Coassembly of Photosystem II and ATPase as Artificial Chloroplast for

Coassembly of Photosystem II and ATPase as Artificial Chloroplast for Light-Driven ATP Synthesis Xiyun Feng, Yi Jia, Peng Cai, Jinbo Fei, and Junbai Li* 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, China S Supporting Information *

ABSTRACT: Adenosine triphosphate (ATP) is one of the most important energy sources in living cells, which can drive serial key biochemical processes. However, generation of a proton gradient for ATP production in an artificial way poses a great challenge. In nature, photophosphorylation occurring in chloroplasts is an ideal prototype of ATP production. In this paper we imitate the light-toATP conversion process occurring in the thylakoid membrane by construction of FoF1-ATPase proteoliposome-coated PSII-based microspheres with well-defined core@shell structures using molecular assembly. Under light illumination, PSII can split water into protons, oxygen, and electrons and can generate a proton gradient for ATPase to produce ATP. Thus, an artificially designed chloroplast for PSII-driven ATP synthesis is realized. This biomimetic system will help to understand the photophosphorylation process and may facilitate the development of ATP-driven devices by remote light control. KEYWORDS: photosystem II, ATP synthase, photosynthesis, biomimetic synthesis, coassembly

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proton gradient for ATPase to rotate and catalyze the synthesis of ATP.27,28 Compared with other photoactive molecules, PSII can be immobilized on functional modified surfaces or encapsulated in reticular sol−gel cavities by covalent or noncovalent interactions.29−35 Herein, inspired by the natural photosynthetic chain in chloroplasts, we construct a novel FoF1-ATPase proteoliposomecoated PSII-based microsphere with a core@shell structure by molecular assembly to imitate the light-to-ATP conversion process occurring in the thylakoid membrane, as shown in Scheme 1A. In detail, hydrogel-like PSII-based microspheres are prepared through coprecipitation of bovine serum albumin (BSA), PSII, and calcium carbonate (CaCO3), followed by glutaric dialdehyde (GA) cross-linking and subsequent removal of the CaCO3 core. Then FoF1-ATPase proteoliposomes are coated on the surface of microspheres mentioned above to build up a simple chloroplast-like light-induced artificial photophosphorylation system. Under red light irradiation, PSII entrapped in the hydrogel-like solid microsphere splits water into protons, which sequentially prompt the rotational catalysis

onstruction of artificial protosubcells or protocells is important to better understand complex biological pathways and design functional biomimetic systems.1−8 In living organisms, adenosine triphosphate (ATP) as the major energy source powers most of the energy consuming processes and regulates many biological pathways. In general, ATP is generated through oxidative phosphorylation and photophosphorylation by proton gradient-driven ATP synthase (FoF1ATPase).9−11 Up to now, much attention has been paid on ensembles of ATPase-based entities by reconstitution of subcellular functional units within synthetic constructs, such as proteoliposomes,12,13 polymersomes,14 and protein or polymerbased microcapsules.15−17 An artificial proton gradient in those systems has been generated by direct pH jump,18 enzymeinduced reaction,19 and light induced proton transfer by transmembrane photoactive bacteriorhodopsin,20−22 calibrated chromatophores,23 or photosensitive artificial porphyrin antennas.24 Light-driven systems provide energy supply by simple remote control of light on/off. Photosystem II (PSII), the only protein complex with the capability of oxidizing water, is driving photophosphorylation in plants. Capturing visible and near-infrared sunlight, PSII drives water splitting into protons, electrons, and oxygen at the Mn4CaO5 cluster.25,26 Protons released and translocated by coupling with the electron-transport chain contribute to the © 2015 American Chemical Society

Received: September 4, 2015 Accepted: November 30, 2015 Published: November 30, 2015 556

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Scheme 1. Schematic Representation of Light-Driven ATP Synthesis: (A) Coassembly of CFoF1-ATPase and PSII; (B) Charge Separation, Electron Transport (Dotted Arrows), Water-Splitting, and Proton Flow in the PSII-Based Microsphere System;a (C) Redox Potential Scheme of the Integrated System Involved in the Electron-Transfer Processes

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In PSII, solar energy captured by the light-harvesting complex (LHC) leads to a series of charge separation and electron-transfer processes. Proton (H+) release and O2 evolution by water oxidation occurs at a Mn4CaO5 cluster. It should be noted that only if F1 subunit of ATPase is in the centrifugal direction (inner-to-outer) toward the exterior solution, ATP synthesis could be possible.

