Photocurrent Generation of Reconstituted Photosystem II on a Self

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Photocurrent generation of reconstituted photosystem II on self-assembled gold film Mariko Miyachi, Shu Ikehira, Daiki Nishiori, Yoshinori Yamanoi, Masato Yamada, Masako Iwai, Tatsuya Tomo, Suleyman I. Allakhverdiev, and Hiroshi Nishihara Langmuir, Just Accepted Manuscript • DOI: 10.1021/acs.langmuir.6b03499 • Publication Date (Web): 19 Jan 2017 Downloaded from http://pubs.acs.org on January 21, 2017

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Photocurrent

generation

of

reconstituted

photosystem II on self-assembled gold film Mariko Miyachi,† Shu Ikehira,† Daiki Nishiori,† Yoshinori Yamanoi,*,† Masato Yamada,‡ Masako Iwai,§ Tatsuya Tomo,‡ Suleyman I. Allakhverdiev,⊥ and Hiroshi Nishihara*,†

†

Department of Chemistry, School of Science, The University of Tokyo, 7-3-1 Hongo, Bunkyo-

ku, Tokyo 113-0033, Japan ‡

Department of Biology, Faculty of Science, Tokyo University of Science, Kagurazaka 1-3,

Shinjuku-ku, Tokyo 162-8601, Japan §

Graduate School of Bioscience and Biotechnology, Tokyo Institute of Technology, Yokohama

226-8503, Japan ⊥

Controlled Photobiosynthesis Laboratory, Institute of Plant Physiology, Russian Academy of

Sciences, Botanicheskaya Street 35, Moscow 127276; Institute of Basic Biological Problems, Russian Academy of Sciences, Pushchino, Moscow Region 142290, and Faculty of Biology, M.V. Lomonosov Moscow State University, Leninskie Gory 1-12, Moscow 119991, Russia

ABSTRACT:

Photosystem II (PSII) modified gold electrodes were prepared by the deposition of PSII reconstituted with platinum nanoparticles (PtNPs) on Au electrodes. PtNPs modified with 1-[15-

ACS Paragon Plus Environment

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(3,5,6-trimethyl-1,4-benzoquinone-2-yl)]pentadecyl

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disulfide

((TMQ(CH2)15S)2)

were

incorporated into the QB site of PSII isolated from the thermophilic cyanobacterium, Thermosynechococcus elongatus. The reconstitution was confirmed by QA-reoxidation measurements. PSII reconstituted with PtNPs was deposited and integrated on a Au(111) surface modified with 4,4’-biphenyldithiol. The cross-section of the reconstituted PSII film on the Au electrode was investigated by SEM. Absorption spectra showed that the surface coverage of electrode was about 18 pmol PSII cm-2. A photocurrent density of 15 nAcm-2 at E = +0.10 V (vs. Ag/AgCl) was observed under 680 nm irradiation. The photoresponse showed good reversibility under alternating light and dark conditions. Clear photoresponses were not observed in the absence of PSII and molecular wire. These results supported the photocurrent originated from PSII and moved to gold electrode by light irradiation, which also confirmed conjugation with orientation through molecular wire.

Introduction Photosynthesis is a photochemical bioprocess that converts solar energy into chemical energy stored in fuel products.1-3 It occurs at two inter-connected light-active centers: photosystem I (PSI) and photosystem II (PSII). The quantum yield of photosynthesis is nearly 100% owing to the superbly adapted electron flow systems that have evolved through natural selection and mutation.4 Photosynthetic components can be isolated from cyanobacteria or algae, and their photoinduced electron-transfer activity can be maintained even after purification. To elucidate and mimic the high performance of natural system, biocomponents have been combined with artificial systems and studied as light-driven devices.5-14 Our laboratory previously reconstituted PSI by exchanging vitamin K1 in the redox cascade of PSI for an artificial molecular wire


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terminated with a gold nanoparticle; as a result, a new bio-photosensor was developed on a FET.15-17 We attempted to reconstitute plastoquinone of PSII with artificial molecules as an extension of this finding. One of the functions of PSII under physiological conditions is to use light energy for oxidizing water into molecular oxygen.18 The reaction occurs via the oxidation of water on a manganese redox center and a series of light-induced electron-transfer reactions through tyrosyl residue, chlorophyll a, pheophytin, and plastoquinone. The X-ray crystal structure of PSII has revealed the locations of the redox center buried within the structure of the transmembrane protein complex.19,20 Recently, reports on photochemical systems which combined PSII with an artificial molecules have been increasing.21-26 Noji et al achieved the electron transfer systems from His-tag modified PSII to gold nanoparticles through Ni-NTA support in 2011.27 Mersch realized a water splitting system by the visible light using modified electrode which absorbed PSII and hydrogenase on porous ITO surface under the external voltage source.28 However, the electron transport efficiency has decreased in comparison with native PSII because these reports are physical adhesion with PSII and electronic conduction materials. Although electron transfer becomes more efficient when PSII of quinone moieties are reconstituted by molecular wire, the light sensitivity of PSII makes it difficult to handle; there are few examples of reorganizing its QB site and studies of its photophysical properties.29 This communication presents a photoelectric conversion system consisting of PSII and molecular wires on a gold electrode. QA reoxidation measurements were used to confirm the reconstitution of the QB site of PSII with the molecular wires. The reconstituted PSII is expected to transfer photo-excited electrons to the PtNPs through (TMQ(CH2)15S)2 molecule. The transfer was evaluated on gold electrodes by photochemical and electrochemical methods. The


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photocurrent of the PSII-nanoparticle hybrid gold electrode was increased by the high surface area of PtNPs which allowed greater deposition of PSII compared with the case of PSII alone. In this system, PtNPs protected with (TMQ(CH2)15S)2, help this communication with the easy connection to SAM on gold surface. In the future, it is extendible to the application of PtNPs to the catalytic reaction.

