Supramolecular Assembly of Photosystem II and Adenosine

Jan 23, 2018 - ... external environment and perform many highly complex functions. ... the proton gradient that can drive ATP synthase to produce ATP...
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Supramolecular Assembly of Photosystem II and Adenosine Triphosphate Synthase in Artificially Designed Honeycomb Multilayers for Photophosphorylation Yue Li, Jinbo Fei, Guangle Li, Haiming Xie, Yang Yang, Jieling Li, Youqian Xu, Bingbing Sun, Jiarui Xia, Xueqi Fu, and Junbai Li ACS Nano, Just Accepted Manuscript • Publication Date (Web): 23 Jan 2018 Downloaded from http://pubs.acs.org on January 23, 2018

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Supramolecular Assembly of Photosystem II and Adenosine Triphosphate Synthase in Artificially Designed Honeycomb Multilayers for Photophosphorylation Yue Li, #,

§, ⊥





§

Jinbo Fei, # Guangle Li, #, Haiming Xie, * Yang Yang, * Jieling Li, #

Youqian Xu, #, ║Bingbing Sun, #, ║ Jiarui Xia, #, ║ Xueqi Fu, ⊥ and Junbai Li, * #, ║ #

Beijing National Laboratory for Molecular Sciences, CAS Key Laboratory of Colloid, Interface

and Chemical Thermodynamics, Institute of Chemistry, Chinese Academy of Sciences, Beijing 100190, China. §

CAS Key Laboratory for Biomedical Effects of Nanomaterials and Nanosafety, National

Center for Nanoscience and Technology, Beijing, 100190, China. ‡

National & Local United Engineering Laboratory for Power Battery Department of Chemistry

Northeast Normal University, Changchun 130024, China. ⊥

College of Life Science, Jilin University, Changchun 130012, China.



University of Chinese Academic of Sciences, Beijing 100049, China.

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*E-mail: [email protected] *E-mail: [email protected]

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*E-mail: [email protected]

ABSTRACT: Plant thylakoid has a typical stacking structure, which is the site of photosynthesis, including light-harvesting, water-splitting and adenosine triphosphate (ATP) production. This stacking structure plays a key role in exchange of substances with extremely high efficiency and minimum energy consumption through photosynthesis. Herein we report an artificially designed honeycomb multilayer for photophosphorylation. To mimic natural thylakoid stacking structure, the multilayered Photosystem II (PSII)-ATP synthase-liposome system is fabricated via Layerby-Layer (LbL) assembly, allowing the three-dimensional distributions of PSII and ATP synthase. Under light illumination, PSII splits water into protons and generates a proton gradient for ATP synthase to produce ATP. Moreover, it is found that the ATP production is extremely associated with the numbers of PSII layers. With such a multilayer structure assembled by LbL one can better understand the mechanism of PSII and ATP synthase integrated in one system, mimicking the photosynthetic grana structure. On the other hand, such an assembled system can be considered to improve the photophosphorylation.

KEYWORDS: Layer-by-layer assembly; Biomimetic synthesis; Supramolecular assembly; Motor protein; Photophosphorylation

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Taking biological systems as an inspiration source, scientists have constructed various complicated supramolecular systems.1-4 Well-ordered structures are often considered to mimic nature’s way of constructing the supramolecular nano- or microarchitectures with special properties for specific biotechnology processes and biodevices.5-11 Especially, bioenergyproduction systems are always attractive from the environment protection point of view, and biosynthesis from photophosphorylation is of particular interest for the effective usage of the photosynthesis. In living organisms, adenosine triphosphate (ATP) is the major energy source to satisfy the energy consuming processes.12-13 ATP can be generated through oxidative phosphorylation and photophosphorylation by proton gradient-driven ATP synthase across the phospholipid membrane.14 Photosystem II (PSII) is the only protein complex with a capability of oxidizing water to run the photophosphorylation that occurs in the thylakoid membrane.15-16 It is also the reaction center of photosynthesis performed with light absorption, water splitting, electron/proton transfer to provide the separated electron (e-) and proton (H+).17-19 Upon light absorption, the produced proton gradients will enable ATP synthase to rotate and catalyze the synthesis of ATP.12 Natural systems with hierarchical structures have the capability to adapt to their external environment and perform many highly complex functions.4 A typical stacking structure of thylakoid grana plays a key role in exchange of substances with extremely high efficiency and minimum energy consumption through photosynthesis.20 As shown in Figure 1 (a), PSII resides mainly in the grana membranes, while ATP synthase resides predominantly outside and the cytochrome b6/f (cyt b6f) complex is distributed approximately evenly between the grana thylakoid and the lamellae thylakoid, where excitation energy is consumed to generate reducing equivalents through photosynthetic electron transport, driving ATP synthesis.20-21 Such a