Figure 1. (A) SDS-PAGE of the purified CFoF1 and PSII complex (BBY). The protein standards are indicated on the left side. (B) DCPIP photoreduction activities of PSII under different conditions: freshly prepared PSII, (B inset) PSII stored after 5 days at 4 °C in dark. Betaine helps to maintain the activity of PSII. Heat and inhibitor (DCMU) treatment are shown as control of inactive PSII. (C) DCPIP photoreduction activities of Mn-depleted PSII. Tris and NH2OH treatment could destroy oxygen evolution complex and Mn cluster of PSII, respectively, which greatly affect electron transport of water oxidation. (D) SEM images of PSII/BSA-loaded CaCO3 particles. (E) TEM images of cross-section of PSII/BSA loaded CaCO3 particles. (F) SEM images of PSII/BSA microspheres after the removal of CaCO3. The insets in (D-F) are images with higher magnification. (G) photoreduction activities of DCPIP in the presence of PSII microspheres before (blue) and after (red) light illumination, the inset is a time-dependent measurement. (H) Action spectra of PSII entrapped in PSII/BSA microspheres.

RESULTS AND DISCUSSION

of FoF1-ATPase proteoliposomes on the shell (Scheme 1B and 1C). By decoupling the natural photophosphorylation pathway, this minimal form of organelle-sized bioreactor makes it possible to bypass cytochrome b6f and photosystem I. This compartmentalization and integration strategy will open a new way for coassembly of multifunctional units and mimicking complex biochemical processes.

PSII and chloroplast FoF1-ATPase (CFoF1-ATPase) as key building blocks were isolated and purified from fresh spinach chloroplasts with minor modifications of reported methods.18,35 Sodium dodecyl sulfate-polyacrylamide gel electrophoresis data (SDS-PAGE) of purified PSII and CFoF1-ATPase are shown in Figure 1A. In detail, isolated PSII consists of the light harvesting 557

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Figure 2. Optical images of PSII-based microspheres. (A, B) CLSM images of PSII microspheres excited at 488 and 559 nm, respectively. The fluorescence signal originates from the combined chlorophylls of PSII. (C) Overlapping of A and B. (D−F) Fluorescence spectra of microspheres at different randomly selected positions obtained by CLSM lambda scanning. (D) PSII complex, (E) BSA microspheres, (F) PSII-based microspheres. There are clear peaks in the range of 650−680 nm in the fluorescence spectra of PSII- and PSII-based microspheres, which can be attributed to the characteristic fluorescence of PSII. (G and H) CLSM images of PSII microspheres modified by Texas red-labeled proteoliposomes excited at 488 and 559 nm, respectively. The fluorescence signal is contributed by the chlorophylls in PSII and Texas-red labered lipid. When excited at 559 nm, the fluorescence intensity of chlorophylls is much lower than the intensity of the fluorescence probe Texas red (about 1/4). (I) Overlapping of G and H. The insets of (A, B, G, and H) are the distributions of the fluorescence intensity along the line indicated in the confocal image.

diameter of about 2−3 μm. From the inset images, one can see that a single microsphere is composed of smaller spheres with diameters of about 50 nm. Its surface is rough with a porous internal structure. Transmission electron microscopy (TEM) images in Figure 1E also reveal cross-sectional features with porosity in good agreement with SEM results. The introduction of GA can help to build a stable reticulate structure inside the porous microspheres by the formation of Schiff’s base covalent bonds between BSA and PSII. To obtain a higher loading and surface enrichment, additional PSII is assembled on the outer shell of the PSII-loaded particles by covalent cross-linking with GA. After removal of the CaCO3 template, the diameter of the PSII microspheres decreases to some degree, as revealed in Figure 1F. The reason could be the collapse of the particles upon drying of BSA-PSII-GA microspheres. More importantly, the microsphere surface becomes much smoother, which facilitates the following liposome modification. In addition, the obtained PSII hybrid microspheres show the well-maintained reticular structure of the CaCO3 template (Figure S5), which is expected to be an ideal scaffold for migration and diffusion of photoinduced active species.34,38 For ATP production, the preservation of PSII activity is of crucial importance. To test the activity of PSII in PSII microspheres, DCPIP photoreduction assays were performed. Under red light illumination, the absorption of DCPIP at 600 nm

