Oxygen-Evolving Porous Glass Plates Containing the Photosynthetic

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Oxygen-Evolving Porous Glass Plates Containing the Photosynthetic Photosystem II Pigment-Protein Complex Tomoyasu Noji, Keisuke Kawakami, Jian-Ren Shen, Takehisa Dewa, Mamoru Nango, Nobuo Kamiya, Shigeru Itoh, and Tetsuro Jin Langmuir, Just Accepted Manuscript • DOI: 10.1021/acs.langmuir.6b02106 • Publication Date (Web): 11 Jul 2016 Downloaded from http://pubs.acs.org on July 14, 2016

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Oxygen-Evolving Porous Glass Plates Containing the Photosynthetic Photosystem II Pigment-Protein Complex Tomoyasu Noji1* • Keisuke Kawakami1 • Jian-Ren Shen2 • Takehisa Dewa3 • Mamoru Nango1 • Nobuo Kamiya1 • Shigeru Itoh4• Tetsuro Jin5 1

The OCU Advanced Research Institute for Natural Science & Technology (OCARINA), Osaka City University, 3-3-138 Sugimoto, Sumiyoshi-ku, Osaka 558-8585, Japan

2

Photosynthesis Research Center, Graduate School of Natural Science and Technology/Faculty of Science, Okayama University, Okayama 700-8530, Japan 3

Department of Frontier Materials, Graduate School of Engineering, Nagoya Institute of Technology, Gokiso-cho, Showa-ku, Nagoya 466-8555, Japan

4

Graduate School of Science, Nagoya University, Furo-cho, Chikusa-ku Nagoya, Aichi 4648602, Japan

5

Research Institute for Ubiquitous Energy Device, National Institute of Advanced Industrial Science and Technology (AIST), 1-8-31, Midorigaoka, Ikeda, Osaka 563-8577, Japan

ACS Paragon Plus Environment

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KEYWORDS artificial photosynthesis, photosystem II, oxygen evolution, porous glass, nanopore

ABSTRACT

The development of artificial photosynthesis has focused on the efficient coupling of reaction at photo-anode and cathode, wherein the production of hydrogen (or energy carriers) is coupled to the electrons derived from water-splitting reactions. The natural photosystem II (PSII) complex splits water efficiently using light energy. The PSII complex is a large pigment-protein complex (20 nm diameter) containing a manganese cluster. A new photoanodic device was constructed incorporating stable PSII purified from a cyanobacterium Thermosynechococcus vulcanus through immobilization within 20- or 50-nm nanopores contained in porous glass plates (PGPs). PSII in the nanopores retained its native structure and high photo-induced water splitting activity. The photocatalytic rate (turnover frequency) of PSII in PGP was enhanced 11-fold compared to that in solution, yielding a rate of 50–300 mol e−/(mol PSII•s) with 2,6-dichloroindophenol (DCIP) as an electron acceptor. The PGP system realized high local concentrations of PSII and DCIP to enhance the collisional reactions in nanotubes with low disturbance of light penetration. The system allows direct visualization/determination of the reaction inside the nanotubes, which contributes to optimize the local reaction condition. The PSII/PGP device will substantively contribute to the construction of artificial photosynthesis using water as the ultimate electron source.


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Introduction The construction of efficient artificial photosynthetic systems is urgently needed for the production of renewable energies, e.g., solar hydrogen and energy carriers. One goal for sustainable artificial photosynthesis systems has been the development of a photo-anode and cathode wherein a water-splitting reaction and a reductive reaction to generate hydrogen (or energy carriers) are well coupled.1 Mimicking the strategy of natural oxygenic photosynthesis is considered a likely promising approach for this purpose. During natural photosynthesis, light reactions are achieved by 2 types of photosystems located on thylakoid membranes.2 Photosystem II (PSII) performs a light-induced water-splitting reaction to supply electrons to Photosystem I (PSI) via electron mediators, whereas PSI produces the energy-rich coenzyme NADPH by its photoreaction.2 PSII, which has been purified from the thermophilic cyanobacterium Thermosynechococcus vulcanus, is very stable pigment-protein complex compared with that from higher plants, and its molecular structure is fully known in detail (a molecular mass of 756 kDa, and a diameter of ~20 nm (Figure 1a) that comprises a dimer of two monomeric units).3-5 A photon absorbed by one of the antenna chlorophyll (Chl a) molecules on antenna moiety is transferred to a special pair of Chl a molecules (P680) in the reaction center moiety, followed by charge separation to generate oxidized P680 (P680+) and a reduced primary electron acceptor plastoquinone (QA−) with the high quantum yield of approximately 90%.6 In vivo, P680+ oxidizes H2O through the function of the Mn4CaO5 cluster via the Kok cycle, whereas QA− transfers an electron to the secondary quinone acceptor QB, which acts as a mobile electron carrier for the cytochrome b6f complex.2 In vitro, the electrons


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produced by PSII can be extracted by the collisional reaction between quinones and artificial electron acceptors such as 2,6-dichloroindophenol (DCIP) in the medium (Figure 1a).7

Figure 1. Immobilization of PSII in porous glass plates (PGPs) with nanopores. Schematic illustrations of PGP (a) and photoreactions of PSII with water (electron donor) and DCIP (electron acceptor) (b). (c) Photograph of a PSII-containing PGP with 50-nm pore diameters (PGP50). The PSII structure was drawn using PyMOL software with the protein database file 3WU2.3

PSII, which can be regarded as a high-performance nanodevice for the photoanodic reaction that generates electrons through water oxidation,1 will be a potential candidate for the catalytic element of photoanodes if it could be properly immobilized on a substrate.8 For this purpose, PSII has been immobilized on various substrates such as gold nanoparticles,9 mesoporous silica materials,10 and IO-mesoITO electrodes.8 PSII was shown to maintain its activity even after being immobilized on these artificial materials; the immobilization provided various benefits as