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distribution of PSII and ATP synthase in the thylakoid can diminish the diffusion of PSII complexes in the grana stacks, meanwhile increasing the proton gradient which can drive ATP synthase to produce ATP.22 In this article, we design and prepare a bio-inspired, supramolecular assembled artificial thylakoid with space-filling multilayered honeycomb substrate. The hierarchically well-defined honeycomb cellulose acetate (CA) is employed as the transparent flexible hydrophilic substrate by using breath figure.23-26 Through simple and efficient layer-by-layer (LbL) assembly technique, driven by electrostatic interaction, molecules with charges can be co-assembled.27-30 A polyethylenimine (PEI) layer is firstly deposited on the CA film, providing a positively charged surface to which the negatively charged PSII adsorbs through electrostatic attraction.

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Subsequent alternating adsorption of PEI and PSII leads to the build-up of a multilayer to mimic the natural thylakoid multilayer structure (Figure 1(a)).20 The multilayer structure can be written as CA/(PEI-PSII)n. To further mimic the natural ATP synthase spacial distribution in thylakoid, a reconstituted ATP synthase-liposome membrane is deposited outside of the constructed multilayer by LbL technique.32 Furthermore, [Fe(CN)6]3- as an electron acceptor2, 33, is employed to transfer separated electron mimicking the function of cyt b6f complexes in natural systems.34 Thus, our light-driven systems provide energy conversion from water to ATP by simple remote control of light on/off. This compartmentalization and integration strategy opens an interesting way for co-assembly of multifunctional units, mimicking complex biochemical processes.

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Figure 1. (a) Natural plant cell, chloroplast, thylakoid stacking structure, and the model of the plant thylakoid membrane with dimensions of the main protein components. (b) Mechanism of fabrication process of cellulose acetate (CA) honeycomb membrane by breath figure, and schematic illustration of the co-assembly of PSII and ATP synthase-liposome through layer-bylayer assembly.

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RESULTS AND DISCUSSION In nature, cellulose, as the main structural component in the cell wall, is usually coated with tightly bound proteins and polysaccharides.35 Cellulose acetate (CA) is the acetate ester of cellulose. It is widely used in biochemical processes due to its high degree of hydrophilicity, high biocompatibility, and relatively low cost36. So we take the porous CA film as a substrate by breath figure method for PSII deposition23. The procedure is illustrated in Figure 1(b). As shown in Figure 2(a,b,d,e), when a solution of 1% CA in a mixture of acetone and dichloromethane (DCM) (1:9 v/v) is applied as the solvents, a well-structured honeycomb film is formed with an average pore diameter of 1-2 µm. DCM evaporates so fast that the temperature on the surface decreases rapidly, causing water vapor to condense on the solution surface. When the droplets grow large enough, they will move downward and form new cavities (Figure 2(c)). After completing evaporation of the organic solvents and the water droplets, the surface and the interior of the films display three-dimensional hierarchical structures with a film thickness of about 4-5 µm (Figure 2(c,f,g)). The cavities provide enough space for the multilayered PSII to assemble, and mimic the natural thylakoid grana structure. In addition, the cavities are connected with each other, forming bridge structures, which mimic the thylakoid lamellae (described in Figure 1(a)). PSII and ATP synthase as key building blocks were isolated and purified from fresh spinach chloroplasts using a published procedure with minor modifications17. Sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE) data of purified PSII and ATP synthase are shown in Figure 3(a). In detail, purified ATP synthase also contains intramembrane Fo complexes (a, b subunit) and the soluble F1 part (α, β, γ, δ, and ε subunit). Isolated PSII consists of the light harvesting complexes (e.g., LHCII) and the reaction center subunits (D1, D2, CP43,

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and CP47 protein, and oxygen-evolving proteins)37-39. PSII and ATP synthase reconstituted in the proteoliposome still retain the good activity (Figure S1, S2).