complexes (e.g., LHCII) and the reaction center subunits (D1, D2, CP43, and CP47 protein, and three oxygen-evolving proteins). Purified CFoF1-ATPase also contains intramembrane Fo complexes (a, b, and c subunit) and the soluble F1 part (α, β, γ, δ, and ε subunit). The quinone analogue 2,6-dichlorophenolindophenol (DCPIP) is a well-known artificial electron acceptor, which can be reduced by PSII in light.34,36 DCPIP photoreduction assays were carried out to measure charge separation and electron transport. The concentration of reduced DCPIP is displayed as a function of illumination time. The rapid decrease of absorbance shown in Figure 1B indicates that the obtained PSII retains a high photoexcited electron generation bioactivity. Moreover, when Mn-depleted PSII are introduced into the system, a large part of this activity is diminished (Figure 1C). These results demonstrate that freshly prepared PSII has a well-maintained Mn-cluster and O2 evolution properties (Figure S1−S4 and Table S1). Since PSII is a membrane-associated protein complex with multisubunits, it is difficult to encapsulate PSII into a nonbiological system maintaining a high bioactivity.31 In this work, by simple coprecipitation, PSII-based microspheres with a high loading capacity were fabricated through a GA covalentcross-linking bioconjugate technique.37,38 Scanning electron microscopy (SEM) images in Figure 1D show the typical morphology of PSII-BSA-CaCO3 hybrid microspheres with a 558

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Figure 3. Light-driven ATP biosynthesis. (A) Photoinduced ATP synthesis with ATPase and PSII coassembled microspheres and a control experiment in the dark. (B) ATP synthesis through on−off cycles of light. (C) Control experiment with PSII in the absence of microspheres, ATPase in the absence of microspheres, inhibitor (DCMU), or uncoupler (CCCP) introduction. (D) Effect of Mn-depleted PSII on ATP synthesis of ATPase-PSII microspheres. All error bars refer to standard deviation (n = 3).

(Figure S9). Hence, the homogeneously distributed fluorescence intensity indicates that PSII is localized uniformly over the microspheres. For construction of a light-driven ATP generating system, CFoF1-proteoliposomes were prepared and incubated with a suspension of monodisperse PSII microspheres.15,16,19 To prove the adsorption of CFoF1-proteoliposomes on the surface of PSII spheres, Texas red-labeled DHPE was added as a fluorescence probe to make liposomes visible. Figure 2H shows continuous fluorescence with red color over the entire shell of the microsphere. Comparing with the yellow spheres in Figure 2C, the green core and red shell structures in Figure 2I imply that proteoliposomes are successfully modified on the protein sphere surface. The activity of the ATPase-PSII microsphere system was measured via the ATP concentration using the luciferin and luciferase assay.16 When luciferase catalyzes the oxidation of luciferin by consuming ATP, a photon is released and bioluminescence occurs. The luminescence intensity is proportional to the amount of ATP present in the system. For comparison, the system was analyzed with and without exposure to red light. It can be seen clearly from Figure 3A that the ATP production distinctly increases with increasing time of light irradiation to about 1100 nmol ATP (mg Chl)−1, while it is nearly unchanged in the dark. Under illumination for 60 min, the rate of ATP production becomes much smaller and it reaches a plateau after about 80 min. This might be due to a loss of PSII activity after long time irradiation. The proof of light-driven ATP production demonstrates that the bioactivities of both proteins are well retained after coassembly, and the ATP synthesis can be simply triggered by light (Figure 3B). This serves as a proof of principle that PSII can be used in the photocatalytic ATPproducing system with the proton source in our designed core@ shell structure.