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well.8-10 For example, PSII immobilized inside internal pores with a 23-nm average diameter in the mesoporous (Santa Barbara Amorphous; SBA) silica material retained its intact structure, maintained the durability of its high oxygen-evolution activity, and acquired increased thermal stability.10 Therefore, the function of the oxygen-evolving complex and the electron-transfer chain are not markedly affected by immobilization on a substrate as far as the structure of PSII is maintained because the Mn4CaO5 cluster and electron transfer chain of PSII are located in the center of the PSII complex. The limited diffusion rate of reactants (phenyl-p-benzoquinone; BQ) reduced by PSII, on the other hand, appeared to limit the overall reaction rate of the immobilized PSII.10 However, it has been difficult to assay the reactions inside nanocavities in experiments using suspensions of micrometer-size PSII-SBA powder. In this study, we realized a direct assay by using a plate-like porous material that can include PSII with excellent transparency. It seems to serve as an effective new platform for reactions of various incorporated photoactive molecules too. Porous glass plates (PGPs) have size-controllable nanopores (3–80 nm) that penetrate the thin plate (Figure 1b). It has a large inner surface area and is transparent in the visible to near infrared light regions.11-14 We have previously reported the generation of a light-induced hydrogenproduction system (named RMH/PGP50) that was constructed in the nanopores of PGPs with 50-nm pore diameters (PGP50) by the introduction of the tris(2,2′-bipyridine)ruthenium(II) complex (Ru(bpy)32+), methyl viologen (MV2+), and [NiFe]-hydrogenase as a photosensitizer, electron mediator, and catalyst, respectively.11 The RMH/PGP50 system efficiently generated hydrogen

powered

by

light

in

the

presence

of

the

sacrificial

electron

donor

ethylenediaminetetraacetic acid. Notably, the activity under aerobic condition measured as the H2-evolution was 3,000-fold higher than that observed in the aerobic solution system without


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PGP.11 This drastic increase of activity was enabled by the use of a high-density photoreaction system of MV that was realized inside the PGP nanocavities. Although MV appeared to function as an artificial co-factor to provide reducing power both for the hydrogenase and for the scavenging of oxygen inside the nanopores, the reaction conditions could be designed only by trial and error owing to the difficulty of direct monitoring of the MV reactions. In this study, we established the efficient reaction conditions of PSII with the electron acceptor DCIP inside the nanocavities in PGPs by directly monitoring the color change of DCIP. To effect this improvement, we constructed a photoanodic reaction system composed of PSII and DCIP in the nanopores inside PGPs of 1-mm thickness with 20- or 50-nm pore diameter (PGP20 or PGP50, respectively). Highly purified PSII with low impurities comparable to crystallizationgrade was used in this study so that we can discuss mechanism of reaction precisely in molecular level. The system yielded a prominently accelerated reaction rate compared to that in the outer medium. The experimental conditions were carefully designed based on the in situ measurements of the DCIP reaction with PSII, and indicated that PGPs are useful as potential platforms for the coupling of photo-anode and cathode systems. The reduced DCIP produced inside the PSII/PGPs will likely be useful as an electron mediator for other systems such as PSI or PSI in association with hydrogen-production catalysts,15-19 assuming the latter can also be incorporated into the same nanocavities in the future.

Materials and Methods Preparation of PSII-immobilized PGPs PGPs of 1 mm × 4 cm × 4 cm in size and an average inner pore diameter of 20 (PGP20) or 50 nm (PGP50) were prepared by acid leaching of phase-separated borosilicate glass.13,14 The


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composition of the mother glass plate of PGP50 was 62.5SiO2-28.3B2O3-9.2Na2O (wt %), whereas that of PGP20 was 62.5SiO2-28.3B2O37.2Na2O-3.0Al2O3 (wt %). The mother glasses were first melted in a platinum crucible. Then, the mother glasses for PGP50 and PGP20 were heated to 610°C for 32 h and 575 °C for 20 h for phase separation, respectively. The phaseseparated glasses were leached in 1N sulfuric acid at 90°C for 2 days. The pore volumes and specific surface areas of the PGPs as estimated by mercury-penetration methods were 0.42 (PGP20) and 0.323 cm3/g (PGP50), and 140 (PGP20) and 35.2 m2/g (PGP50), respectively.11,13,14 The light-receiving areas of PGP20 and PGP50 were 7.83 and 8.4 cm2/g, respectively. The isolation of PSII from T. vulcanus was performed as described previously.3 To immobilize PSII in PGPs, a stock solution of PSII was diluted to give 1.0 mg Chl/ml with a 20 mM MESNaOH (pH 6.5) buffer containing 20 mM NaCl, 20 mM CaCl2, and 0.4 M sucrose (medium A) together with 0.1% final concentration (w/v) of n-dodecyl-β-D-maltoside (DDM). The resulting solution containing PSII was then stirred on ice for 5 min. The solution was diluted 2-fold in medium A, followed by dilution of the DDM concentration to 0.03% (w/v), using Amicon Ultra15, 50,000 MWCO filtration devices (Millipore Co., Billerica, MA, USA). The final concentration of PSII was adjusted to 0.15 mg Chl/ml by adding medium A containing 0.03% (w/v) DDM. PGPs with adsorbed PSII and DCIP (PSII-DCIP/PGPs) were prepared as follows. Fragmented PGPs were immersed in the aforementioned solution of PSII containing 2 mM DCIP at 150 mg PGP/ml and equilibrated for 30 min at 25°C in the dark. The resulting PSII-DCIP/PGPs were carefully washed with medium A. PGPs adsorbed with either DCIP (DCIP/PGPs) or PSII (PSII/PGPs) were prepared in a similar manner. PSII/PGPs were used to measure the fluorescence yield of Chl as described below. The absorption spectra of PSII in PGPs measured


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in medium A were corrected for the effect of PGP-dependent light scattering by subtracting the spectra of PGP alone. The amount of PSII immobilized inside the PGPs was estimated by measuring the absorbance at 674 nm of PSII-containing PGPs using an extinction coefficient of the PSII dimer of 5.1 (µM•cm)-1.20 Absorption spectra were acquired with a double-beam spectrophotometer (UV-3100PC, SHIMADZU, Kyoto, Japan).

