Photosystem II–Gold Nanoparticle Conjugate as a Nanodevice for the

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Photosystem II
Gold Nanoparticle Conjugate as a Nanodevice for the Development of Artificial Light-Driven Water-Splitting Systems Tomoyasu Noji,† Hiroyuki Suzuki,‡ Toshiaki Gotoh,† Masako Iwai,§,^ Masahiko Ikeuchi,§ Tatsuya Tomo,‡,|| and Takumi Noguchi*,† †

Division of Material Science, Graduate School of Science, Nagoya University, Furo-cho, Chikusa-ku, Nagoya, 464-8602, Japan Department of Biology, Faculty of Science, Tokyo University of Science, Kagurazaka 1-3, Shinjuku-ku, Tokyo, 162-8601 Japan § Department of Life Sciences (Biology), The University of Tokyo, Komaba 3-8-1, Meguro, Tokyo 153-8902, Japan PRESTO, Japan Science and Technology Agency (JST), 4-1-8 Honcho Kawaguchi, Saitama 332-0012, Japan

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‡

bS Supporting Information ABSTRACT: Photosystem II (PSII) is an enzyme that performs efficient light-driven water oxidation to provide electrons necessary for CO2 fixation in photosynthesis. In this study, we have for the first time generated PSII
gold nanoparticle (GNP) conjugates dispersed in a solution aiming at applications in artificial photosynthesis. PSII core complexes from the thermophilic cyanobacterium Thermosynechococcus elongatus, in which a His-tag was introduced into the C-terminus of CP47, were immobilized on GNPs with a 20 nm diameter via nickel-nitrilotriacetic acid, orienting the electron acceptor side to the gold surface. Optical analysis showed that four to five PSII dimers are bound to a single GNP, which was confirmed by transmission electron microscopy. The PSII immobilized on GNP retained O2 evolution activity comparable to that of free PSII. The PSII
GNP conjugate will be a useful nanodevice for the development of artificial systems for light-driven water splitting into O2 and H2. SECTION: Biophysical Chemistry

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evelopment of efficient artificial photosynthesis systems, which directly produce fuels such as H2 and CH4 utilizing sun light, is crucial to address the energy and environmental problems that mankind faces today.1
3 In particular, a system for light-driven water splitting into H2 and O2 is the most attractive one because water is an abundant resource on Earth, and H2 is a clean, carbon-free fuel. Although considerable efforts have been made to develop inorganic photocatalysts for water splitting4
6 since the discovery of UV-induced H2 and O2 production on TiO2 about 40 years ago,7 further improvements of the quantum efficiencies and the availability of visible light are necessary for practical use. On the other hand, utilization of natural photosynthetic proteins in the construction of artificial photosynthesis is an alternative attractive approach,8 because of the high quantum efficiencies near 100% in their excitation and electron transfer reactions, which have been optimized during evolution for over 3 billion years.9 In plants and cyanobacteria, photosystem II (PSII) has a function of light-driven water oxidation to evolve O2,10
12 which exactly corresponds to the anode reaction of water splitting. PSII protein complexes have been immobilized on the planar or nanostructured surface of a gold electrode.13
16 Observation of photocurrents in these systems suggested the functionality of the PSII proteins, although direct detection of O2 evolution has not been achieved. On the other hand, PSI corresponds to a cathode, and r 2011 American Chemical Society

together with ferredoxin as a mediator and ferredoxin NADP+ oxidoreductase (FNR) as a catalyst, it performs NADP+ reduction to produce NADPH as a reducing power. Although PSI originally does not have a function of H2 formation, it has been demonstrated that Pt nanoparticles17
21 or hydrogenase22,23 attached on the electron acceptor side of PSI can generate H2 under visible light illumination. The drawback of these H2 production systems using PSI is that electrons are donated from sacrificial reagents such as ascorbate. Thus, to enable lightinduced H2 production by overall water splitting, coupling with a device that donates electrons from water with a high efficiency is essential. In this study, we have generated the conjugate of a gold nanoparticle (GNP) with PSII complexes as a nanodevice for the development of artificial light-driven water-splitting systems. The PSII core complexes from the thermophilic cyanobacterium Thermosynechococcus elongatus, in which a His-tag was introduced to the C-terminus of the CP47 subunit, were immobilized on a GNP with a diameter of 20 nm via nickel-nitrilotriacetic acid (Ni-NTA), orienting the electron acceptor side to the GNP surface (Figure 1). The PSII core complexes from T. elongatus are Received: August 26, 2011 Accepted: September 9, 2011 Published: September 09, 2011 2448

