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Enhanced Bioenergy Transformation by Metal-Free Electrozyme-Based Mitochondrion Nanoarchitectonics

2022; Chinese Chemical Society; Volume: 5; Issue: 7 Linguagem: Inglês

10.31635/ccschem.022.202202066

2096-5745

Tonghui Wang, Jinbo Fei, Zhenzhen Dong, Xia Xu, Weiguang Dong, Junbai Li,

Electrochemical Analysis and Applications

Open AccessCCS ChemistryRESEARCH ARTICLES12 Aug 2022Enhanced Bioenergy Transformation by Metal-Free Electrozyme-Based Mitochondrion Nanoarchitectonics Tonghui Wang, Jinbo Fei, Zhenzhen Dong, Xia Xu, Weiguang Dong and Junbai Li Tonghui Wang Beijing National Laboratory for Molecular Sciences (BNLMS), CAS Key Lab of Colloid, Interface and Chemical Thermodynamics, Institute of Chemistry, Chinese Academy of Sciences, Beijing 100190 University of Chinese Academy of Sciences, Beijing 100049 , Jinbo Fei Beijing National Laboratory for Molecular Sciences (BNLMS), CAS Key Lab of Colloid, Interface and Chemical Thermodynamics, Institute of Chemistry, Chinese Academy of Sciences, Beijing 100190 University of Chinese Academy of Sciences, Beijing 100049 , Zhenzhen Dong Beijing National Laboratory for Molecular Sciences (BNLMS), CAS Key Lab of Colloid, Interface and Chemical Thermodynamics, Institute of Chemistry, Chinese Academy of Sciences, Beijing 100190 University of Chinese Academy of Sciences, Beijing 100049 , Xia Xu Beijing National Laboratory for Molecular Sciences (BNLMS), CAS Key Lab of Colloid, Interface and Chemical Thermodynamics, Institute of Chemistry, Chinese Academy of Sciences, Beijing 100190 University of Chinese Academy of Sciences, Beijing 100049 , Weiguang Dong Beijing National Laboratory for Molecular Sciences (BNLMS), CAS Key Lab of Colloid, Interface and Chemical Thermodynamics, Institute of Chemistry, Chinese Academy of Sciences, Beijing 100190 and Junbai Li *Corresponding author: E-mail Address: [email protected] Beijing National Laboratory for Molecular Sciences (BNLMS), CAS Key Lab of Colloid, Interface and Chemical Thermodynamics, Institute of Chemistry, Chinese Academy of Sciences, Beijing 100190 University of Chinese Academy of Sciences, Beijing 100049 https://doi.org/10.31635/ccschem.022.202202066 SectionsSupplemental MaterialAboutAbstractPDF ToolsAdd to favoritesDownload CitationsTrack Citations ShareFacebookTwitterLinked InEmail Herein, we couple a synthetic electrozyme in a supramolecule-assembled nanoarchitecture to achieve enhanced bioenergy transformation by mimicking mitochondrial oxidative phosphorylation. Different from the natural counterpart, the metal-free electrozyme is a semiconducting polymer deposited on an electrode. The well-matched electrocatalytic property of the electrozyme permits oxidization of reduced nicotinamide adenine dinucleotide (NADH) to release protons under a much lower electric potential. As a consequence, the generated proton gradient drives rotary catalysis of adenosine 5′-triphosphate (ATP) synthase reconstituted in a lipid membrane to produce ATP. Remarkably, electrochemical bioenergy conversion of NADH to ATP is accomplished with much higher efficiency in such a bio-like system compared with the natural mitochondria. This work integrates synthetic and natural catalytic chemistry to facilitate enhanced bioenergy transformation, thereby greatly improving prospects in ATP-fueled bioapplications. Download figure Download PowerPoint Introduction Supramolecular assembly is an emerging strategy to construct bio-like architectures in a bottom-up way by integrating natural and synthetic building blocks.1–8 This strategy not only provides the precise regulation for deeply understanding molecular mechanisms of biological activities, but also gives a powerful route toward enhancing biological functions.9–12 Specifically, much effort has been made to fabricate chloroplast- or mitochondrion-mimic systems with hierarchical structures by supramolecular assembly for producing the bioenergy currency adenosine 5′-triphosphate (ATP).13,14 In these systems, substrates such as glucose and water were oxidized to yield protons, and transmembrane proton gradients were established to drive ATP synthase for ATP synthesis. Moreover, abiotic chemical reactions based on self-assembly monolayer chemistry and boric ester chemistry have been developed to drive ATP production.15,16 However, ATP in mitochondria is involved with the biological conversion of energy-rich chemicals such as reduced nicotinamide adenine dinucleotide (NADH). It remains a great challenge to achieve the bioenergy transformation with high efficiency in an artificial fashion. To address this issue, we envision rationally coupling a robust synthetic catalyst with high performance in a biomimic architecture with a well-defined structure. Recently, enzyme-like nanocatalysts, termed nanozymes, have been widely explored due to their vital significance in biological applications such as biosensors, bioimaging, and biocatalysis.17–21 Outstandingly, these synthetic enzymes show obvious advantages of catalytic activities with high stability, when compared with the natural counterparts. The electrochemical oxidation of NADH has been investigated extensively by using various synthetic catalysts including carbon nanotubes,22 graphene,23 quinones,24 phenothiazines,25 and metals.26 In particular, phenothiazine derivatives have attracted much attention because of similar aromatic structures or functions to natural flavin mononucleotide, which is the primary redox cofactor for NADH oxidation in Complex I.27 Nevertheless, the catalytic bioapplications are heavily limited in biofuel cells or electrochemical analysis.28,29 It is rational to hypothesize that these electrocatalysts can serve as artificial enzymes (electrozymes) to facilitate biological transformation. In this communication, we explore supramolecular assembly on an electrode to achieve improved bioenergy transformation of NADH to ATP. As shown in Figure 1a,b, during the natural aerobic cell respiration process, NADH is oxidized through a proton-coupled electron transfer reaction with flavin mononucleotide in Complex I, and the electrons are then accepted by iron-sulfur clusters.30,31 In our design (Figure 1c,d), a phenothiazine molecule (Azure B), with a similar molecular structure to flavin mononucleotide, is electrodeposited on the surface of a glassy carbon electrode (GCE) to form the metal-free semiconducting polymer (Poly (Azure B), PAB). After that, proteoliposomes containing ATP synthase are spread through fusion to construct the mitochondrion-mimicking nanoarchitecture. In the absence of oxygen, PAB possesses NADH oxidase-like activity, enabling catalytic oxidation of NADH to produce NAD+, proton, and electron. As a consequence, a transmembrane proton gradient is established to activate ATP synthase to yield ATP from ADP and inorganic phosphate (Pi). Meanwhile, the electrons are transferred by electrode under a much lower applied voltage. To the best of our knowledge, this is the first study to realize highly efficient bioenergy transformation of NADH to ATP and simultaneous generation of two cofactors NAD+ and ATP in a single hybrid architecture. Figure 1 | (a) The structure of respiratory Complex I from Thermus thermophilus with bound NADH (PDB:3IAM, PDB = Protein Data Bank). (b) Proton generation and electron transport during NADH oxidation by Complex I in the process of mitochondria oxidative phosphorylation. (c) Schematic illustration of the biohybrid architecture on a GCE. It couples synthetic and natural enzymes to achieve bioenergy transformation of NADH to ATP by mimicking natural oxidative phosphorylation. (d) Proton generation from NADH oxidation catalyzed by PAB as the electrozyme. Rib, ribose; ADP-Rib, adenosine 5-diphosphate-ribose. Download figure Download PowerPoint Experimental Methods Chemicals and materials Azure B was purchased from Sigma-Aldrich, Burlington, Massachusetts, United States. β-Nicotinamide adenine dinucleotide, reduced