Figure 2. (a) Optical image and SEM images of honeycomb CA film with a (b) lower and (d,e) higher resolution. (c) cross-section of honeycomb CA film. (h-k,n,o) SEM images of (h,i) CA/(PEI/PSII)1 (j,k) CA/(PEI/PSII)5 (n,o) CA/(PEI/PSII)5/PEI/ATP synthase-liposome. The inset is the relevant optical image. (l,m) CLSM images of depth scan of PSII-CA film excited at 559nm. The fluorescence signal originates from the combined chlorophylls of PSII. (f,g,p) AFM images of (f,g) CA honeycomb film and (p) CA/(PEI/PSII)5/PEI/ATP synthase-liposome Since PSII is a membrane-associated protein complex with multiple subunits, it is difficult to transfer PSII to a non-biological system while maintaining a high bioactivity. In this work, positively charged PEI and negatively charged PSII, both of which are biocompatible, are alternately adsorbed on the CA film to mimic natural stacking grana22 (Figure 1(b)). The deposition of PSII on the CA film is proven by confocal laser scanning microscope (CLSM) image in Figure 2(l,m). PSII is adsorbed on the bottom and on the bridges of the honeycomb.

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Furthermore, with increasing number of polyelectrolyte layers, the honeycomb holes are gradually filled up with PSII after 5 bilayers. As we know, the ATP synthase is usually located in a relatively flat membrane22. Obviously, compared with CA/(PEI/PSII)1, CA/(PEI/PSII)5 forms a flatter surface for ATP synthase-liposome to spread on (see Figure 2(h,i) and Figure 3(j,k)). Similar structures can be observed from Supplementary Figure 2(a-d). The amount of PSII can be estimated by total chlorophyll (chl) concentration in 80% acetone solution. In the calculation: total chlorophyll (µg mL-1)=(8.02×(A663-A720)+20.21×(A645-A720))×sample dilution.17 In Figure 3(b,c), with each deposition cycle of the PEI-PSII bilayer, the light absorbance increases gradually, indicating the increasing amount of PSII is deposited. PSII-CA film shows a similar peak position indicating the ability of light-harvesting is maintained on assembled film (Figure 3(d)). The inset in Figure 3(d) is the optical image of PSII-CA film immersed in water, showing the flexibility and the penetrability of it.

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Figure 3. (a) SDS-PAGE of the purified PSII and ATP synthase. (b) UV-vis spectra of CA/(PEI/PSII)n films with varying numbers of LbL depositions on CA film after sonication in water. (c) Adsorption amount of PEI-PSII multilayered films with varying numbers of LbL deposition on CA membranes. (d) UV-vis spectra of PSII dissolved in water (red line) and PSII adsorbed on the CA honeycomb film by PEI (black dash). The inset is the optical image of PSII enriched CA flexible film immersed in water. (e) Photo-reduction activities of DCPIP in the presence of PSII with varying numbers of CA/(PEI/PSII)n films. (f) Fluorescence excitation spectra of pyranine buffer from PSII-CA film immersed at different illumination times with Em=513nm. To test the activity of PSII-CA film, 2,6-dichlorophenolindophenol (DCPIP) photo-reduction assays were performed40. It is employed as an electron acceptor. The reduction of DCPIP demonstrates the capability of oxidizing water of PSII-CA film. Moreover, with each deposition cycle of the PEI-PSII bilayer the light absorbance increases gradually, the rate of DCPIP reduction increases significantly before 5-6 bilayers (Figure 3(e)). Protons are the products from the decomposition of water by PSII catalysis. To detect the protons, we employ a pH-sensitive probe, 8-hydroxyprene-1,3,6-trisulphonic acid (pyranine)41. For pyranine, the excitation wavelength of 460 nm will decrease with the reduction of pH (emission (Em) wavelength=513 nm). The generation of acid induced by PSII is monitored with time (Fig. 3(f)). It shows that with increasing time the intensity at 466 nm decreased dramatically, demonstrating that the environment with light sensitive PSII is becoming more acidic, and further illustrating the ability of PSII to split water. The same result can also be obtained by another fluorescence assay in Figure S3, which shows the fluorescent images of the PSII-CA film after being mixed with 10 µM pyranine probe. Light from 635nm excitation (Ex)