Reduction of the primary electron acceptor plastoquinone QA as monitored by the yield change of flash-induced Chl a fluorescence on PSII The light-induced electron transfer activity required to reduce QA in PSII by the electrons from the water splitting system was monitored by measuring the flash-induced yield of Chl fluorescence which is almost proportional to QA- content. The fluorescence yield was measured with a pulse-amplitude-modulation (PAM) fluorometer (PAM-101; Walz, Effeltrich, Germany),21,22 equipped with an excitation flash unit (FL-103/E; Walz) and a modulation measurement light (1.6 kHz) unit (PAM-103; Walz) with a saturation level of 0.8–1.0 of the excitation flash at an intensity level 3 of the modulation light. PSII/PGP20 or free PSII suspended in medium A containing 0.03% (w/v) DDM at concentrations equivalent to approximately 0.03 mg Chl/ml, was placed in a glass cuvette with a light path length of 2 mm. The fluorescence yields of PSII/PGP20 over long periods were measured by providing saturated flash pulses sequentially every 1 min for ~60 min.

Observation of DCIP photoreduction in PSII-DCIP/PGP50 under low-light conditions Color changes of DCIP inside the PSII-DCIP/PGP50 in medium A were monitored by capturing photographic images under weak illumination from white fluorescent lamps at 4 µmol/(m2•s) on the plate surface. The color changes were monitored with a digital camera (SP-


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590UZ; Olympus Imaging Corp., Tokyo, Japan) after removing the PSII-DCIP/PGPs from the reaction medium. The reactions were allowed to proceed further by replacing the complexes into the medium.

Measurement of PSII activity in PSII-DCIP/PGPs under intense light The photoreduction activities of PSII-DCIP/PGPs induced by intense light were measured at 25°C by monitoring the changes of the DCIP absorbance inside PSII-DCIP/PGPs with a spectrophotometer through optical fibers (EPP2000-VIS-50, StellarNet, Inc., Tampa, FL, USA), without stirring of the reaction medium. Light from a 550-W halogen lamp passing through a layer of water (14 cm) and optical filters (an HA50 heat-cut [Hoya Candeo Optronics, Toda, Japan] and a Toshiba Y-43 UV-cut [Toshiba, Tokyo, Japan]), provided white light in the range of 430–750 nm to the PSII-DCIP/PGPs immersed in medium A. Alternatively, the light was passed through reaction media containing free PSII (30 µg Chl/ml) and DCIP (0.1, 0.3, 0.5, 0.7, 0.9, or 1.1 mM) in medium A containing 0.03% (w/v) DDM. The optical path lengths of the cuvettes containing the reaction medium for PSII-DCIP/PGPs and the solution system of free PSII were 2 and 1 mm, respectively. The absorption spectra of PSII-DCIP/PGPs were measured with a UV-3100PC spectrophotometer prior to the irradiation. Although the PSII complexes used in this study were primarily in the dimeric form.3 if not indicated otherwise, the PSII activities were expressed on the basis of PSII monomer units in this study. Releases of PSII and DCIP from the PSII-DCIP/PGPs were not detectable during the measurements.

Double reciprocal plot of the rate versus DCIP concentration in the lower concentration range


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To determine the value of the Michaelis constant of the DCIP reduction and Vmax values, the DCIP reduction activity of PSII was measured in the low DCIP concentration range. Accordingly, cuvettes of 10-mm optical path lengths containing the reaction medium were irradiated by the white light as previously described with stirring.

pH dependence of oxygen-evolution activity Oxygen evolution was measured at 25°C using a Clark-type electrode (Rank Brothers, Ltd., Cambridge, England), as reported.10 Red light from a 550 W halogen lamp passed through a UVcut and red-pass filter (Y43, Toshiba, Tokyo, Japan), a heat-cut filter (HA-50, Hoya, Saitama, Japan), and a 14-cm water layer, and then illuminated a 1 cm-diameter reaction vessel containing 1 ml of reaction mixture. The reaction mixture contained 20 mM MES-NaOH (pH 6.5), 20 mM CaCl2, 20 mM NaCl, 0.4 M sucrose, and 3 µg Chl/ml PSII, together with 0.5 mM phenyl-pbenzoquinone (BQ) as the external electron acceptor.

Results and Discussion Adsorption of PSII and DCIP into the PGP nanopores The dry PGPs are opaque white due to the light scattering from the nanopores.11 When immersed in PSII solution, PGP50 acquired a strong green color and became transparent by incorporating water and PSII into the nanopores. A representative green fragment of plate containing PSII is shown in Figure 1c. The fragment showed the dense green color of PSII especially in the regions beneath the plate surfaces. Figure 2 shows the distribution of PSII in 1-mm thick PGPs as measured by detecting Chl fluorescence, using a confocal laser-scanning microscope. The cross-sectional profiles of


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fluorescence intensity clearly showed that PSII was preferentially adsorbed within ~0.2-mm depth from the surfaces in the 1-mm total thicknesses of PGP20 or PGP50.

Figure 2. Fluorescence-intensity profiles of PSII across PGP20 (solid line) and PGP50 (broken line). The fluorescence of PSII at 680 nm was measured using a confocal laser-scanning microscope. The distance from the surface of a 1-mm PGP is shown. The inset shows a crosssectional photographic image of PSII/PGP50.