disodium salt (NADH), and β-nicotinamide adenine dinucleotide (NAD+) were bought from Aladdin, Shanghai, China. Texas red-labeled 1,2-dihexadecanoyl-sn-glycero-3-phosphoet hanolamine, triethylammonium salt (Texas red-DHPE), dimyristoylphosphatidylcholine (DMPC), and dimyristoylphosphatidylglycerol (DMPG) were obtained from Avanti in powder form. All the other reagents were purchased from Sigma-Aldrich without further purification. Deionized water (18.2 MΩ·cm, Millipore) was used in all the experiments. Treatment and modification of GCE The GCE (2 cm × 2 cm) was used as the working electrode. The electrode was mechanically polished with alumina powders (1.0, 0.3, and 0.05 μm) to a mirror finish and then washed by water before use. The PAB-modified electrode was prepared by cyclic sweeping from −0.60 to +1.30 V (vs Ag/AgCl) at 100 mV s−1 in Britton–Robinson buffer solution (pH 9.7) containing 0.5 mM Azure B for 20 cycles according to a previous report.32 Subsequently, the electrode modified with PAB was washed thoroughly with water. All electrochemical experiments were performed on a three-electrode setup using an electrochemical analyzer (CHI 660E, Beijing, China) under nitrogen atmosphere at room temperature (25 °C). The Ag/AgCl reference electrode was employed, and a platinum foil was used as the counter electrode. Isolation of ATP synthase from spinach chloroplasts ATP synthase was isolated and purified from spinach chloroplasts.33 In detail, fresh leaves were picked from spinach bought at market and placed at 4 °C overnight in black plastic after washing with water. Spinach leaves (400 g) were shredded and then homogenized in a precooled blender together with 500 mL of homogenization buffer solution 1 (pH 8.0, 100 mM Tricine-NaOH, 2 mM MgCl2, 0.4 M sucrose). The leaf fragment mash was filtered through 8 layers of cotton gauze. The filtrate was centrifuged at 10,600 × gmax for 30 min to collect chloroplasts. Next, the precipitant was resuspended in 180 mL of hypotonic buffer solution 2 (pH 8.0, 10 mM Tris–HCl, 0.5 mM MgCl2) to burst the chloroplasts, stirred for 15 min, and centrifuged at 16,900 × gmax for 15 min. The precipitant was dispersed in high ionic strength buffer solution 3 (pH 8.0, 10 mM Tris–HCl, 0.5 mM, MgCl2 0.4 M sucrose, 150 mM NaCl) and centrifuged at 16,900 × gmax for 25 min to collect thylakoid membranes. The precipitant was resuspended in suspension buffer solution 4 (pH 8.0, 50 mM Tricine-NaOH, 0.2 mM MgCl2, 0.4 M sucrose) and the total volume was adjusted to about 12 mL. The chlorophyll concentration of thylakoid membranes suspension was determined and adjusted to 5 mg Chl/mL with buffer solution 4. Then dithiothreitol (DTT) powder was added to the suspension to a final concentration of 50 mM, followed by stirring until DTT was completely dissolved. Next, an equal volume of extraction buffer solution 5 (pH 8.0, 20 mM Tricine-NaOH, 5 mM MgCl2, 200 mM sucrose, 400 mM (NH4)2SO4, 50 mM DTT, 60 mM N-Octyl-β-d-glucopyranoside, 25 mM sodium cholate, 2 mM Na2ATP) was mixed and gently stirred for 30 min at 4 °C to solubilize membrane protein. Solubilized protein was separated from the membranes by centrifugation at 208,000 × gmax for 60 min. (NH4)2SO4 was gradually added to the supernatant containing ATP synthase with stirring until turbidity appeared. The precipitate was collected by centrifugation at 12,000 × gmax for 10 min and then resuspended in 5 mL of redissolution buffer solution 6 (pH 7.2, 30 mM NaH2PO4, 2 mM MgCl2, 0.5 mM Na2EDTA, 4 mM n-dodecyl-β-d-maltoside, 200 mM sucrose) to get the preliminary extract of ATP synthase. It was added into the sucrose density gradients (20, 28, 36, 44, 52, and 60% w/v) containing sucrose and buffer solution 7 (pH 7.2, 30 mM NaH2PO4, 2 mM MgCl2, 0.5 mM Na2EDTA, 4 mM n-dodecyl-β-d-maltoside) and then centrifuged at 242,000 × gmax for 14 h at 4 °C. The 44% sucrose layer rich in ATP synthase was collected and quickly frozen in liquid nitrogen. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) on 12% polyacrylamide gels was used to identify the subunits of ATP synthase. Reconstitution of ATP synthase with liposomes DMPG and DMPC (1∶9 by mass) were mixed to prepare liposomes by the thin-film dispersion method. The size of liposomes was controlled through a phospholipid extruder (Avanti, Birmingham, Alabama, United States) with a filter membrane (200 nm). Then 420 μL of liposomes (10 mg/mL) were mixed with 80 μL of ATP synthase (2.68 mg/mL), 80 μL of Triton X-100 (10%) for solubilization, and 580 μL of buffer solution (pH 8.0, 10 mM Tricine-NaOH, 20 mM NaCl, 5 mM MgCl2) at 4 °C and stirred for 1 h, followed by removal of Triton X-100 by Biobeads SM-2.34 Next, the incorporation of ATP synthase and liposomes led to the formation of proteoliposomes. The activity of ATP synthase was evaluated by using a pH jump. Spreading of ATP synthase on PAB modified electrode and evaluation of ATP synthesis Proteoliposomes were immobilized on the surface of the PAB-modified electrode by co-incubation for 1 h at room temperature.35 After removing excessive proteoliposomes that were not adsorbed on the surface with water, the electrode was immersed into an electrochemical cell (containing 10 mM Tricine-NaOH, 2.5 mM MgCl2, 5 mM NaH2PO4, 0.2 mM ADP, 10 mM NADH) as the working electrode. A continuous potential of +100 mV (vs Ag/AgCl) was applied to oxidize NADH and produce H+ under stirring. ATP production activity was analyzed by the luciferin–luciferase system (ENLITEN ATP Assay System, Promega, United States) in a biological & physical chemiluminescence (BPCL) ultra-weak luminescence analyzer (BPCL-GP15, Institute of Biophysics, Chinese Academy of Sciences). Aliquots (30 μL) were removed from the cell at certain time intervals to quantify the amount of ATP production as a function of time. Characterization Electrochemical experiments were performed on a three-electrode setup using an electrochemical analyzer (CHI 660E, Beijing, China). Atomic force microscopy (AFM) was carried out by JPK NanoWizard (Bruker, Berlin, Germany) operated in QITM mode. Scanning electronic microscopy images were obtained by a S-4800 (Hitachi, Chiyoda, Tokyo, Japan) at 10 kV. Cryo-transmission electronic microscopy (Cryo-TEM) images were collected using a Themis 300 (ThermoFisher Scientific, Waltham, Massachusetts, United States) at 300 kV. Confocal laser scanning microscopy (CLSM) images were acquired by ONOA Olympus FV500 (Shinjuku, Tokyo, Japan) using a 60× oil-immersion objective. Energy dispersive X-ray spectroscopy (EDX, INCA Oxford, England) was applied to identify the elemental ratio. X-ray photoelectron spectra (XPS) were obtained by using ESCALAB 250XiXPS (ThermoFisher Scientific, United States). Size distribution and ζ potential of liposomes and proteoliposomes were measured by a Malvin, Zetasizer Nano ZS. UV–vis spectroscopy was measured by UV-2600 spectrophotometer (Shimadzu, Kyoto, Tokyo, Japan). The ζ potential of the electrode was measured using a SurPASS 2 electrokinetic analyzer for solid surface analysis (Anton Paar, Graz, Austria). Results and Discussion In a typical experiment, the electrozyme (PAB) was fabricated by electropolymerization of Azure B (AB) on the surface of GCE. Figure 2a shows the successive cyclic voltammograms (CVs) in a scan range from −0.60 to +1.30 V (vs Ag/AgCl), revealing the formation process of PAB. In detail, there two redox peaks appearing at −0.20 and −0.41 V in the first cycle, which are assigned to the reversible redox reaction of AB (Figure 2b, I). With increasing scan cycles, a pair of new reversible redox peaks at +0.03 and −0.24 V appear. The peak currents increase gradually and the anodic peak potential shifts in the positive direction while the cathodic peak potential moves to a more negative value. This is assigned to redox reactions of polymers. Along with the cycling number increase, the PAB thin film gradually forms to change the electrical conductivity of the working electrode, leading