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Figure 1. A schematic model of a PSII
GNP conjugate. The dimers of PSII complexes with a His-tag at the C-terminus of the CP47 subunit are immobilized on a GNP with a diameter of 20 nm via Ni-NTA, orienting the electron acceptor side to the gold surface. The proteins of individual PSII monomers are distinguished by red and blue colors, and chlorophylls are expressed in green. The PSII structure was deduced from the PDB data (PDB entry: 3ARC) by Umena et al.35

suitable material for the construction of artificial systems because of their high thermal stability.24 This is the first report of the formation of PSII
GNP conjugates dispersed in a solution aiming at applications in artificial photosynthesis except for the previous transmission electron microscopy (TEM) studies utilizing GNPs (∼2.5 nm diameter) as a probe to identify the location of the His-tagged PsbH subunit in a PSII complex.25,26 We have confirmed successful formation of the PSII
GNP conjugates by optical and TEM analyses and directly detected lightdriven O2 evolution proving their functionality. Oxygen-evolving PSII core complexes of T. elongatus with a (His)6-tag at the C-terminus of the CP47 subunit were prepared as described previously.27 The O2 evolution activity of this PSII complexes was 2500 μmol O2 (mg Chl)
1 h
1. The dimeric form of the obtained PSII complexes was confirmed by highresolution clear native electrophoresis.28,29 For the preparation of PSII
GNP conjugates, an aqueous GNP (20 nm diameter) solution was first mixed with 3,30 -dithiobis[N-(5-amino-5-carboxypentyl)-propionamide-N0 , N0 -diaceticacid] dihydrochloride (dithiobis(C2-NTA)), and the obtained C2-NTA
GNP was washed with a 10 mM NaH2PO4/NaOH buffer (pH 7.0) containing 0.06% ndodecyl-β-D-maltoside (DM) (buffer A). The C2-NTA
GNP solution was then suspended in buffer A in the presence of 0.2 mM NiSO4 to obtain Ni-NTA
GNP followed by washing with buffer A. The His-tagged PSII complexes suspended in a 10 mM PIPES buffer (pH 7.0) containing 50 μM CaCl2 and 0.06% DM was added to the Ni-NTA
GNP solution, and the mixture was incubated at 4 C for 2 h in the dark. The obtained PSII
GNP conjugate, in which PSII was immobilized to Ni-NTA
GNP via the His-tag, was washed with buffer A (The details of the experimental procedures are described in the Supporting Information).

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Figure 2. (A) UV
vis absorption spectra of free GNP (black), NiNTA
GNP (green), PSII
GNP (red), and free PSII complexes (blue). Inset: An expanded view of the spectrum of PSII
GNP in the Chl Qy region. (B) The Chl Qy band of the PSII
GNP after baseline correction (red solid line) in comparison with that of free PSII complexes with an adjusted intensity (blue dotted line). The baseline correction of the spectrum of the PSII
GNP was performed by subtraction of the spectrum of free GNP with an appropriate factor.

Figure 2A shows the absorption spectra of the solutions of free GNP (black line) and Ni-NTA
GNP (green line), and PSII
GNP (red line) together with the absorption spectrum of free PSII complexes (blue line). The concentration of free GNP solution was 1.2 nM, and the Ni-NTA
GNP and PSII
GNP, whose spectra were obtained during the preparation processes, were suspended in buffer A with the same volume (0.9 mL) as the initial GNP solution. The concentration of dimeric PSII complexes in the free PSII solution was 50 nM. The large band at 525 nm of the GNP solution originates from surface plasmon absorption,30,31 and it has been known that the peak position are influenced by the refraction index of the medium surrounding GNP.32 Upon binding of Ni-NTA (Figure 2A, green line), the peak position was red-shifted by 3 nm concomitant with some band broadening. This tendency became more significant with a red shift by 8 nm upon further binding of PSII proteins. The decreases in the peak intensities in Ni-NTA
GNP and PSII
GNP in comparison with original GNP may be ascribed to the band broadening as well as some sample loss by centrifugation and resuspension procedures. The red shifts of the peak positions suggest that Ni-NTA and His-tagged PSII complexes were successfully assembled on the GNP surface. In addition to the GNP band, the PSII
GNP conjugate exhibited bands at 674 and 437 nm originating from the Chl Qy and 2449

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Figure 3. The TEM images of free GNP (A) and the PSII-GNP conjugate (B). The samples were negatively stained with 2% phosphotungstic acid. Red arrows indicate the images of the PSII complexes immobilized on a GNP.

