-1 exhibited the best catalytic effect, forming 1.2 μmol of hydrogen gas within 2 hours, with a TON of 120. Compared to the NI artificial enzyme without Se, the catalytic efficiency of Sec-1 was 3.9 times higher than that of NI, indicating that introducing Se atoms into the catalytic center can significantly optimize the antioxidant properties and catalytic ability of the artificial enzyme.

[0069] To further investigate the catalytic stability of the artificial enzyme under oxygen conditions, the same batch of artificial enzymes was divided into two parts. One part was stored in nitrogen, and the other part was exposed to air for 7 days. After 7 days, the photocatalytic activity of the artificial enzymes under the two different treatments was tested simultaneously at pH=9. The results are as follows: Figure 10 The results showed that after seven days of storage under oxygen and carbon dioxide conditions, the catalytic efficiency of different artificial enzymes decreased slightly, but there was no significant change. Sec-1 still had the best hydrogen evolution efficiency, which corresponds to the previous circular dichroism chromatogram results. That is, under oxygen conditions, not only does the structure of the artificial enzyme not change, but its catalytic efficiency also does not change significantly.

[0070] 2.4 Electrochemical characterization of artificial enzymes To further clarify the catalytic mechanism of artificial enzymes, we used protein membrane voltammetry (PFV) to investigate their electrochemical properties. During the test, the artificial enzyme, in a sub-monolayer form, covers the electrode surface, allowing for precise adjustment of the electrode potential to provide the driving force for the catalytic reaction. Direct electron transfer (ET) occurs between the electrode and the active site of the artificial enzyme. The catalytic activity can be detected by the instrument as a steady-state Faraday current, the magnitude of which is proportional to the reaction rate. The results allow us to determine the differences in reaction rates among different artificial enzymes.

[0071] The PFV was used to investigate the electron-transferring ability of different artificial enzymes. The specific experimental procedures are as follows: (1) Use nitrogen to continuously blow the electrolytic cell solution of the electrochemical cell to completely remove all oxygen and ensure that all electrochemical experiments are carried out under oxygen-free conditions; (2) The experiment was conducted using an Ivium-Vertex.C potentiostat, with the Ag / AgCl electrode as the reference electrode, the platinum mesh as the counter electrode, and the glassy carbon electrode as the working electrode; (3) For the first test, the electrode surface should be polished with 1.0 μm, 0.3 μm and 0.05 μm alumina powder / paste in sequence. After polishing each particle size of alumina, the electrode surface should be checked to see if it is uniform until the electrode surface is as smooth as a mirror. For subsequent tests, the electrode surface can be polished with 0.05 μm alumina powder / paste. After polishing, the electrode surface should be ultrasonicated with ultrapure water / anhydrous ethanol / water for 15 seconds in sequence until the alumina is completely removed. Then, the electrode surface should be air-dried at room temperature. (4) After the working electrode is polished, 2 μL of 2wt% Nafion / isopropanol solution is slowly added to the surface of the glassy carbon electrode using a micropipette. The solvent is evaporated at room temperature to obtain the desired modified electrode. In order to eliminate the memory effect of the electrode, step three needs to be repeated after each measurement. (5) Add 5 μL of artificial enzyme solution to the electrode to form an enzyme film; (6) Place the electrode into the electrolytic cell, connect the electrode clamp correctly and start the test. Purge the solution with nitrogen gas throughout the test to ensure that the test environment is oxygen-free. (7) All potentials are referenced to SHE and calculated as E(SHE) = E(Ag / AgCl) + 205 mV.

[0072] The results of PFV are as follows Figure 11As shown, the voltammetric curve for Sec-1 has the steepest slope, while that for NI has the steepest. The other artificial enzymes exhibit similar slopes. The results obtained from the electrochemical tests are consistent with the photocatalytic hydrogen evolution efficiency of the artificial enzymes at pH=9. This indicates that the difference in catalytic efficiency among artificial enzymes lies in the different electron transfer efficiencies of their sequences. Sec-1 possesses the best electron transfer efficiency, thus exhibiting the highest catalytic efficiency. Furthermore, it can be seen from... Figure 11 The observed voltammetric curves showed a distinct reduction peak but no oxidation peak, indicating that the artificial enzyme primarily catalyzes H+. + Restored to H2.