decreases by up to 40%, with the rate of DCPIP reduction about 120 μmol DCPIP (mg Chl)−1 h−1, as shown in Figure 1G. Also, the action spectra of PSII display the characteristics of pigment composition and relative energy migration efficiency for photochemical reaction centers.39 As shown in Figure 1H, PSII microspheres exhibit relatively higher DCPIP reduction rates in the 400−500 and 600−700 nm range, respectively. This indicates that entrapped PSII in microspheres has the same photoexcitation properties as PSII has alone (Figure S6). Hence, after immobilization, PSII still retains its capability of oxidizing water. Confocal laser scanning microscopy (CLSM) was employed to directly observe PSII microspheres before and after modification of CFoF1-ATPase proteoliposomes. As shown in Figure 2A and 2B, the PSII spheres exhibited a significant autofluorescence when excited at 488 and 559 nm. This could be attributed to chlorophyll contained in the PSII and the C−N bonds in the Schiff’s bases formed during the cross-linking reaction between amino groups in proteins (BSA and PSII) and aldehyde groups in GA.40,41 This autofluorescence is of importance for detecting and monitoring the distribution and location of this PSII-based energy conversion system. To further locate the spatial distribution of PSII, lambda scanning of CLSM was performed. When excited at 405 nm laser, the emission spectra of PSII have a strong signal in the range of 650−680 nm (Figure 2D and S7). Noting that no obvious fluorescence signal can be observed for BSA microspheres in the same range (Figure 2E and S8), it can be concluded that the signal around 680 nm in PSII-microspheres originates from PSII. Scanning spectra of randomly selected areas of PSII-based microspheres were also obtained to image the PSII position in two-dimensional scope (Figure 2F). A specific signal at 650−680 nm appears in the whole view of PSII-based microspheres, which can be attributed to the characteristic fluorescence of PSII. When displayed as xz and yz sections, the 3D reconstitution image of PSII microspheres also shows the same behavior at different sections 559

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have been added in a Triton X-100 solubilized CFoF1-ATPase buffer solution (10 mM Tricine pH 8.0, 20 mM NaCl and 5 mM MgCl2), allowing the protein-detergent micelles to incorporate, followed by three slow removal cycles of Triton X-100 by Biobeads SM-2. This leads to the formation of CFoF1-proteoliposomes. The final concentration of CFoF1 and lipid were 150 nM and 5 mg mL−1. 3% Texas Red-DHPE (w/ w) were used as a fluorescent label to make proteoliposomes visible. Preparation of PSII microspheres. Equal volumes of 0.33 M Na2CO3 and CaCl2 containing 5 mg mL−1 BSA and 0.2 mg mL−1 PSII were rapidly mixed in a beaker under vigorous stirring at room temperature. The products were washed three times, followed by dispersion in 0.025% glutaraldehyde (GA) in 20 mM MES for 3 h at 4 °C in dark containing 30% ethanediol. After three times washing with 20 mM MES solutions, the particles were suspended in PSII solutions containing 0.03% dodecyl maltoside for 6 h, followed by washing with 20 mM MES buffer. 0.1 M Na2EDTA in 20 mM MES buffer solution (pH 6.5) was used to remove the CaCO3 templates. PSII spheres were obtained and stored at 4 °C in dark for further analysis. For control experiments the same amount of active PSII was replaced by Mn-depleted PSII. CLSM images were taken with an Olympus FV 1000 confocal system (Olympus, Japan), for lambda scanning the step size is 10 nm, resolution is 20 nm. Photoinduced ATP synthesis activity. Photoinduced ATP synthesis activity was measured by a bioluminescence assay (ENLITEN ATP Assay System, Promega, USA) using a BPCL ultra weak luminescence analyzer. The procedure used here was modified from our previous methods.15,16 The reaction mixture contained PSII microsphere/ ATPase-liposome complex buffer solution, 0.2 mM ADP, 5 mM NaH2PO4, 2.5 mM MgCl2, 2 mM K3[Fe(CN)6] and deionized water. All solutions were equilibrated at room temperature in dark for 1 h and then illuminated using a Xe lamp (HAMAMAZU, model E7536, P 150 W) with a red filter (λ> 550 nm) and heat-cut filter (λ < 800 nm). Before exposure and at subsequent time points 5 μL aliquots were removed from the reaction to quantify the amount of ATP production.