Experimental Section Materials. All chemical reagents and solvents were used as purchased unless otherwise stated.

2,3,6-Trimethyl-1,4-benzoquinone

(TMQ)30

and

2-[2-(2-

methoxyethoxy)ethoxy]ethanethiol (TEGSH)31 was prepared according to the literature. MilliQ was purified with YAMATO AUTOPURE WD500 or MILLIPORE ELIX 3 UV. HPLC grade ethanol, chloroform, and dichloromethane were used for solvent and cleaning solution. Buffer solution is composed of 25% glycerol, 50 mM MES (pH 6.5), 10 mM MgCl2, 5 mM CaCl2 and 0.04% n-dodecyl-β-maltoside. Gold surface with a thickness of 100 nm deposited on natural mica was used as electrode. All reactions were carried out under air unless otherwise noted. The isolated PSII and reconstituted PSII were stored in buffer solution at −80 °C before use. Measurement. NMR spectra were measured with a JEOL AL-400 spectrometer and a Bruker DRX-500 spectrometer, and referenced to tetramethylsilane (0.00 ppm) as an internal standard. Transmission Electron Microscope (TEM) images were taken at 200 kV with a HITACHI HF-2000. Absorption spectra were measured with a JASCO V-630 spectrometer. Photocurrent and CV were measured using an electrochemical analyzer (ALS 750A, BAS Inc.). Nanoparticles and PSII were centrifuged with a HITACHI himac CS150GXII and a CF15RXII.


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Ultrafiltration was conducted with Merck Millipore Amicon Ultra–0.5mL Centrifugal Filters Ultracel–100K. As a light source for action spectrum of PSII-reconstituted Au electrode, ASAHI SPECTRA MAX-302 Xe lamp was used. The light was monochromatized through suitable band pass filters. Chlorophyll fluorescence kinetics were measured by a Photon Systems Instruments Fast Fluorometer FL 3500-F with QA-reoxidation mode. Synthesis

of

2-(15-bromopentadecyl)-3,5,6-trimethyl-1,4-benzoquinone

(TMQ(CH2)15Br). To a 1 L round-bottom flask were added 2,3,6-trimethyl-1,4-benzoquinone (TMQ, 3.84 g, 25.6 mmol), 16-bromohexadecanoic acid (10.0 g, 29.8 mmol), silver nitrate (6.83 g, 40.2 mmol), ammonium persulfate (9.23 g, 40.4 mmol), acetonitrile (200 mL), and water (200 mL). The reaction mixture was heated to reflux for 12 h. The solution was then cooled to rt, organic layer was separated, and the aqueous layer was extracted with diethyl ether. The combined extracts were washed with brine and dried over Na2SO4. The solvent was evaporated under a reduced pressure, and the residue was purified by column chromatography on silica gel (hexane/ethyl acetate = 100/1) to give the desired compound as yellow powder (5.98 g, 53%). Mp: 37.6−40.1 °C. 1H NMR (500 MHz, CDCl3) δ 3.41 (t, 2H, J = 7.0 Hz), 2.46 (t, 2H, J = 7.0 Hz), 2.02 (s, 3H), 2.01 (s, 6H), 1.85 (quin, 2H, J = 7.2 Hz), 1.42−1.25 (m, 24H). 13C NMR (125 MHz, CDCl3) δ 187.9 (C=O), 187.2 (C=O), 144.5 (Cq), 140.4 (Cq), 140.3 (Cq), 140.0 (Cq), 34.1 (CH2), 32.8 (CH2), 29.9 (CH2), 29.6 (CH2), 29.5 (CH2), 29.41 (CH2), 29.40 (CH2), 28.8 (CH2), 28.7 (CH2), 28.1 (CH2), 26.6 (CH2), 12.34 (CH3), 12.32 (CH3), 12.1 (CH3). FAB−MS m/z 438 ([M+H]+). FAB−HRMS Calcd for C24H39O2Br: 438.2133. Found: 428.2148 ([M+H]+). Synthesis

of

1-[15-(3,5,6-trimethyl-1,4-benzoquinone-2-yl)]pentadecyl

disulfide

((TMQ(CH2)15S)2). A 500 mL round-bottom flask was charged with TMQ(CH2)15Br (5.01 g, 11.4 mmol), thiourea (4.33 g, 56.8 mmol), and ethanol (200 mL). The mixture was heated to


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reflux under nitrogen. After 26 h, tetraethylenepentamine (4.3 mL, 23 mmol) was added. The reaction mixture was refluxed again. After 26 h, the mixture was quenched with water, extracted with ethyl acetate, washed with brine, and dried over Na2SO4. The solvent was evaporated under a reduced pressure, and the residue was purified by column chromatography on silica gel (hexane/ethyl acetate = 50/1) to give the desired product as yellow powder (1.30 g, 29%). Mp: 48.2−50.0 °C. 1H NMR (500 MHz, CDCl3) δ 2.68 (t, 4H, J = 7.5 Hz), 2.46 (t, 4H, J = 7.5 Hz), 2.02 (s, 6H), 2.01 (s, 12H), 1.85 (quin, 4H, J = 7.3 Hz), 1.42−1.20 (m, 48H).