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wavelength is used to trigger PSII to decompose water and generate protons. It shows that the surrounding of the membrane is becoming brighter and brighter, implying that local proton gradients increased with the increased illumination time. Based on the naturally specific structure that the ATP synthase residing predominantly outside of PSII, an artificial system of PSII multilayers covered with ATP synthase is structured. As shown in Figure 2(n,o,p) and Figure S4, ATP synthase-proteoliposome is reconstituted and deposited on the relatively flat PSII-CA film to form a sticky film. To further prove the adsorption of ATP synthase-liposome outside of PSII multilayers, Rhodamine B-labeled ATP synthase-liposome is added as a fluorescence probe to make liposomes visible by CLSM. Figure S5 shows that green signal (only from PSII) does not match with the red signal (from PSII and ATP synthase-liposome) at the same depth, implying that Rhodamine B-labeled ATP synthaseliposome is deposited outside of PSII successfully.

Figure 4. (a) Schematic illustration of the artificial designed system in this study. (b) Photoinduced ATP synthesis (nM (mg Chl-1)) of CA/(PEI/PSII)n/PEI/ATP synthase-liposome films.

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Count were measured by a bioluminescence assay (ENLITEN ATP Assay System, Promega, USA) using a BPCL ultra weak luminescence analyzer. (c) Photo-induced ATP synthesis (nM cm-2) of different bio-devices. (d) Redox potentials and the electron transfer process in artificial integrated system. (e) ATP synthesis rate of CA/(PEI/PSII)n/PEI/ATP synthase-liposome film. (f) Photo-induced ATP synthesis of CA/(PEI/PSII)n/PEI/ATP synthase-liposome through on−off cycles of light. Insets are the relevant optical images of CA/PEI/(PEI/PSII)5/PEI/ATP synthaseliposome film without/ with illumination (550-800 nm). All error bars refer to standard deviation (n = 3). Figure 4(a) shows that the mechanism illustration of our artificial thylakoid. The ATP concentration is measured by the luciferin and luciferase assay17, 40. As shown in Figure 4, the ATP production distinctly increases with increasing time of light irradiation. In particular, with each deposition cycle of the PEI-PSII bilayer the ATP production increased gradually. During the first to fifth deposition cycle, the mesosubstrate provides a high enough space to integrate PSII and maintain property proton for photophosphorylation (Figure 2,3). Gradually, honeycombs are filled up with PSII, and less bilayers are deposited on the planar surface. The ATP production distinctly increases steadily with increasing time of light irradiation over 700 nM ATP cm-2 or over 70000 nM ATP (mg Ch-1) for 7 bilayers (Figure 4b,4c), while it is nearly unchanged compared with 5 bilayers. The trend of the changes in residual [Fe(CN)6]3- (see Figure S6) matches with the production of ATP (Figure 4(c)). In addition, compared with 5 bilayers, with 7 bilayers has lower ATP production with the statistics of unit chlorophyll of PSII, implying bilayers more than 5 cannot increase the output in this system (Figure 4(b)), and the sample with 5 bilayers has the most effective structure. We hypothesize that with 5 bilayers the cavities are filled fully enough and there is no excessive stacking outside. Furthermore, under