to potential shift and current increase. The results are consistent with the previous report.36 A layer of golden yellow film was deposited on the surface of the electrode ( Supporting Information Figure S1a). In contrast, only a couple of redox peaks corresponding to the redox reactions of AB monomers are observed when the potential sweep is confined within the region of −0.60 to +0.50 V ( Supporting Information Figure S1b). It indicates that the electropolymerization of AB monomers is not initiated at the upper-limit voltage of 0.5 V. These findings confirm the construction of electrozyme with the voltage-gated feature. Consistent with the previous findings,37–41 we propose a possible structure and formation mechanism of PAB (Figure 2b, II and Supporting Information Figure S2). Figure 2 | (a) CV of 0.5 mM AB in Britton–Robinson buffer solution (pH 9.7) at 100 mV s−1 from −0.60 to +1.30 V (vs Ag/AgCl). (b) I: Reversible redox reaction of AB monomer. II: Possible structure of PAB (taking trimer as an example). (c) CV of PAB-modified GCE in a phosphate buffer solution (pH 7.4) at 20 mV s−1. (d) AFM images of bare GCE and electrozyme modified GCE surface. (e) UV–vis absorption spectra of 0.025 mM AB solution, ITO substrate coated with AB, and ITO substrate modified with electrozyme. (f) UV–vis absorption spectra of electrozyme-ITO as a function of scan cycles. Download figure Download PowerPoint When the modified electrode was put in a phosphate buffer solution (pH 7.4), two couples of redox peaks were observed in the CV experiment (Figure 2c). The peak I is attributed to redox reaction of PAB having the same redox center as AB. The peak II at more negative potential is ascribed to the monomer-type conjugation structure of AB existing in PAB.42 These results reveal the electrochemical property of PAB and further confirm the formation. As a comparison, only a couple of redox peaks appear for the electrode modified at the upper potential of +0.50 V ( Supporting Information Figure S3), which is assigned to electrochemical reaction of AB adsorbed on the electrode. In addition, the CV signal of AB monomer is much lower than that of PAB, which implies that AB monomer is adsorbed in small amounts on the electrode surface under control conditions. Therefore, electropolymerization for synthesis of the electrozyme is superior to surface adsorption. Morphological characterization of electrozyme was carried out by AFM. After modification with electrozyme, the surface roughness of the electrode is reduced (Figure 2d). It is more conducive to fusion of proteoliposomes in the following steps. Meanwhile, the results of EDX and XPS analysis ( Supporting Information Figures S4 and S5) show the existence of N and S elements from PAB, further verifying electrozyme deposited on the surface of GCE. To facilely investigate UV–vis absorption properties of electrozyme, indium-tin oxide (ITO) substrate was used to prepare electrozyme-ITO by electropolymerization under the same condition. For the AB solution, there is a main peak at 646 nm and a shoulder peak at 600 nm (Figure 2e). The latter corresponds to the dimer species of AB above a critical monomer solution.43 For the PAB film, two peaks are observed at 670 and 610 nm. Compared with the AB solution, redshifts appear, which are attributed to higher conjugation after electropolymerization. Moreover, the peak at 610 nm, ascribed to the corresponding multimer, shows relatively higher absorbance than the main peak at 670 nm. In contrast, it is difficult to observe these changes in the case of the monomer, which only exhibits a redshift due to aggregation of the monomer. These results further confirm the formation of polymers rather than aggregates. Furthermore, UV–vis absorption spectra as a function of scan cycles were recorded to monitor the electropolymerization process (Figure 2f). The result reveals the absorbances at 670 and 610 nm intensify along with the increasing scan cycle times. It means that the