[0073] In recent years, hydrogen, as an emerging antioxidant therapy, has demonstrated significant effects in alleviating oxidative damage in various disease models, particularly showing remarkable therapeutic potential in diabetic wound repair. For example, HTON-containing photocatalytic hydrogen evolution hydrogels can efficiently evolve hydrogen in situ while consuming glucose, alleviating oxidative stress damage at the wound site and promoting diabetic wound healing. Light-driven hydrogen evolution nanoliposomes, under 660nm laser irradiation, can generate hydrogen in situ and release insulin, clearing excess ROS while lowering blood glucose at the wound site. Thus, photocatalytic hydrogen evolution hydrogels exhibit good efficacy in diabetic wound repair, demonstrating their significant potential in locally alleviating oxidative stress and promoting tissue regeneration. Building upon this foundation, future research could further develop intelligent hydrogel systems with excellent biocompatibility, encapsulating optimized Se-containing hydrogenase artificial enzymes. This would effectively isolate the artificial enzyme's activity from oxygen inhibition and enhance its stability and catalytic efficiency in complex physiological environments. Through continuous in-situ hydrogen production, it is expected to expand its precision medicine applications in chronic wound repair, inflammation regulation, and the treatment of oxidative stress-related diseases.

[0074] The above embodiments only describe a portion of the specific implementation methods of the present invention in detail, and are not limited to the embodiments disclosed herein. Furthermore, the substantive content protected by the present invention is not limited thereto. Any other modifications, equivalent substitutions, improvements, etc., made based on the principles and techniques of the present invention without departing from its design scope are all within the protection scope of the present invention.

Claims

1. A polypeptide for the self-assembly of nickel-selenium artificial hydrogenase, characterized in that, The polypeptide is selected from any one of NI, Sec-1, Sec-3, Sec-5, and Sec-12, wherein the amino acid sequence of NI is shown in SEQ ID No.1, the amino acid sequence of Sec-1 is shown in SEQ ID No.2, the amino acid sequence of Sec-3 is shown in SEQ ID No.3, the amino acid sequence of Sec-5 is shown in SEQ ID No.4, and the amino acid sequence of Sec-12 is shown in SEQ ID No.

5.

2. A polypeptide self-assembled nickel-selenium artificial hydrogenase, characterized in that: The artificial hydrogenase is a polypeptide-nickel complex with a binuclear coordination center formed by the self-assembly reaction of a polypeptide with nickel ions in a buffer solution; the amino acid sequence of the polypeptide is selected from any sequence in SEQ ID NO. 1 to 5 of claim 1; the main chain of the polypeptide provides two strongly coordinating amide groups; and the polypeptide cysteine ​​and selenocysteine ​​residues provide two side chain thiols and / or selenools, which together with Ni 2+ Coordination occurs, forming a dual-core coordination center.

3. A method for preparing a polypeptide self-assembled nickel-selenium artificial hydrogenase, characterized in that, Includes the following steps: Step 1: Design an artificial hydrogenase polypeptide sequence, wherein the polypeptide sequence is selected from any one of SEQ ID No. 1-5 as described in claim 1; Step 2: Synthesize artificial hydrogenase polypeptides. The five polypeptides described in Step 1 are synthesized using a standard solid-phase polypeptide synthesis method. Step 3: Preparation of self-assembled artificial hydrogenase. The five peptides described in Step 2 undergo self-assembly reactions with nickel ions in a buffer solution to form peptide-nickel complexes with binuclear coordination centers. The main chain of the peptide provides two strongly coordinating amide groups, and the cysteine ​​and selenocysteine ​​residues of the peptide provide two side-chain thiols and / or selenools, which, together with Ni... 2+ Coordination occurs, forming a dual-core coordination center.

4. The method for preparing the polypeptide self-assembled nickel-selenium artificial hydrogenase according to claim 3, characterized in that, The standard solid-phase polypeptide synthesis method described in step 2 uses Wang-resin as the solid-phase support.

5. The method for preparing the polypeptide self-assembled nickel-selenium artificial hydrogenase according to claim 3, characterized in that, The buffer solution in step 3 contains 25 mM HEPES, 100 mM NaCl and 3.75 mM TCEP, and the pH value of the buffer solution is 8.

5.

6. The method for preparing the polypeptide self-assembled nickel-selenium artificial hydrogenase according to claim 3, characterized in that, The final concentration of the polypeptide in step 3 is 0.75 mM, and the nickel ions are from the NiCl2 solution with a final concentration of 1.65 mM.

7. The method for preparing the polypeptide self-assembled nickel-selenium artificial hydrogenase according to claim 3, characterized in that, The self-assembly reaction time described in step 3 is 2 hours at 50°C and 48 hours at room temperature.

8. The application of the polypeptide self-assembled nickel-selenium artificial hydrogenase of claim 2 in hydrogen production, characterized in that, The photosensitizer and the sacrificial electron donor coexist with the nickel-selenium artificial hydrogenase self-assembled by the polypeptide in the same reaction system, and synergistically achieve the catalytic hydrogen evolution of protons in aqueous solution under visible light irradiation.

9. The application of the polypeptide self-assembled nickel-selenium artificial hydrogenase according to claim 8 in hydrogen production, characterized in that, The photosensitizer is Eosin Y, and the sacrificial electron donor is triethanolamine.

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