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illumination for 40 min, the rate of ATP production becomes much lower and reaches a plateau after about 60 min (Figure 4(e)). This might be due to the loss of PSII activity after long irradiation time. It can also be considered as a light-driven film in one hour (Figure 4(f)). It serves as a proof of principle that PSII and ATP synthase can be integrated into one artificially designed system for ATP production. In order to illustrate the special benefit of honeycomb film device, we have added a series of contrast experiments. It can be seen from Figure S7 in Supporting Information, a simple LbL device can also generate ATP, however not as much as honeycomb-LbL device performs (Figure S7(b)). An additional data in Figure S7(a) shows the adsorption of PSII in honeycomb-LbL film is much higher. Other than the higher adsorption capability, honeycomb-LbL film still performs better (Figure S7(c)), which may mean an ordered honeycomb structure is essential for the proton storage and further for the ATP generation. An additional experiment is shown in supporting information to compare two common electron acceptor, 2,5-dichloro-1,4-benzoquinone (DCBQ) and ferricyanide. As shown in Figure S8, both of them exhibit similar results in this system. To further prove that the increase of ATP concentration results from the proton gradientcoupled ATP synthesis, we introduced an uncoupler (carbonyl cyanide-mchlorophenylhydrazone, CCCP) as a control experiment. CCCP can dissipate the H+ gradient generated on the two sides of the membrane and thus inhibit phosphorylation.17 As shown in Fig. 4(c), when CCCP is added to the system, no ATP is produced. When the integrated PSII-ATP synthase system is incubated with DCMU (PSII inhibitor)17 for one hour before light exposure, the ATP synthesis is effectively suppressed (Fig. 4(c)). Moreover, other control experiments in which either ATP synthase or PSII is absent shows the same results. All these results demonstrate that the process of ATP synthesis is primarily attributed to light-induced proton

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gradient-linked rotary catalysis of ATP synthase and occurs only in the presence of all components. Such a light-sensitive CA/(PEI/PSII)n/PEI/ATP synthase-liposome system mimics the stacking structure of thylakoid grana for photophosphorylation. CONCLUSIONS In conclusion, our work describes a bio-inspired artificial thylakoid containing PSII which can provide a proton gradient for the ATP effective synthesis with ATP synthase-liposome membrane. Such a bio-inspired artificial thylakoid is designed to mimic natural thylakoid structure and the three-dimensional distributions of protein complexes. The well-ordered CA honeycomb provides a high surface area to integrate PSII and to maintain a sufficient proton gradient to drive the production of ATP by ATP synthase. With each deposition cycle of the PEIPSII bilayers, the light absorbance and the energy conversion efficiency increased gradually. We demonstrate that 5 bilayers give the optimal structure. Under light illumination, the PSII-ATP synthase co-assembled system can produce ATP with high efficiency (over 70 µM (mgChl-1)). It offers an artificial thylakoid through the integration of PSII and ATP synthase according to the natural structure and three-dimensional distributions. The procedure provides a simple and efficient strategy to assemble bioinspired system. EXPERIMENTAL SECTION Materials. Polyethylenimine (PEI, Mw∼60,000), cellulose acetate (CA, acetyl content=39.8%, average Mn∼30,000), 3-(3, 4-dichlorophenyl)-1, 1-dimethylurea (DCMU), glutaraldehyde (GA), 2,6-dichlorophenolindophenol (DCPIP) were purchased from Sigma-Aldrich. Acetone and dichloromethane (DCM) were supplied by the Shanghai Chemical Regents Co. (Shanghai, China). Dimyristoylphosphatidylcholine (DMPC) and dimyristoylphosphatidylglycerol (DMPG)

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were purchased from Avanti in powder form. Bioluminescence assay was purchased from Promega. All other chemicals were purchased from Sigma-Aldrich without further purification. Preparation of CA honeycomb films. Firstly, the predetermined CA was dissolved in a mixture of acetone and DCM (1:9 v/v) with vigorous stirring to make the precursor solution with the concentration of 1%. Then 40 µL of precursor solution was allowed to drop and cast on the glass substrate. Finally, the glass substrate was transferred into the sealed container under a preset relative humidity and temperature. The relative humidity was regulated and was set at 85 % via applying saturated potassium nitrate solution. The sealed container was placed in a water bath of 55 °C. After the solvent has evaporated completely, the CA films were taken and dried overnight to remove the remaining solvent. The CA films can be peeled off easily after immersing into water. The honeycomb structure in the polymer films were observed by a S-4800 scanning electron microscopy (SEM) and by atomic force microscopy (AFM). Reconstitution of ATP synthase and proteoliposome. Liposomes were prepared from a mixture of DMPC and DMPG (9:1 by mass). Briefly, liposomes have been added in a Triton X100 solubilized ATP synthase buffer solution (20 mM Tricine pH 8.0, 40 mM NaCl and 5 mM MgCl2), allowing the protein-detergent micelles to interact, followed by three slow removal cycles of Triton X-100 by Biobeads SM-2. This leads to the formation of ATP synthaseproteoliposome.42 LbL assembly of artificially designed system. A standard LbL assembly procedure was used to prepare the film supported on the CA substrate. The following process is at 4°C in dark. At the beginning, the CA membrane was firstly soaked in a PEI aqueous solution (2 mg mL−1 in 0.15M NaCl) for 30 min, rinsed with Milli Q water, and dipped into the PSII solution (0.5 mg mL-1 in