thickness of PAB film can be controlled by electropolymerization. Furthermore, the electrocatalytic activity of PAB as electrozyme for NADH oxidation was evaluated. As shown in Figure 3a, in the presence of NADH, there is a distinct increase in the anodic currents of peak I at +0.10 V (vs Ag/AgCl) due to more reduction of PAB on the electrode surface by NADH. Next, Figure 3b reveals much more obvious staircase responses of anodic current at +0.10 V to NADH in the chronoamperometry of PAB electrode, compared with the bare electrode. In addition, Supporting Information Figure S6 shows the oxidation overpotential of NADH is reduced by about 0.54 V after modification with PAB. These findings clearly reveal that PAB possesses a high electrocatalytic performance of NADH oxidation, which is illustrated in Figure 3c. Figure 3 | (a) CV of PAB modified GCE in the absence and presence of 5 mM NADH in phosphate buffer solution (pH 7.4) at 20 mV s−1. (b) Chronoamperometry executed at +0.10 V versus Ag/AgCl under N2 atmosphere in phosphate buffer solution (pH 7.4) upon increasing the NADH concentration in 2 mM steps. (c) Schematic illustration of NADH oxidation in bare GCE and PAB-GCE. (d) UV–vis absorption spectra of NADH at different time under +0.10 V versus Ag/AgCl and N2 atmosphere in phosphate buffer solution (pH 7.4). (e) The decrease of NADH concentration under catalytic oxidation. (f) The corresponding proton production. Download figure Download PowerPoint Quantitative NADH oxidation by the electrozyme was monitored by UV–vis absorption spectroscopy. Figure 3d shows, before the reaction, there are two absorption peaks at 340 and 260 nm. The former corresponds to the n-π* transition of dihydronicotinamide in NADH, while the latter is assigned to the π-π* transition of adenine ring. Along with the reaction time, the absorbance at 340 nm decreases while that at 260 nm increases, which indicates NADH is oxidized into NAD+, as in the previous report.44 It is noted that NAD+ has no absorbance at 340 nm ( Supporting Information Figure S7). Moreover, the absorbance at 340 nm of NADH exhibits a good linear fit with the concentration ( Supporting Information Figure S8). Thus, it is rational to choose the absorbance to investigate the quantitative concentration change of NADH during catalytic reaction (Figure 3e). To be specific, the consumption rate of NADH is around 0.27 μM·min−1. In contrast, in the absence of the electrozyme, there is no evident decrease of the NADH concentration. The consumption of NADH and generation of NAD+ and proton follow a strict stoichiometric ratio. That is, the electrochemical oxidation of a NADH is accompanied by release of a proton.45 Therefore, the corresponding proton production rate can be calculated as 0.27 μM·min−1 (Figure 3f). As a consequence, a proton gradient is generated as the driving force for ATP synthesis. After isolation and purification from spinach, the structural integrity of ATP synthase was confirmed by SDS-PAGE ( Supporting Information Figure S9a). The protein concentration was measured as 2.68 mg·mL−1 using the standard curve by the Bradford method ( Supporting Information Figure S9b). ATP synthase was then reconstituted in the liposomes composed of DMPC and DMPG to form proteoliposomes ( Supporting Information Figure S9c). Cryo-TEM was used to characterize the structure of assembled proteoliposomes. As shown in Supporting Information Figure S9d,e, white arrows highlight the lipid bilayer (5.2 nm) and a yellow arrow depicts a single ATP synthase located in the lipid bilayer with the F1 domain outward-oriented.46 Dynamic light scattering measurements were performed to reveal their size distributions ( Supporting Information Figure S9f). Importantly, Supporting Information Figure S9g shows that photon count increases after directly introducing transmembrane proton gradient, indicating ATP synthase maintains bioactivity after purification. Then, the proteoliposomes were incubated with electrozyme modified