Soret transitions, respectively (expanded view of the Qy region is presented in Figure 2A inset). The presence of these Chl bands confirms that PSII protein complexes were in fact bound to GNP. The Qy band of the PSII
GNP conjugate after background correction (Figure 2B, red line) was virtually identical to the Qy band of free PSII (Figure 2B, dotted blue line), suggesting the intactness of the PSII complexes after immobilization on the GNP. The number of PSII dimers bound to one GNP in the PSII
GNP conjugate was estimated using the plasmon absorption band (Figure 2A) and the Chl spectrum of the methanol extract (data not shown). The concentration of GNP in the PSII
GNP solution was calculated with the assumption that the area intensity of the GNP absorption, after removing the contribution of scattering, is unchanged upon binding of PSII. The concentration of PSII was calculated from that of Chl and the information from the X-ray crystallographic structure in which 70 Chls are bound to a PSII dimer.33
35 It was estimated that 4.7 PSII dimers, i.e., four to five dimers, on average were bound to a single GNP. This value was virtually unchanged even when the incubation time of the mixture of PSII and Ni-NTA
GNP was prolonged from 2 to 12 h. Figure 3 shows the TEM images of free GNP (A) and the PSII
GNP conjugate (B), which were negatively stained with phosphotungstic acid. In each image, a GNP with a 20 nm diameter was clearly shown as a black sphere. In addition, white “shadows” were observed around the GNP in the PSII-GNP conjugate (Figure 3B, red arrows), whereas there were no such shadows around the free GNP. The PSII dimer has a dimension of 20  13  11 nm (Figure 1).33
35 Thus, the white shadows with 10
20 nm sizes are reasonably explained by binding of several PSII dimers to one GNP, consistent with the estimation by optical analysis. The orientation of the PSII complexes in the PSII
GNP conjugate was studied by examining the occupancy of the His-tag of PSII in the conjugate using Ni-affinity chromatography. PSII complexes with His-tags occupied by Ni-NTA will be passed through the Ni-affinity column. The amounts of the PSII or PSII
GNP samples eluted from the column were estimated by detecting the absorbance at 674 (PSII Qy) or 533 (GNP) nm. In the case of free His-tagged PSII, only 8% of the sample was eluted, indicating that most of the PSII complexes were bound to

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the Ni-affinity column with free His-tags. By contrast, 91% of the PSII
GNP conjugate was eluted. This result indicates that basically all of PSII complexes are bound to Ni-NTA
GNP via the His-tag at the C-terminus of CP47, which is located on the electron acceptor side of PSII. Thus, it is concluded that PSII core complexes are successfully immobilized on GNP orienting its electron acceptor side to the gold surface. The O2 evolution activities of the PSII
GNP conjugate was measured at 25 C using a Clark-type O2 electrode in a 20 mM Mes buffer (pH 6.5) containing 20 mM CaCl2, 20 mM NaCl, and 0.4 M sucrose in the presence of 0.5 mM phenyl-p-benzoquinone (PpBQ) and 2 mM ferricyanide as exogenous electron acceptors. Illumination was performed with continuous red light at 661 nm from a diode laser (90 mW at the head), which excites the Qy absorption of Chl avoiding the large plasmon absorption of GNP. The O2 evolution activity of the PSII
GNP conjugate was estimated to be 575 ( 80 μmol O2 (mg Chl)
1 h
1. This is the first case of the direct detection of O2 evolution from PSII proteins immobilized on a metal surface. Free PSII complexes incubated in buffer A also showed similar O2 evolution activities of 580 μmol O2 (mg Chl)
1 h
1. Thus, it can be concluded that PSII complexes almost fully retained the O2 evolution capability even after immobilization on GNP. The above O2 activities of free PSII and PSII
GNP conjugates in buffer A were lower than the original activity of the PSII complexes (2500 μmol O2 (mg Chl)
1 h
1). This is probably ascribed to the absence of Ca2+ and Cl
in this buffer. Free PSII in buffer A supplemented with 2.5 mM CaCl2 and NaCl recovered a high activity of 2460 μmol O2 (mg Chl)
1 h
1. It has also been reported that the O2 activity of cyanobacterial PSII core complexes is decreased when incubated in buffers without CaCl2.36,37 The absence of salts in the buffer for the PSII
GNP conjugates was required to avoid aggregation of GNPs, which are stabilized in a solution by electrostatic repulsion. Further approaches such as using functional polymers assembled around GNPs to stabilize colloidal nanoparticles38,39 will solve the aggregation problem in a high-salt solution and maximize the O2 evolution activity. In conclusion, PSII
GNP conjugates, in which the electron acceptor side of PSII is oriented to GNP, were successfully generated using His-tagged PSII complexes and Ni-NTA assembled GNPs. Four to five PSII dimers were immobilized on a single GNP, retaining a moderate O2-evolution activity under visible-light illumination. Thus, the obtained PSII
GNP conjugate is a new nanodevice that has a function of light-driven water oxidation. Further binding of enzymes or inorganic catalysts to this device will realize construction of semiartificial systems in which electrons from water can be used to reduce various substances. In particular, light-driven nanosystems that perform overall water splitting into O2 and H2 will be constructed by hybridization of the PSII
GNP conjugate with PSI complexes coupled with a Pt nanoparticle17
21 or hydrogenase22,23 as a catalyst for H2 production. In such hybridization systems, in which the donor side of PSI complexes is bound to the GNP surface,23,40,41 electrons can be transferred from PSII to PSI though a GNP.