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C NMR (125

MHz, CDCl3) δ 187.9 (C=O), 187.2 (C=O), 144.5 (Cq), 140.4 (Cq), 140.3 (Cq), 140.0 (Cq), 39.1 (CH2), 29.9 (CH2), 29.62 (CH2), 29.58 (CH2), 29.53 (CH2), 29.50 (CH2), 29.4 (CH2), 29.22 (CH2), 29.19 (CH2), 28.8 (CH2), 28.5 (CH2), 26.6 (CH2), 12.34 (CH3), 12.32 (CH3), 12.1 (CH3). FAB−MS m/z 782 (M+). FAB−HRMS Calcd for C48H78O4S2: 782.5342. Found: 782.5323 (M+). Synthesis

of

Amphiphilic

platinum

nanoparticles

partially

attached

with

(TMQ(CH2)15S)2. A 1 L round-bottom flask was charged with hexachloroplatinic (IV) acid (0.29 g, 0.70 mmol), TEGOH (4.2 g, 26 mmol) and water (450 mL). Under vigorous stirring, sodium borohydride (0.155 g, 4.1 mmol) dissolved in water (10 mL) was dropped. TEGSH (4.2 g, 23 mmol) was added to the mixture and the mixture was stirred for 5 min. Then ethanol (300 mL) was added to the mixture. The mixture was extracted with dichloromethane and concentrated under a reduced pressure. After the filtration with membrane, amphiphilic platinum nanoparticles were obtained as black powder. To a dispersion of platinum nanoparticles in dichloromethane obtained by the above method was added (TMQ(CH2)15S)2 (10 mg, 12 µmol) and stirred vigorously for 24 h at room temperature. Hexane was added to the mixture until precipitation appeared. Then the precipitation was collected with membrane filter to give


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(TMQ(CH2)15S)2 attached PtNP 2 as black powder (22 mg). The resulting particles were characterized by TEM and determined to be 4.4 ± 1.2 nm in diameter. PSII reconstitution with a platinum nanoparticles. Initially, QB-free PSII was prepared by modified method based on the literature.32 PSII (final concentration was 14 µM) was incubated in buffer solution at 4 °C with sodium dithionite (final concentration was 100 mM) and benzyl viologen dichloride (final concentration was 30 µM) for 1 d. Then the mixture was washed with water by ultrafiltration (14000 × g, 10 min, 4 °C) three times and recovered (1000 × g, 3 min, 4 °C) to give QB-free PSII. A centrifugation tip was charged with QB-free PSII (final concentration was 2.2 µM), (TMQ(CH2)15S)2 attached platinum nanoparticle (final concentration was 6.6 µM) and buffer solution (final volume was 1.0 mL). After the incubation for 1 d at 4 °C, reconstituted PSII dispersion was obtained. Preparation of reconstituted PSII film electrode. Au surface was annealed with a hydrogen flame prior to use. This treatment gave Au(111) surface comprising hundreds of nanometer wide Au single-crystal grains. The preparation was initiated with the fabrication of a SAM consisting of 4,4’-biphenyldithiol, by immersing an Au/mica electrode into a chloroform solution of 4,4’-biphenyldithiol (0.1 mM) for 12 h. The modified electrode was rinsed with chloroform and water and dried under nitrogen flow. Bis(sulfosuccinimidyl) sodium salt (BS3) in buffer solution (1 mg/mL, 10 µL) was mixed with 2 in buffer solution (77 µg chl/mL, 50 µL). The mixture was immediately drop-casted onto the SAM modified Au substrate and reacted for 20 h under dark at 4 °C. The modified surface was rinsed with water and electrochemical measurement was conducted.


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Preparation of gold electrode for control experiments. (a) In the absence of PSII: SAM consisting of 4,4’-biphenyldithiol on gold electrode was prepared by above-mentioned method. PtNP dispersion was drop-casted onto the SAM modified Au substrate and reacted for 20 h under dark at 4 °C. In this process, PtNP was immobilized on Au. The electrode was rinsed with water before the electrochemical measurement. (b) In the absence of molecular wire: Au(111) surface was prepared by above-mentioned method. Native PSII (77 µg chl/mL, 50 µL) was mixed with buffer solution (10 µL), and the mixture was drop-casted onto Au substrate and reacted for 17 h under dark at 4 °C. The modified surface was rinsed with water and electrochemical measurement was conducted. Absorption spectra measurement for estimating the concentration of PSII. To PSII dispersion (10 µL), acetone/water (4/1) (1 mL) was added. After centrifugation (15000 rpm, 5 min, 4 °C), the supernatant was transferred to the cell and absorption spectra were measured. Concentration of chlorophyll a (CChl) in 80% aqueous acetone solution is calculated by the equation (1) using absorbance A according to the literature.33 CChl = 13.71 × A663.6 − 2.85 × A646.6