To establish that the increase of ATP concentration is definitely due to the proton gradient-coupled ATP synthesis, we introduced an uncoupler (carbonyl cyanide-m-chlorophenylhydrazone, CCCP) for a control experiment. CCCP is an H+ ionophore that dissipates the H+ gradient generated on the two sides of the membrane and thus inhibits phosphorylation.42 If CCCP with the final concentration of 30 μM was added to the system, no ATP was produced. When the intact PSII-ATPase hybrid system was incubated with 20 μM DCMU (PSII inhibitor) for an hour before light exposure, ATP synthesis was effectively suppressed. Other control experiments including the absence of either ATPase or PSII spheres showed the same results (Figure 3C). Moreover, Mn-depleted PSII (treated by Tris-washing or NH2OH) was also introduced to verify the characteristics of PSII photochemistry, and no significant amount of ATP was generated (Figure 3D). All these results demonstrate that the process of ATP synthesis is primarily attributed to light-induced proton gradient-linked rotary catalysis of CFoF1 ATPase and only occurs in the presence of all components. It should be mentioned that these ATPase-PSII spheres are robust when stored at 4 °C in dark and retain at least 50% functionality of photodriven ATP synthesis for 3 weeks (Figure S10). It is suggested that immobilization of PSII in a protecting BSA-GA matrix accounts for long time stability and the hydrogel cores can support the ATPase-liposome. This light and uncoupler-sensitive ATP synthesis system mimics the photophosphorylation process in chloroplasts.

CONCLUSIONS In conclusion, the present work demonstrates a minimal chloroplast-like light-to-chemical energy (ATP) conversion model by simple coassembly of CFoF1-proteoliposomes and PSII in core@shell microspheres. The reticulate hydrogel structure formed inside the porous microspheres not only preserves the activity of PSII, but also provides support for ATPase-liposomes. Under light illumination, the PSII-ATPase hybrid system uses water entrapped in the hydrogel microspheres as the primary proton source and generates the proton gradient for ATPase to produce ATP. Therefore, by taking advantage of the renewable resources (solar energy and water), this minimal chloroplast-like model successfully achieved lightto-ATP conversion. This suggests microreactor applications in light-triggered ATP-driven micro-/nanodevices. It offers alternative and facile strategies to construct and manage multiple enzymes in more complex systems, even to realize the coassembly of functional systems in artificial simplified organelles. This may have great implications in fabrication of miniature energy harvesting and conversion devices, that are promising, if energy in the form of ATP is required and especially useful in designing and developing integrated, multifunctional structures with intelligent functionalities.

ASSOCIATED CONTENT S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsnano.5b05579. Isolation and characterization of CFoF1-ATPase and PSII (Figures S1−S10) (PDF)

AUTHOR INFORMATION Corresponding Author

*E-mail: [email protected]. Notes

The authors declare no competing financial interest.

ACKNOWLEDGMENTS The authors acknowledge the financial support from the National Basic Research Program of China (973 program, 2013CB932802), the National Natural Science Foundation of China (Project No. 21433010, 21320102004, 21321063, 21303221 and 21573248), and the National key foundation for exploring scientific instrument (2013YQ16055108). We also wish to thank Prof. Michael Grunze for critically reading the manuscript.

EXPERIMENTAL SECTION Materials. Sodium carbonate (Na2CO3), calcium chloride (CaCl2), 3-(3, 4-dichlorophenyl)-1, 1-dimethylurea (DCMU), glutaraldehyde (GA), 2, 6-dichlorophenolindophenol (DCPIP) were purchased from Sigma-Aldrich. Dimyristoylphosphatidyl choline (DMPC) and dimyristoylphosphatidylglycerols (DMPG) were purchased from Avanti in powder form. Texas Red-DHPE was purchased from Life. All other chemicals were purchased from Sigma-Aldrich without further purification. Reconstitution of CFoF1-liposome. Liposomes were prepared from a mixture of DMPC and DMPG (9:1 by mass). The reconstitution of CFoF1 into liposomes was described previously.12 Briefly, liposomes

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