Figure 3 shows the raw absorption spectra of PSII in PGP20 (solid black line), PSII in PGP50 (solid red line), PGP20 (dashed black line), and PGP50 (dashed red line) in a medium A containing 20 mM sodium ascorbate. The absorption of DCIP was eliminated by the presence of ascorbate to evaluate the amount of PSII immobilized in PGPs. The inset shows the absorption spectra, corrected for the effect of PGP-dependent light scattering, of PSII after its immobilization inside PGPs. The spectral profiles of PSII in PGP20 (solid black line) and PGP50


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(solid red line) were identical to that of PSII in solution (green line) after normalization at their peaks, suggesting that PSII maintains almost intact core antenna pigments of Chl a and carotenoids, as well as an intact structure inside the nanoporous environments. The amounts of adsorbed PSII in the PGPs were estimated to be 0.58 and 2.73 nmol PSII/g PGP for PGP20 and PGP50, respectively (Table 1) from the absorbance at 674 nm. (PSII contents are expressed on the basis of the monomer unit hereafter). It is likely that the apparent binding affinities of PSII to the nanopores depend on the pore size because the quantity of PSII bound to PGP50 was larger than that bound to PGP20. It seems that the small pore diameter of PGP20, which is comparable to the outer size of the PSII dimer (20 × 13 × 11 nm),3 limited the affinity and/or accessibility of PSII to the pores. When PSII/PGPs were soaked again in a solution containing DDM without PSII, no desorption of PSII was observed in either case suggesting the tight binding of PSII to the nanopores in the PGP surface regions. It has been suggested that the trans-membrane hydrophobic region of photosystem I has better affinity to the pore wall in a mesoporous silica material.23 PSII might be also immobilized by the interaction between the trans-membrane hydrophobic region of PSII and the inner surface of PGPs. The amount of DCIP immobilized in PSII-DCIP/PGPs was estimated from difference spectra of the absorbance of PSII-DCIP/PGPs before and after adding ascorbate. The amount of DCIP per unit weight of PGP, the DCIP concentration within the pore, and the amount of DCIP per unit of light-receiving area were almost the same for PGP20 and PGP50 (Table 1). This result also indicated that the small molecule DCIP was able to penetrate deeper into the inner pores of PGPs. DCIP adsorbed onto PGP20 and PGP50 both at the surface and the deeper central regions (Figure 6b).


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Table 1. Quantity of PSII and DCIP adsorbed in the pores inside PGPs

a

and photoreduction

activities

Pore volume

Photoreaction system

3

PSII

Lightreceiving area

(cm /g PGP)

(cm2/g PGP)

nmol/ µM g PGP

PGP20 b, f

0.42

7.8

0.58

PGP50 b, f

0.32

8.4

Solution c, f

-

Solution c, f

-

DCIP

TOF

nmol/ cm2 e

Ratio of DCIP/ PSII

(mol e/[mol PSII •s])

pmol/ cm2 e

µmol/ mM g PGP

1.4d

73

0.22

0.52d

28

380

300

2.73

8.5d

330

0.20

0.6d

23

70

50

-

-

0.94

94

-

0.3

30

320

28

-

-

0.94

94

-

0.5

50

530

34

a The amount of adsorbed PSII and DCIP in PGPs was estimated from the absorbance of the PSII-DCIP/PGPs. The nanopore concentrations and densities of PSII and DCIP per lightreceiving area were estimated from the pore volumes and specific surface areas of the PGPs. b A 1-mm thick plate was used. c A quartz cell with a 1-mm light path was used. d Concentration inside the pores, without considering the heterogonous distribution of PSII in nanopores. e Density per light-receiving area. PSII concentration was expressed as the monomer concentration. f The standard deviation of photoreaction systems in PGP20, PGP50, and solution were 20%, 40%, and 7%, respectively.


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Figure 3. Absorption spectra of PSII-DCIP/PGP20 (solid black line), PSII-DCIP/PGP50 (solid red line), PGP20 (dashed black line), and PGP50 (dashed red line) in medium A containing 20 mM sodium ascorbate. In the measurement DCIP was fully reduced by adding ascorbate to eliminate its contribution to the absorption spectra. The inset shows the absorption spectra of PSII in PGP20 (black), PSII in PGP50 (red), and PSII dissolved in medium A containing 0.03% (w/v) DDM (green line). All the spectra were corrected for the effect of PGP-dependent light scattering and normalized with respect to the 674 nm peak height.

Primary charge-separation activity of PSIIs bound inside the PGP nanopores To test the integrity of the photochemical function of PSIIs in PGPs, the yield of charge separation in PSII was estimated by the change of fluorescence yield of Chl a, which is known to almost proportional to the extent of QA reduction,22 using a PAM fluorometer. The fluorescence yield was monitored by the modulated (at 1.6 kHz) excitation light, which was attenuated to be weak enough to induce no appreciable charge separation. The fluorescence yields in the closed


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and open state traps of the PSII reaction center are known to be high and low, respectively, and are designated as Fm and Fo levels. These levels can be measured with and without the overlaid irradiation of a saturated-intensity single-turnover flash, respectively. The yield of charge separation, then, can be evaluated as the normalized ratio (Fm – Fo)/Fm.22 Figure 4a and b show the changes of the fluorescence yield as measured for PSII in solution and for the PSII/PGP20 system, respectively. The strong flash increases the fluorescence yield to the maximum Fm level by producing QA−.22 Subsequent charge recombination in the dark re-oxidizes QA− (Figure 4d),24 and decreases the fluorescence yield to the low Fo level again.22 It has been reported that the quantum yield of charge separation was decreased, and the kinetics of QA− re-oxidation were slowed by denaturing the oxygen-evolving complex of PSII.25 The yield of charge-separation of PSII for PSII/PGP20 was calculated as 0.89 ± 0.04, which is comparable to that for PSII in the solution system (0.91 ± 0.03). The kinetics of QA− re-oxidation in PSII/PGP20 were similar to those of PSII in solution. These results indicated that the activity and structure of PSII were well maintained in the nanoporous environment. Similar flash-responses could be repeated for over 60 min (Figure 4c), suggesting that multiple cycles of charge separation and recombination occurred in the PSII that were immobilized in the PGP nanopores. This result resembles that reported for the other porous silica materials that contained photosynthetic membrane proteins such as RC (purple bacterial reaction center)-FSM,26 and PSI-NAM.23