(nmol chl/mL) (1)

A663.6 = absorbance at 663.6 nm A646.6 = absorbance at 646.6 nm Because one PSII molecule has 35 chlorophyll a molecules, concentration of PSII (CPSII) was calculated by the equation (2).34,35 CPSII = CChl × 1/35

(nmol/mL) (2)


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Chlorophyll fluorescence kinetics measurement (QA-reoxidation). A measurement cell was charged with reconstituted PSII or native PSII dispersion (final concentration was 3 µg chl/mL) and buffer solution (final volume was 2.0 mL). The fluorescence beyond 700 nm was measured by FL3500’s QA-reoxidation mode. The wavelength of the continuous actinic light was 630 nm and of the measuring pulse was 617 nm. Before measurement, the sample was incubated for 5 min under dark. After the measurement of reconstituted PSII, 3-(3,4-dichlorophenyl)-1,1dimethylurea (DCMU) was added as DMSO solution (final concentration was 10 µM and DMSO/ water ratio was 1/100). Then DCMU added sample was incubated under dark and measured by the same way. Photocurrent measurements. Photocurrent measurements were carried out using a reconstituted PSII film Au/mica electrode (electrode area: 0.264 cm2) as a working electrode, a Pt wire as a counter electrode, and an Ag/AgCl wire in saturated KCl aq as a reference electrode in a photoelectrochemical cell. All experiments were carried out in 0.1 M NaCl / 40 mM buffer solution (pH 6.5). As for the sacrificial reagent, 1,5-diphenylcarbazide (DPC) in DMSO solution (100 mM) was added to the measurement solution to the final concentration of 1 mM. The cell was sealed and deoxygenized by argon bubbling for 15 min. The single wavelength light and visible light of longer than 400 nm were generated from a xenon lamp and provided with a bandpass filter. The electrode potential and photocurrent was measured using an electrochemical analyzer (ALS 750A, BAS Inc.). Calculation of external photo-electric conversion efficiency. The reconstituted PSII film electrode was fixed at +0.10 V vs. Ag/AgCl in sat. KCl under the measurements. At the potential, no anodic current induced by the direct oxidation of DPC in the dark was observed. The external quantum yield of photoelectric conversion, Φ, was calculated using equation (3):


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Φ = ne / np (3) where ne is the mole of electrons that flows in the circuit per unit time (in mol s-1) and np is the mole of photons irradiated to the electrode per unit time (in mol s-1). ne and np were calculated using equations (4) and (5): ne = i / F (4) np = Wλ / NA hc (5) where i is the photocurrent flow, F is the Faraday constant (9.65 × 104 C mol-1), W is the photon flux of incident light (in J s-1), λ is the wavelength of the irradiated light (6.80 × 10-7 m), NA is the Avogadro constant (6.02 × 1023 mol-1), h is the Planck constant (6.63 × 10-34 Js) and c is the velocity of light (3.00 × 108 m s-1). i was calculated using equation (6): i = iL − iD (6) where iL is the average light current for the first cycle (5 s) and iD is the average dark current just before the illumination of light. A photon counter (8230E, ADC Corporation) was employed for the quantification of W. For every sample, W was measured independently, and a typical value for W was 29 mW. Quantification of PSII immobilized on Au substrate. The reconstituted PSII film electrode was scratched off with a spatula, followed by extracted with acetone/water (4/1) (1 mL). After centrifugation at 15000 rpm for 10 min, the supernatant was analyzed by UV-vis absorption spectroscopy for estimating the amount of chlorophyll a.


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Calculation of turn over frequency (TOF). The reconstituted PSII film electrode was fixed at 0.1 V vs. Ag/AgCl in sat. KCl at the measurements. At the potential, no anodic current induced by the direct oxidation of DPC in the dark was observed. The turn over number, TOF (in s-1), was calculated using equation (7): TOF = ne- / nPSII (7) where ne- is the mole of electrons that flows in the circuit per unit time (in mol s-1) and nPSII is the mole of PSII immobilized on the electrode per unit time (in mol). The value of ne- was calculated using equation (2) and ne- was acquired by UV-vis analysis, as described above. Differential pulse voltammetry. Differential pulse voltammetry (DPV) was carried out to study the oxidation potential of DPC using a glassy carbon working electrode, a Pt wire counter electrode, and an Ag/AgCl in saturated KCl solution reference electrode in a standard onecompartment cell. NaCl in buffer solution (0.1 M) was used for an electrolyte solution. DPC in DMSO (100 mM) was added into the measuring solution (1/100, v/v) to the final concentration of 1 mM. The solution was degassed with argon bubbling for 5 min before the measurement.