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20mM MES buffer PH 6.5) for another 30 min. After 30 min in the dark, the electrode was rinsed with a buffered solution (20 mM MES, pH 6.5) to remove weakly bound PSII from the surface before immersing the electrode into PEI. The surface needs to be dried between the different assembly steps. The above process was repeated to finally obtain the layer-by-layer assembled film on demand. The film was soaked into an PEI aqueous solution (2 mg mL−1 in 0.15M NaCl), immersed in the MES buffer for 15min, and then moved to the ATP synthaseliposome solution for another 30 min and finally rinsed with Milli-Q water to obtain the CA/(PEI/PSII)n/PEI/ATP synthase-liposome film. AFM, SEM and Olympus FV-1000 CLSM were used to study the layer-by-layer assembled films. Generation of proton gradient. PSII enriched CA film (CA/(PEI/PSII)n) was soaked in 10 µM pyranine (TCI, Ex=406 nm, Em=513 nm) solution(10 µM pyranine, 6 µM DCBQ, 200 µM K3[Fe(CN)6]). Laser light at 635 nm excitation wavelength was used to induce PSII for watersplitting. Experiments were carried out by reading the increase of pyranine green fluorescence (Ex=405 nm) by confocal microscopy. The software, Image J, is employed to show the statistics of the changes. Additional fluorescence assays were employed to test the change of proton concentration by a fluorescence spectrophotometer (F-4500, HITACHI). The generation of PSII induced acid during red light exposure (550 -800 nm) was monitored at different times. The PSII enriched film was soaked into pyranine buffer (10 µM pyranine, 6 µM DCBQ, 200 µM K3[Fe(CN)6]). Excitation spectra of pyranine at different reaction times were scanned from 380 to 500 nm at an emission wavelength of 513 nm. Photo-induced ATP synthesis. The CA/(PEI/PSII)n/PEI/ATP synthase-liposome film was soaked in a mixed reaction buffer solution (0.2 mM ADP, 5 mM NaH2PO4, 2.5 mM MgCl2, 2 mM K3[Fe(CN)6], 30mM NaCl, 10mM Tricine±NaOH pH 8.0). The reaction solution (500 µL)

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containing the film was equilibrated at 4oC in the dark for 30 min and then illuminated using a Xe lamp (550-800 nm). To further prove that the increase of ATP concentration results from the proton gradient-coupled ATP synthesis, we introduced an uncoupler (carbonyl cyanide-mchlorophenylhydrazone, CCCP) as a control experiment. 30 µM CCCP or 200 µM DCMU is added to the system for one hour before light exposure as the control experiments. ASSOCIATED CONTENT Supporting Information. This file includes Supplementary Method and Figure S1 to S8. The authors declare no competing financial interest. AUTHOR INFORMATION Corresponding Author * [email protected] * [email protected] * [email protected] ORCID Junbai Li: 0000-0001-9575-3125 Yang Yang: 0000-0002-1535-718X Haiming Xie: 0000-0002-7653-4071 Jinbo Fei: 0000-0002-8675-7412 Author Contributions

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Y. L., J. F., G. L., H. X., J. L., Y. X., B. S., J. X., and X. F. performed the experiments. J. L., Y. Y., H. X. and Y. L. designed the study and advised on manuscript preparation. All authors contributed to writing the paper. ACKNOWLEDGMENT The authors gratefully acknowledge the financial support for this research by the National Natural Science Foundation of China (No. 21433010, 21320102004, 21573248, 21503236 and 21673056). Y. Y. and H. X. particularly thank the Open Project of National & Local United Engineering Lab for Power Battery, Northeast Normal University (No.130017503); J. F. particularly thanks the Youth Innovation Promotion Association of CAS (No. 2016032) and Institute of Chemistry, CAS (No. Y6290512B1). We thank Prof. Shu Wang for kindly providing clark oxygen electrode. REFERENCES (1)

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