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Figure 4. Flash-induced reduction and dark re-oxidation of the electron acceptor plastoquinone QA in PSII. (a) PSII in a solution system; and (b) in PGP20. Changes of the Chl fluorescence yield, which represents the QA− amount, upon saturating-intensity flash excitation was measured with a PAM fluorometer as described in Materials and Methods. Fo and Fm represent the lowest and highest fluorescence yields, indicating the open- and closed-states of PSII, respectively. (c) Changes of Chl fluorescence yields in PSII/PGP20 during sequential (every 1 min) excitation of flashes for ~60 min. (d) Reactions in PSII after the flash excitation.24

Reduction of DCIP by PSII PSII in the medium efficiently reduced DCIP under illumination (Figure 5). The activity increased with the increase of concentration of DCIP up to 0.9 mM, indicating the rather low affinity of DCIP to PSII (Figure 5a). Although a higher activity was expected at the higher DCIP concentrations based on the enhancement effect in the lower concentration range, the activity decreased above 0.9 mM (turnover frequency [TOF] = 36 ± 1 mol e−/[mol PSII •s]; Figure 5a). Although a straight line was not obtained in the double reciprocal plot of the rates versus DCIP concentration, extrapolation of the dependency in the lower (< 0.15 mM) DCIP concentration


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range (Figure 5b) to the infinite value gives the Km and Vmax values for DCIP to be 1.3 ± 0.5 mM and 220 ± 40 mol e−/(mol PSII •s), respectively. The decrease in activity in the higher concentration range, which was rather significant, can be assumed to be caused by the attenuation of the excitation light intensity screened by the high concentration of DCIP, because the transmittance of the solution at 674 nm is only 20% at 0.9 mM DCIP even with the 1-mm light path, as seen from the scale on the right hand ordinate. The photoactivity of isolated PSIIs at the high DCIP concentrations, therefore, is limited by the light-shielding effect of DCIP, and can never attain the high TOF value of 100–400 s-1 detected in intact PSII in vivo.27 However, if the shielding effect is avoided, we can expect the higher rates at the higher DCIP concentrations; therefore, it is of interest to determine how the photoreduction of DCIP by PSII is modified inside PGPs.


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Figure 5. (a) Dependence of the DCIP-reduction activity of PSII on the DCIP concentration (left axis) and the transmittance at 674 nm of the DCIP solutions (right axis). White light was irradiated onto a cuvette with 1-mm optical path length containing the reaction medium without stirring. (b) The double reciprocal plot of the rates versus DCIP concentrations in the concentration range. A straight line was drawn for the points at DCIP concentrations lower than 0.15 mM, where a reaction medium in a cuvette with 10-mm optical path length was irradiated with stirring. The red and green circles represent the rates for PSII-DCIP/PGP20 and PSIIDCIP/PGP50, respectively, versus the concentration values of DCIP assumed as described in text. The horizontal broken line represents Vmax.


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Visualization of light-induced reduction of DCIP in PSII/DCIP/PGP The photoreduction of DCIP by PSII in PGP can be detected as the color change of DCIP even by the naked eye. Figure 6a shows a top view of the PSII-DCIP/PGP50 complex prior to irradiation. PGP50 exhibited the green color of PSII on the surface regions on both sides but not in the central region, whereas it showed the blue color of the oxidized form of DCIP both on the edge and in the central regions (Figure 6b, ii′). A small molecule such as DCIP, thus, was able to penetrate into the nanopores in the central region of PGP50 almost freely. Therefore, the poor binding of PSII in the central region appears to arise from the difficulty of PSII in penetrating into the nanopores even though the pore diameters are comparable or a little larger than the PSII diameter. The situation seems to be comparable to a car traffic jam in a network of narrow roads. In contrast, DCIP might instead function like a motorcycle that can proceed deeper into the network even among the large PSII inside the narrow pores.


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Figure 6. Photographic images of a PSII-DCIP/PGP50 under light excitation. (a) Top view of the plate fragment before illumination. (b) Color changes of PSII-DCIP/PGP50 induced by light excitation with a white fluorescence lamp at the intensity of 4 µmol m-2 s-1. (i) and (i′), top and cross-sectional views of a dry PGP50 without PSII and DCIP, respectively. (ii-iv), Images of a PSII-DCIP/PGP50 conjugate at the position indicated by the circle in (a) after sequential irradiations for 0 (ii), 30 (iii), and 60 (iv) min. (ii′-iv′), Cross-sectional images at corresponding times to those above. The blue color in the oxidized DCIP (ii and ii′) can be seen to have gradually vanished from the illumination concomitant with the photoreduction of DCIP by PSII (iii′ and iv′).


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The DCIP gradually lost its blue color after illumination with a fluorescent lamp at the low intensity of 4 µmol/(m2•s) for 0, 30, and 60 min (Figure 6b, ii-iv). The cross-sectional images (Figure 6b, ii′-iv′) showed that the DCIP blue color was bleached rapidly in the surface regions containing high PSII density (Figure 6b, iii′), and more slowly in the central region (Figure 6b, iv′). After 60 min illumination, all DCIP in the surface and center of PGP lost its blue color, indicating its full reduction (Figure 6b, iv′). However, DCIP reduction occurred more slowly in the central regions where there was a lesser quantity of PSII. We can, therefore, assume that the PSII reduces DCIP molecules directly in the surface regions and, then, electrons were transferred to the DCIP molecules in the central region either by slow DCIP diffusion or by an exchange of electrons between DCIP molecules in the nanopores. Similar fast and slow DCIP reduction/oxidation patterns were also observed with PSII-DCIP/PGP20 (data not shown).