Results and Discussion (TMQ(CH2)15S)2 was designed as a structural analogue of plastoquinone. It was connected to monodispersed amphiphilic PtNPs, to which 2-[2-(2-methoxyethoxy)ethoxy]ethanol (TEGOH) and

2-[2-(2-methoxyethoxy)ethoxy]ethanethiol (TEGSH) were bound through a

ligand-exchange reaction on the particle surface (Figure 1(a)).36,37 TEM investigation of PtNPs revealed particles to be between 2 nm and 8 nm without aggregation. Detailed statistical analysis


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showed the diameter of the PtNPs to be 4.4 ± 1.2 nm, which is equivalent to about 2900 Pt atoms and 360 surfactant molecules per nanoparticle (Figure 1(b)).38 The average number of TEGOH (or TEGSH) and (TMQ(CH2)15S)2 molecules on the particle surface were found to be 360 and 7, respectively, by integrating the 1H NMR spectrum although the number of TEGOH and TEGSH could not be separately determined by 1H NMR due to the overlapped peaks of 2-[2-(2methoxyethoxy)ethoxy]ethyl groups (Figure S5). The approach to the preparation of PSII reconstituted with PtNPs is depicted in Figure 1(c). PSII was isolated from cyanobacterium species, Thermosynechococcus elongatus BP-1.39 Plastoquinone QB was selectively removed by reductive treatment, which also removed the oxygen-evolving moiety (Mn4), allowing QB-free PSII to have the molecular wire incorporated into the QB site to give reconstituted PSII (2).


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Figure 1. (a) Structures of plastoquinone and PtNPs modified with (TMQ(CH2)15S)2 (1) (b) TEM image and size histogram of 1. Scale bar is 20 nm. (c) Schematic of the reconstitution process between PSII and 1. Reaction conditions: (i) sodium dithionite, benzyl viologen dichloride, 1 d, 4 °C. (ii) 1, 1 d, 4 °C.


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A chlorophyll fluorescence kinetics study (QA-reoxidation measurement) confirmed the reconstitution in 2 (Figure 2).40,41 QB-free PSII showed slow fluorescence decay because of the inhibition of QA reoxidation by recombination with P680. Fluorescence decay increased after the reconstitution. The variation in fluorescence intensity data of reconstituted PSII was mainly attributed to scattering by the PtNPs. Adding 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) to this system also showed slow fluorescence intensity decay indicating that QA to QB electron transfer was blocked.42 This observation indicated that the PtNPs were replaced with DCMU. As a result, PSII was reconstituted with PtNPs and photo-excited electrons at P680 were transported to the PtNPs through the electron transfer pathway in PSII.


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Figure 2. Results of QA-reoxidation measurements for QB-free PSII (green), reconstituted PSII (blue), and reconstituted PSII + DCMU (black).


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Forming SAMs with well-chosen head groups is a sophisticated tool for designing functional surfaces at the molecular level. The photocurrent generation with reconstituted PSII was studied further with PSII immobilized on a mercapto-functionalized Au electrode (Figure 3(a)). The attachment of PtNPs to thiol units, followed by crosslinking PSII units with BS3 (bis(sulfosuccinimidyl)suberate sodium salt), yielded an integrated electrically connected electrode. Overall, the α,ω-dithiol SAM was expected to connect PtNPs with the Au electrode, and BS3 was also intended to crosslink the PSII molecules through the amide bonds on their Nterminals. Cross-sectional information of the electrode has been investigated by SEM. We previously reported cross-sectional TEM observation of photosynthetic component on ITO by direct cutting with ultramicrotome,43 however it is difficult to prepare cross-sectional ultra-thin film (< 20 nm) of soft gold electrode for TEM observation with ultramicrotome or focused ion beam. The crosssectional SEM image of the PSII-gold interface showed that material 2 was uniformly immobilized on the gold surface with a thickness of ca. 80 nm (Figure 3(b)). The amount of PSII immobilized on the Au electrode was estimated by measuring the amount of chlorophyll a. The reconstituted PSII film electrode was scratched off with a spatula, and extracted with acetone/water in a ratio of 80/20.44 After centrifugation, absorption spectrum showed the coverage to be 18 pmol PSII cm-2, which was comparable to be ca. ten layers’ coverage (Figure 3(c)). Based on the size of PSII,45,46 ten layer thickness was in agreement with the results from cross-sectional SEM observation (ca. 90−100 nm).


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Figure 3. (a) Schematic representation of the reconstituted PSII film on a Au electrode. Reaction conditions: (i) 4,4’-biphenyldithiol, rt, 12 h. (ii) 2, BS3, 4 °C, 20 h. (b) Crosssectional SEM image of the PSII film on a Au electrode. (c) Absorption spectrum after the scratch off, extraction with acetone/water, and centrifugation of reconstituted PSII film on Au electrode.