’ ASSOCIATED CONTENT

bS

Supporting Information. Details of experimental procedures. This material is available free of charge via the Internet at http://pubs.acs.org.

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’ AUTHOR INFORMATION Corresponding Author

*Address: Division of Material Science, Graduate School of Science, Nagoya University, Furo-cho, Chikusa-ku, Nagoya, 464-8602, Japan. Phone: +81-52-789-2881. Fax: +81-52-789-2883. E-mail address: [email protected]. Present Addresses

^ Center for Biological Resources and Informatics, Tokyo Institute of Technology, Yokohama 226
8501, Japan.

’ ACKNOWLEDGMENT This study was supported by Grants-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology (21370063, 23108706, and 23657099 to T.N., 21570038 and 22370017 to T.T., and 23370014 to M.I.) and by grants from JST PRESTO (T.T.) and GCOE (M.I.) programs. ’ REFERENCES (1) Kalyanasundaram, K.; Graetzel, M. Artificial Photosynthesis: Biomimetic Approaches to Solar Energy Conversion and Storage. Curr. Opin. Biotechnol. 2010, 21, 298–310. (2) McConnell, I.; Li, G.; Brudvig, G. W. Energy Conversion in Natural and Artificial Photosynthesis. Chem. Biol. 2010, 17, 434–447. (3) Gust, D.; Moore, T. A.; Moore, A. L. Solar Fuels via Artificial Photosynthesis. Acc. Chem. Res. 2009, 42, 1890–1898. (4) Osterloh, F. E. Inorganic Materials as Catalysts for Photochemical Splitting of Water. Chem. Mater. 2008, 20, 35–54. (5) Kudo, A.; Miseki, Y. Heterogeneous Photocatalyst Materials for Water. Chem. Soc. Rev. 2009, 38, 253–278. (6) Maeda, K.; Domen, K. Photocatalytic Water Splitting: Recent Progress and Future Challenges. J. Phys. Chem. Lett. 2010, 1, 2655–2661. (7) Fujishima, A.; Honda, K. Electrochemical Photolysis of Water at a Semiconductor Electrode. Nature 1972, 238, 37–38. (8) Esper, B.; Badura, A.; R€ogner, M. Photosynthesis as a Power Supply for (Bio-)Hydrogen Production. Trends Plant Sci. 2006, 11, 543– 549. (9) Blankenship, R. E. Early Evolution of Photosynthesis. Plant Physiol. 2010, 154, 434–438. (10) Hillier, W.; Messinger, J. Mechanism of Photosynthetic Oxygen Production. In Photosystem II: The Light-Driven Water:Plastoquinone Oxidoreductase; Wydrzynski, T., Satoh, K., Eds.; Springer: Dordrecht, The Netherlands, 2005; pp 567
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