electrode to construct the bio-like system (Figure 4a). CLSM confirms the fusion and spreading of proteoliposomes on the electrode. A Texas red-labeled lipid acts as the fluorescent probe ( Supporting Information Figure S10). High density and uniform red fluorescence localizes on the region with proteoliposomes (Figure 4b). In contrast, no fluorescence appears over the untreated region. AFM images further reveal that the morphology features change after spreading of the proteoliposomes ( Supporting Information Figure S11). The increase of surface roughness is attributed to the existence of ATP synthase embedded into lipid bilayer. Furthermore, ξ potentials of proteoliposomes and PAB-GCE are −27.6 and +11.7 mV, respectively ( Supporting Information Figure S12), which demonstrates that electrostatic interaction drives the fusion of proteoliposomes on the electrozyme-modified electrode. Figure 4 | (a) Schematic illustration of construction of the natural-artificial hybrid system. (b) CLSM image of the functional electrode surface after adsorption of proteoliposomes labeled with Texas Red, excited at 559 nm. (c) Time-dependent ATP production with and without electrozyme. Download figure Download PowerPoint The ATP production in such assembled architecture was measured by using the luciferin–luciferase ATP kit.47 As shown in Figure 4c, ATP is synthesized steadily over time in the presence of electrozyme at +0.10 V (vs Ag/AgCl). In detail, ATP production has a relatively rapid growth at the early stage and reaches a stable value of 320 nM at 140 min. More intuitively, the average synthesis rate reaches the maximum value of 5.16 nM·min−1 at 20 min. Considering the total amount of ATP synthase on the electrode, the corresponding oxidative phosphorylation efficiency is calculated as 215 nmol ATP·min−1·mg−1, higher than those in natural mitochondria ( Supporting Information Table S1).48,49 Then, it decreases due to plausible dissociation of the bio-like architecture during the long-term electrochemical reaction. As a comparison, under the same conditions, ATP production is hardly detected in the absence of electrozyme. Furthermore, it should be noted that other phenazine and phenoxazine molecules are potential candidates with similar properties for electrocatalytic oxidation of NADH.50 The relevant chemical structures are shown in Supporting Information Figure S13. Thus, the strategy could be extended to mimic oxidative phosphorylation for bioenergy transformation by constructing synthetic electrozyme. Conclusion We constructed a mitochondrion-like complex through supramolecular assembly to achieve bioenergy transformation by mimicking oxidative phosphorylation occurring in mitochondria. The metal-free electrozyme possesses similar chemical structure to the active center in natural NADH oxidase, which enables NADH oxidation with high efficiency at a lower applied voltage. Subsequently, proton is generated to establish a transmembrane proton gradient in the assembled architecture. As a result, ATP was produced through rotary catalysis of ATP synthase with an average rate up to 215 nmol·min−1·mg−1, higher than those from natural mitochondria. The highly efficient bioenergy transformation of NADH to ATP was achieved by integrating synthetic and natural enzymes. The key cofactors NAD+ and ATP were coupled in single hybrid architecture, which holds great promise in wide applications such as biocatalysis, biosensors, and biosynthesis. Supporting Information Supporting Information is available and includes data referred to in the paper ( Figures S1–S13 and Table S1). Conflict of Interest There is no conflict of interest to report. Funding Information This work was supported by the National Natural Science Foundation of China (grant nos. 221930301, 21961142022, 22072160, and 21872150). J.F. particularly thanks to Institute of Chemistry, CAS (grant no. Y6290512B1). References 1. Kashiwagi D.; Shen H. 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