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A constant photocurrent was generated with a sacrificial reagent (1,5-diphenylcarbazide; DPC) under continuous visible light irradiation.47,48 The redox potential was estimated as E = +0.31 V (vs. Ag/AgCl) by the measurement of DPV in MES buffer solution (Figure S6). The dependence of the photocurrent on electrode potential was measured between −0.3 V and +0.3 V (Figure 4(a)). Photocurrent was not generated below E = −0.20 V (vs. Ag/AgCl), a value almost equal to the redox potential of plastoquinone in the QB site.49 Positive photocurrent was observed above −0.20 V. We used an electrode potential of +0.10 V throughout following experiments owing to the stability of the background. The photocurrent action spectrum was consistent with the absorption spectrum of PSII (Figure 4(b)), showing that the photocurrent response from the PSII-immobilized electrode was mainly due to the photoexcitation of PSII. (TMQ(CH2)15S)2 does not contribute to the photocurrent because absorption peak is located far from 680 nm (Figure S7). These results suggest that photo-excited electrons from PSII were transferred to the Au substrate via (TMQ(CH2)15S)2 and PtNP connected to the QB site. Accordingly, the PSII reconstitution succeeded when plastoquinone in the QB site was exchanged for a PtNP modified with (TMQ(CH2)15S)2. The photoresponse of the PSII-modified gold electrode under illumination was clear and showed high reproducibility (Figure 4(c)). The quick response of the photocurrent was probably due to material 2 being anchored at thiol binding sites on the SAM and smooth electron injection from PSII to the gold electrode. A photocurrent density of 15 nAcm-2 was observed under 680 nm irradiation (29 mWcm-2) in the presence of DPC as a sacrificial reagent. The current density represents an improvement by a factor of 3.6 over the performance of the system without BS3. Considerable amount of PSII was peeled off from the gold electrode in the absence of cross-


18

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Langmuir

linkers. The role of cross-linker is to strengthen the structure of material and to avoid the detachment of PSII from the electrode. No appreciable photocurrent was detected by each lightto dark transition in a control experiment without PSII (Figure S8). The turn over frequency was calculated to be 2 e- (PSII)-1 s-1, comparable to that previously reported for an electrode modified with PSII.50 External quantum yield was calculated to be Φ = 9.2 × 10-7 according to the equations (3)−(6) in experimental section. The calculated TOF and Φ was not so high for five layers of PSII. In the consideration of its quantum yield, there is a possibility that all PSII may not participate in the photocurrent generation. Partially PSII on the electrode surface was not reconstituted with molecular wire and was not able to work as a photosensitizer. Besides, since there are ten layers of PSII on the electrode with no addition of electrical conductor to this system, some of PSII may lack of electrical contact to the electrode. PtNP can work as an electron mediator, but the number of PtNPs seems not enough for making an electrical pathway between protein complexes of PSII. Exposure to discontinuous illumination (alternating 10 sec periods of light and dark three times) resulted in little photocurrent change (≈ 15 nAcm-1) with the rapid reverse current flow following each light to dark transition. Accordingly, the molecular wire 1 was not removed from QB site during the photo irradiation. The control experiment without PSII did not display clear photocurrent (Figure S8). Another control experiment, which measured photocurrent of PSII casted on gold electrode (without SAM and molecular wires), did not also show photocurrent at all either (Figure S9). The photo-electron conversion was achieved due to the direct connection between the QB site of PSII and the gold surface through the molecular wire 1. Pt−S bond formation in reconstitution system contributed to the stabilization of modified electrode and the enhancement of photocurrent.


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Figure 4. Operation of the PSII−PtNPs−Au electrode. (a) Photostimulated photocurrent generated by the PSII−PtNP−Au electrode. Illumination time 10 s. (b) Photocurrent action spectrum of PSII modified Au electrode (black dots) and absorption spectrum of PSII (solid line). (c) Photocurrent response of PSII modified electrode under 680 nm irradiation. Blue line: in the presence of BS3. Red line: in the absence of BS3. Potential applied: E = +0.10 V (vs. Ag/AgCl), optical power: W = 29 mWcm-2.


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Conclusion In summary, we developed a reconstituted PSII system by introducing PtNPs via a biomimetic molecular wire in the QB site to replace plastoquinone. Strong evidence for successful reconstitution was provided by the QA-reoxidation measurements and the applied potential vs. photocurrent of the reconstituted PSII film on a gold electrode. This direct coupling strategy afforded high light-induced photocurrent generation than that achieved using a comparable Au electrode without PSII and molecular wire. Although water oxidation complex, Mn cluster (Mn4), is depleted during the process of QB removal in the existing state, photocurrent can be generated without the sacrifice reagent if Mn complex can be reconstitute in future.

ASSOCIATED CONTENT Supporting Information. The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.langmuir. Copies of the

1

H and

13

C NMR spectra, UV-vis absorption spectrum of

(TMQ(CH2)15S)2, differential potential voltammetry (DPV) of DPC, and results of control experiments (PDF)

AUTHOR INFORMATION


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Corresponding Author * To whom correspondence should be addressed. TEL: +81-3-5841-4346. FAX: +81-3-58418063. E-mail: [email protected]; [email protected]. Notes The authors declare no competing financial interest.

ACKNOWLEDGMENT The present work was financially in part supported by CREST from JST (H.N.), Tokyo Kasei Chemical Promotion Foundation (Y.Y.), Nippon Sheet Glass Foundation for Materials Science and Engineering (Y.Y.), Precise Measurement Technology Promotion Foundation (Y.Y.), Russian Science Foundation (No. 14-14-00039; S.I.A.), Grant-in-Aids for Scientific Research (S) (No. 17726220801; H.N. M.M. and T.T.), Grant-in-Aids for Scientific Research (C) (No. 15K05604; Y.Y.), and Scientific Research on Innovative Areas “Molecular Architectonics: Orchestration of Single Molecules for Novel Functions” (area 2509, Nos. 26110505, 26110506, 16H00957, and 16H00958; H.N. and Y.Y.) from the Ministry of Education, Culture, Sports, Science, and Technology, Japan.