DCIP reduction activity of PSII in PGP nanopores under continuous illumination The extent of photoreduction of DCIP by PSII in PGP was determined by the decrease of the DCIP absorption band. The peaks of the absorption bands of DCIP in reaction medium, PGP20, and PGP50 were detected at 600, 544 and 565 nm, respectively (Figure 7a). The different peak wavelengths suggest that the nanopores in PGPs elicited different local pH values or different binding conditions for DCIP because the absorption band of DCIP in outer medium shifted depending on the medium pH (data not shown). The pH range inside PGP20 and PGP50 was estimated to be 5.5–6.0, based on the absorption peak wavelengths of DCIP/PGPs. The local pH inside PGPs thus seemed to be lower than that in the outer bulk solution owing to the interaction with silanol groups (Si-OH) abundant on the PGP surfaces as has been reported for SBA, which inside pH was estimated to be lower by 0.1–0.2 units compared to that in the outer medium.10


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The shift, however, did not seem to affect oxygen evolution activity by itself in reaction medium at pH 6.5 because the oxygen-evolving activity of PSII of T. vulcanus was almost independent of the medium pH at pH 5.5–6.5 (Figure 7b). Blue color of DCIP inside PGPs was eliminated after PSII-DCIP/PGPs or DCIP/PGPs were soaked in medium A containing 20 mM sodium ascorbate so that the colorimetric ability of DCIP was maintained even inside PGPs.

Figure. 7. (a) Absorption spectrum of DCIP in solution (black line), in PGP20 (red line), and in PGP50 (blue line). (b) pH dependence of the oxygen-evolution activity of PSII.

The absorption of DCIP (at 0.3 mM) decreased continuously (black square in Figure 8) upon the illumination of the medium containing PSII, exhibiting the reducing activity of 14 mol DCIP/(mol PSII•s) with TOFs of 28 mol e−/(mol PSII •s) as listed in Table 1. Approximately 40% of DCIP was reduced within 1 s in PGP20 (red squares in Figure 8) and PGP50 (green squares in Figure 8). PSII complexes in 1-mm thick PGPs were located within a ~0.2-mm depth from both surfaces (i.e. 40% of the 1-mm thickness) as shown in Figure 2. Therefore, these


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results indicated that the reduction of DCIP was complete by 1 s in the region containing high PSII density. The DCIP reduction was fast at the beginning but then became markedly slower in both PGP20 and PGP50 (Figure 8 and inset). The rate constants for the fast phase in the surface regions were estimated to be about 300 and 50 mol e−/(mol PSII•s) for PGP20 and PGP50, respectively (Table 1). The rates in the PGPs, thus, were 1.8–11 fold higher compared to the steady state rate in the solution. The activity is also similar to or higher than that reported for PSII immobilized on IO-mesoITO (TOF = 48 mol e−/(mol PSII •s)).8 These results indicate that the PSII/PGP system provides a more effective photoreaction field than does the PSII solution system. We also measured oxygen concentrations in the medium outside of PSII-DCIP/PGPs using an oxygen electrode (Figure S1, Supporting Information) during light irradiation to directly monitor oxygen evolution. The amount of evolved oxygen nearly equaled that of reduced DCIP determined by the spectroscopic method. This result suggested that the function of the Mn4CaO5 cluster of PSII was maintained and that electrons extracted from water via PSII reduced DCIP. The rapid oxygen evolution achieved in pores within PSII-DCIP/PGPs could not be detected directly due to the slow responsivity of oxygen electrode and the limited oxygen exchange occurring at the interface between the outer medium and the pores inside PGPs. Therefore, the oxygen-evolution activity of PSII-DCIP/PGPs measured by an oxygen electrode was underestimated at a value of 20–30 mol e−/(mol PSII•s). We repeatedly measured the oxygenevolution activity of PSII-DCIP/PGP50. After the second measurement, DCIP was supplied to PSII-DCIP/PGP by immersing PSII-DCIP/PGP50 into medium A containing 2 mM DCIP for 30 min. The oxygen-evolution activity at the fourth measurement was approximately 70% that of the first measurement. The turnover number of PSII-DCIP/PGPs was estimated to be ≥2000 mol


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e-/mol PSII by the end of the fourth measurement. Thus, the durability of PSII-PGPs can be estimated to be ~104 mol e-/mol PSII as reported for PSII immobilized in SBA.10

Figure. 8. Photoreduction of DCIP in PSII-DCIP/PGPs and in PSII solution under illumination. Red and green lines represent the absorption changes of DCIP in PSII-DCIP/PGP20 (monitored at 544 nm) and PSII-DCIP/PGP50 (at 565 nm), respectively. A black line represents the lightinduced absorption change in the PSII solution at 600 nm in medium A that contained 0.03 mg Chl/ml of PSII, 0.03% (w/v) DDM, and 0.3 mM DCIP (monitored at 600 nm). Light path lengths were 1-mm (thickness) in PGP and 1 mm in PSII solution. The inset shows the time courses for longer periods. The horizontal broken line represents the absorbance when 40% of the DCIP immobilized on PSII-DCIP/PGPs is reduced. The solid line represents the absorbance after DCIP was completely reduced.