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(3) Govindjee, Bioenergetics of Photosynthesis, Academic Press, NY, 1975. (4) Emerson, R.; Chalmers, S. V.; Cederstand, C. Some Factors Influencing the Long-Wave Limit of Photosynthesis. Proc. Natl. Acad. Sci. USA 1957, 43, 133−143. (5) Yamanoi Y.; Nishihara, H. Assembly of nanosize metallic particles and molecular wires on electrode surfaces. Chem. Commun. 2007, 3983−3989. (6) Yamanoi, Y.; Nishihara, H. Solar-energy conversion technology using photosynthetic functionality of cyanobacteria. Kobunshi 2007, 56, 835−838. (7) Yamanoi, Y.; Miyachi, M.; Nishihara, H. Modification of Electrode Interfaces with Nanosized Materials for Electronic Applications. In Molecular Architectonics: The Third Stage of Single Molecule Electronics, Ogawa T. Ed.: Springer: New York, in press. (8) Samanta, D.; Sarkar, A. Immobilization of bio-macromolecules on self-assembled monolayers: methods and sensor applications. Chem. Soc. Rev. 2011, 40, 2567−2592. (9) Gooding, J. J.; Darwish, N. The rise of self-assembled monolayers for fabricating electrochemical biosensors-an interfacial perspective. Chem. Rec. 2012, 12, 92−105. (10) Gorka, M.; Schartner, J.; van der Est, A.; Rögner, M.; Golbeck, J. H. Light-Mediated Hydrogen Generation in Photosystem I: Attachment of a Naphthoquinone–Molecular Wire–Pt Nanoparticle to the A1A and A1B Sites. Biochemistry 2014, 53, 2295−2306. (11) Lubner, C. E.; Körzer, P.; Silva, P. J. N.; Vincent, K. A.; Happe, T.; Bryant, D. A.; Golbeck, J. H. Wiring an [FeFe]-Hydrogenase with Photosystem I for Light-Induced Hydrogen Production. Biochemistry 2010, 49, 10264−10266.


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(18) Diner, B. A.; Yocum, C. F.; Andersson, B.; Arntzen, C. J.; Pakrasi, H. B.; Kyle, D. J.; Ohad, I.; Sétif, P.; Mathis, P.; Wollman F.-A. Photosystems I and II: Structure, Proteins, and Cofactors. In Encyclopedia of Plant Physiology, Staehelin, L. A.; Arntzen, C. J. Eds., vol. 19, pp. 422−495, Springer, Berlin. (19) Umena, Y.; Kawakami, K.; Shen, J.-R.; Kamiya, N. Crystal structure of oxygen-evolving photosystem II at a resolution of 1.9 Å. Nature 2011, 473, 55−60. (20) Suga, M.; Akita, F.; Hiarata, K.; Murakami, H.; Nakajima, Y.; Shimizu, T.; Yamashita, K.; Yamamoto, M.; Ago, H.; Shen, J.-R. Native structure of photosystem II at 1.95 Å resolution viewed by femtosecond X-ray pulses. Nature 2014, 517, 99−103. (21) Feng, X.; Jia, Y.; Cai, P.; Fei, J.; Li, J. Coassembly of Photosystem II and ATPase as Artificial Chloroplast for Light-Driven ATP Synthesis. ACS Nano 2016, 10, 556−561. (22) Yehezkeli, O.; Tel-Vered, R.; Wasserman, J.; Trifonov, A.; Michaeli, D.; Nechushtai, R.; Willner, I. Integrated photosystem II-based photo-bioelectrochemical cells. Nat. Commun. 2012, 3, 1741/1−1741/7. (23) Badura, A.; Guschin, D.; Esper, B.; Kothe, T.; Neugebauer, S.; Schuhmann, W.; Roegner, M. Photo-Induced Electron Transfer Between Photosystem 2 via Cross-linked Redox Hydrogels. Electroanalysis 2008, 20, 1043−1047. (24) Badura, A.; Esper, B.; Ataka, K.; Grunwald, C.; Woell, C.; Kuhlmann, J.; Heberle, J.; Roegner, M. Light-Driven Water Splitting for (Bio-)hydrogen Production: Photosystem 2 as the Central Part of a Bioelectrochemical Device. Photochem. Photobiol. 2006, 82, 1385−1390.


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(25) Yehezkeli, O.; Tel-Vered, R.; Michaeli, D.; Nechushtai, R.; Willner, I. Photosystem I (PSI) / Photosystem II (PSII)-Based Photo-Bioelectrochemical Cells Revealing Directional Generation of Photocurrents. Small 2013, 9, 2970-2978. (26) Giardi, M. T.; Koblizek, M.; Masojidek, J. Photosystem II-based biosensors for the detection of pollutants. Biosens. Bioelectron. 2001, 16, 1027−1033. (27) Noji, T.; Suzuki, H.; Gotoh, T.; Iwai, M.; Ikeuchi, M.; Tomo, T.; Noguchi, T. Photosystem II-Gold Nanoparticle Conjugate as a Nanodevice for the Development of Artificial Light-Driven Water-Splitting Systems. J. Phys. Chem. Lett. 2011, 2, 2448−2452. (28) Mersch, D.; Lee, C. Y.; Zhang, J. Z.; Brinkert, K.; Fontecilla-Camps, J. C.; Rutherford, A. W.; Reisner, E. Wiring of Photosystem II to Hydrogenase for Photoelectrochemical Water Splitting. J. Am. Chem. Soc. 2015, 137, 8541−8549. (29) Kato, M.; Zhang, J. Z.; Paul, N.; Reisner, E. Protein film photoelectrochemistry of the water oxidation enzyme photosystem II. Chem. Soc. Rev. 2014, 43, 6485−6497. (30) Hu, P.; Huang, S.; Xu, J.; Shi, Z. J.; Su, W. Construction of Substituted Benzene Rings by Palladium-Catalyzed Direct Cross-Coupling of Olefins: A Rapid Synthetic Route to 1,4Naphthoquinone and Its Derivatives. Angew. Chem. Int. Ed. 2011, 50, 9926−9930. (31) Foos, E. E.; Snow, A. W.; Twigg, M. E.; Ancona, M. G. Thiol-terminated di-, tri-, and tetraethylene oxide functionalized gold nanoparticles: a water-soluble, charge-neutral cluster. Chem. Mater. 2002, 14, 2401−2408.


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(32) Mieghem, F. J. E.; Nitschke, W.; Mathis, P.; Rutherford, A. W. The influence of the quinone-iron electron acceptor complex on the reaction centre photochemistry of Photosystem II. Biochim. Biophys. Acta Bioenerg. 1989, 977, 207−214. (33) Porra, R. J.; Thompson W. A.; Kriedemann P. E. Determination of accurate extinction coefficients and simultaneous equations for assaying chlorophylls a and b extracted with four different solvents: verification of the concentration of chlorophyll standards by atomic absorption spectroscopy. Biochim. Biophys. Acta Bioenerg. 1989, 975, 384−394. (34) Loll, B.; Kern, J.; Saenger, W.; Zouni, A.; Biesiadka, J. Towards complete cofactor arrangement in the 3.0 A resolution structure of photosystem II. Nature 2005, 438, 1040−1044. (35) Ferreira, K. N.; Iverson, T. M.; Maghlaoui, K.; Barber, J.; Iwata, S. Architecture of the photosynthetic oxygen-evolving center. Science 2004, 303, 1831−1838. (36) Yang, J.; Lee, J. Y.; Too, H. Size effect in thiol and amine binding to small Pt nanoparticles. Anal. Chim. Acta 2006, 571, 206−210. (37) Bigall, N. C.; Haertling, T.; Klose, M.; Simon, P.; Eng, L. M.; Eychmueller, A. Monodisperse Platinum Nanospheres with Adjustable Diameters from 10 to 100 nm: Synthesis and Distinct Optical Properties. Nano Lett. 2008, 8, 4588−4592. (38) Hostetler, M. J.; Wingate, J. E.; Zhong, C. J.; Harris, J. E.; Vachet, R. W.; Clark, M. R.; Londono, J. D.; Green, S. J.; Stokes, J. J.; Wignall, G. D.; Glish, G. L.; Porter, M. D.; Evans, N. D.; Murray, R. W. Alkanethiolate Gold Cluster Molecules with Core Diameters from 1.5 to 5.2 nm: Core and Monolayer Properties as a Function of Core Size. Langmuir 1998, 14, 14−30.


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Table of Contents Graphic and Synopsis


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Langmuir (a)

Plastoquinone (QB)

1 (b)

250 200

Counts

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42

150 100 50 0 1 2 3 4 5 6 7 8 9 1011

Diameter [nm]

(c)

Native PSII QA

QB-free PSII QB

Pheo P680

(i)

QA

Reconstituted PSII

(ii)

Pheo

Pt

Pheo P680

P680 YZ

QA

YZ

Mn4


YZ 2

Langmuir

1.1

Fluorescence intensity (a.u.)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42

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0.9 0.7 0.5 0.3 0.1

-0.1

0.0001

0.001

0.01

t (s)

0.1

1


10

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Langmuir

(a) (i)

(ii)

Au

Au

Au electrode

SAM modified Au electrode

SAM Au Reconstituted PSII film on Au electrode

(b)

Reconstituted PSII film

Au 100 nm

0.07

(c)

0.06

Absorbance

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

0.05 0.04 0.03 0.02 0.01 0 600

620 640 Plus Environment 660 680 ACS Paragon Wavelength (nm)

700

720

(a)

Photocurrent (μA/cm2)

Page 34 of 35

6 4 2 0 -2 -0.4

-0.2 0 0.2 0.4 Applied potential (V) vs. Ag/AgCl in sat. KCl

(b)

Absorption (a.u.)

Photocurrent (a.u.)

640

660 680 Wavelength (nm)

(c)

30

Photocurrent (nA/cm2)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42

Langmuir

8

ON

OFF

ON

OFF

700

ON

OFF

25 20 15 10 5 ACS Paragon Plus Environment

0 0

5

10

15 20 Time (s)

25

30

35

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1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42

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DPC+

DPC

light

e-

Pt

Pt

Pt

Mercaptofunctionalized Au substrate


Photocurrent Generation of Reconstituted Photosystem II on a Self

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