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In PGP20 and PGP50, the local concentration of PSII in the nanopores could be estimated to be 1.4 µM and 8.5 µM, respectively, The former was comparable to that in the solution system (at 0.94 µM), and the latter 9 times higher. The local concentration of PSII in the surface regions might be ~2.5 times higher when the inhomogeneous distribution of PSII across the PGP plate, as seen in Figure 2, is taken into account. However, the PSII concentration difference does not interpret the large difference in the observed reduction rates. Therefore, the enhanced seems to have occurred by another reason. The quantities of DCIP per unit of light-receiving area for the PGP20, PGP50, and aqueous solution systems were 28, 23, and 30 nmol/cm2, respectively (Table 1). However, the effective DCIP concentrations inside the small pores of PGP20 and PGP50 can be estimated to be higher at 0.52 mM and 0.60 mM, respectively (Table 1). If we extrapolate the dependency at the lower concentration range in Fig. 5(b), DCIP-reduction activity of PSII in solution should reach to 67 mol e-/(mol PSII•s) at 0.56 mM DCIP if the decline due to the light shielding effect of DCIP can be avoided somehow. Actually the DCIPreduction activity of PSII in solution at 0.5 mM DCIP was measured to be 34 mol e-/(mol PSII•s) suggesting the light shielding by DCIP of 51% (= 34/67 × 100), as estimated from the 44% transmittance of 0.5 mM DCIP solution. The light-shielding effect of DCIP, thus, well interprets the activity loss in the higher DCIP concentration range. The DCIP-reduction activity measured with PSII-DCIP/PGP50 (50 mol e-/(mol PSII•s), a green circle in Figure 5(b)) was 75% of 67 mol e-/(mol PSII•s) value predicted for the nonshielding condition. This percentage is similar to a transmittance value of 69% measured at 0.23 mM DCIP (Figure 5(a)), at a concentration in PSII-DCIP/PGP50 estimated with thickness of 1 mm and the amounts of DCIP per unit of light-receiving area (23 nmol/cm2) as listed in Table 1.


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This result supports the idea that the enhancement of the DCIP photoreduction rate in PGP occurred from the increase of the local concentrations of DCIP with low screening effect. Another experimental value in PSII-DCIP/PGP20 at 300 ± 60 mol e-/(mol PSII•s) is also shown as a red circle in Figure. 5(b). The high value was almost comparable to Vmax = 220 ± 40 mol e−/(mol PSII •s) obtained in Figure. 5(b). Therefore, it seems to be concluded that if the light-shielding effect of DCIP is avoided, the activity at the high concentration reaches to the catalytic maxima. It is noted that the light-shielding by the blue-shifted absorbance band of DCIP in PSII-DCIP/PGP20 should be lower because of lower overlap with Chl (Figure 7(a)). This high activity indicates that increasing the collision/association rate between DCIP and PSII because the value of the activity over 67 mol e-/(mol PSII•s) predicted for the non-shielding condition. Thus, it is concluded that the photoreduction activity of PSII in PGP can be increased inside nanopores by the high local DCIP concentration. The high local DCIP concentration inside nanopores enables the high activity with little light-shielding effect and improvement of the collision/association rate.

Figure 9 shows a model that explains the higher activity of PSII in PGP. The reaction rate of DCIP with PSII is expected to be higher at the higher DCIP concentrations because of the rather low affinity of an artificial acceptor DCIP to PSII (Km = 1.3 mM, Figure 5b). The high concentration of DCIP, on the other hand, absorbs the light at 674 nm that excites the Qy band of PSII. Transparency of the solution containing 0.5-0.6 mM DCIP was 44-37% at 674 nm (Figure 5(a)). The shielding-effect by DCIP decreased the DCIP-photoreduction activity to 51% (Figure 5(a) and Figure 9). On the other hand, in the PGP system overall transparency of the


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system was 60-70% even at 0.5-0.6 mM local concentration of DCIP around PSII in nanopores. This facts is understood by Beer-Lambert law: absorbance is not changed if light-pass length is shortened to a half with the twice condensation. High activity of PSII-DCIP/PGPs, thus, comes from the solute condensation with the shorter light-pass length. The DCIP-photoreduction rate per unit of pore volume (∼210 µM s-1), which was calculated from the observed rate, pore volume, and the light-receiving area was, therefore, ~16-fold larger than that in the solution system (13 µM s-1; Figure 10). This result indicates that nanocavities in the thin layer in PGPs provide a special environment favorable for the photoreduction of DCIP by PSII by increasing the local concentration of DCIP and PSII.


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Figure 9. Proposed enhancement mechanism of the photoreductive activity of PSII-DCIP/PGPs. The DCIP concentration is estimated to be 0.2-0.3 mM and 0.5-0.6 mM when the transmittance is 60–70% and 44-37%, respectively, at 674 nm (light-path length of 1 mm). For the PGP systems, the DCIP concentration inside the PGP pores was estimated to be 0.5–0.6, although the apparent overall concentration was 0.2–0.3 mM DCIP. Therefore, the photoreductive activity of PSII in PGPs increased because the situation makes the local concentrations of PSII and DCIP inside the PGPs higher than those in homogenous solution that same amount of PSII and DCIP.


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The TOFs were the photoreduction activities of entities 1, 2, and 3 listed in Table 1 and Figure 5(a). The reduction rate of DCIP per pore volume of PSII-DCIP/PGPs was estimated to be ∼16fold greater than of that of the corresponding PSII solution.

It has been reported that the activity of PSII-mediated oxygen-evolution was not enhanced in nanopores in SBA or in a sol-gel system10,15 in which the activities were determined by the timecourses of light-induced oxygen production detected by an oxygen electrode placed in the outer medium, using artificial electron acceptors such as BQ or DCIP. The overall activities of these PSII-SBA conjugate systems seemed to be limited by the exchange rate of molecular oxygen or electron acceptors between the nanopores in SBA and the outer medium.10 The situation might be similar with the sol-gel system15 and PSII-DCIP/PGPs (Figure S1, Supporting Information).15 On the other hand, the fast reduction rate of DCIP detected in the present study was not limited by the slow diffusion rate of reactants and products because the activities in situ in the nanopores were optically measured directly. It is, therefore, expected that the collisional/association reactions is markedly accelerated if the whole process were carried out inside the same nanopores. For example, the light-driven hydrogen production system inside nanopores in PGPs has worked efficiently 3000-times higher than compared with that of solution under aerobic condition by improvement of the electron transfers among the dense redox components inside nanopores.11 The high activity of PSII-DCIP/PGP20 comes from not only decreasing the shielding-effect by DCIP but also accelerating the collisional/association reactions. It is interesting to use the quinone acceptors without light-shielding effect, instead of DCIP.


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It can be calculated that the PSII in PGP20 or PGP50 turns over ~300 or ~50 times, respectively, for the 2-electron-reduction of DCIP during the 1-s illumination. Then, the oxygenevolution rates in PGP20 and PGP50 would be ~60 and ~40 µmol O2/(m2•s), respectively. This system, therefore, would be expected to generate a robust photocurrent of 1.5–2.3 mA/cm2 if assembled on electrodes, which is almost comparable to or higher than the photocurrent of 0.93 mA/cm2 detected with PSII immobilized on IO-mesoITO.8 Because PGPs can serve as electrodes by introducing TiO2 into nanopores28, the PSII-DCIP/PGP system might be useful as a photoanode in the construction of artificial photosynthesis devices. The oxygen-evolution rate of the PSII-DCIP/PGP system, therefore, is approximately 3-fold that reported as the maximum hydrogen-evolution rate (13 µmol H2/[m2•s]) of RMH/PGP50.11 Therefore, PGPs are useful photoreaction fields of both photo-anodes and photo-cathodes.

Conclusion In this study, a high activity of PSII in PGP nanopores was demonstrated via the color change of DCIP, which coexisted with PSII inside the nanopores. The results extend the possibility shown in our previous study, which indicated a high activity of PSII immobilized in the small particles of SBA.10 Here, PSII immobilized in PGP nanopores was shown to perform the chargeseparation reaction with a high quantum yield of 89%, comparable to that realized in a solution system. The photocatalytic reduction rate of DCIP by PSII was enhanced 1.8–11 fold under nanoporous environments in PGPs, under which the local DCIP concentration can be increased substantially with low disturbance on the light penetration. The reduction rate of DCIP in the nanocavities, thus, can be 16-fold greater than that in solution owing to the high local concentrations of PSII and DCIP in the nanopores that effectively increase the collision/association rate between DCIP and PSII. It is anticipated that the PSII-DCIP/PGP


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system will also perform well in donating electrons to other mediators or electron-utilizing systems such as PSI or PSI combined with hydrogen-production catalysts16-19, especially if they are incorporated into the same nanopores. The PSII-DCIP/PGP system developed in this studywill be useful to produce solar hydrogen using the electrons supplied from water.

AUTHOR INFORMATION Corresponding Author *E-mail: [email protected]

Author Contributions T.N. prepared PSII/PGPs and PSII-DCIP/PGPs, and performed all experiments. K.K., J.-R.S., and N.K. prepared PSII. T.J. prepared and characterized PGPs. T.N., T.D., N.M., and S.I. analyzed the data and prepared the manuscript. All authors approved of the final version of the manuscript.

Funding Sources This study was supported by funding from the Takahashi Industrial and Economic Research Foundation, Iwatani Naoji Foundation, the Japan Prize Foundation, and the Koyanagi Foundation.


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ACKNOWLEDGMENTS All authors thank Dr. Yusuke Daiko and Dr. Tetsuo Yazawa for preparing the porous glass plates. ASSOCIATED CONTENT Supporting information is available free of charge via the Internet at http://acs.pubs.org.

ABBREVIATIONS BQ, phenyl-p-benzoquinone; Chl, chlorophyll; DCIP, 2,6-dichloroindophenol; DDM, n-dodecylβ-D-maltoside; IO-mesoITO, a macroporous inverse opal ITO electrode; ITO, indium tin oxide; MV, methyl viologen; P680, a special pair of Chl a molecules of PSII; PAM, a pulse-amplitudemodulation; PGP, porous glass plate; PSI, photosystem I; PSII, photosystem II; QA, the primary electron acceptor plastoquinone; QB, the secondary quinone acceptor; SBA, Santa Barbara Amorphous; TOF, turnover frequency

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24. Zimmermann, K.; Heck, M.; Frank, J.; Kern, J.; Vass, I. Zouni, A. Herbicide Binding and Thermal Stability of Photosystem II Isolated from Thermosynechococcus elongatus. Biochim. Biophys. Acta 2006, 1757, 106-114 25. Semin, B.K.; Davletshina, L.N.; Ivanov, I.I.; Rubin, A.B.; Seibert, M. Decoupling of the Processes of Molecular Oxygen Synthesis and Electron Transport in Ca2+-Depleted PSII Membranes. Photosynth. Res., 2008, 98, 235-249 26. Oda, I.; Iwaki, M.; Fujita, D.; Tsutsui, Y.; Ishizaka, S.; Dewa, M.; Nango, M.; Kajino, T.; Fukushima, Y.; Itoh, S. Photosynthetic Electron Transfer from Reaction Center Pigment-Protein Complex in Silica Nanopores. Langmuir 2010, 26, 13399-13406 27. Dismukes, G.C.; Brimblecombe, R.; Felton, G.A.; Pryadun, R.S.; Sheats, J.E.; Spiccia, L.; Swiegers, G.F. Development of Bioinspired Mn4O4-Cubane Water Oxidation Catalysts: Lessons from Photosynthesis. Acc. Chem. Res. 2009, 42, 1935-1943 28. Lin, H.; Jin, T.; Dmytruk, A.; Saito, M.; Yazawa, T. Preparation of a porous ITO electrode. J. Photochem. and Photobiol. A: Chem. 2004, 164, 173-177

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Oxygen-Evolving Porous Glass Plates Containing the Photosynthetic

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