Two photon absorption energy transfer in the light-harvesting complex of photosystem II (LHC-II) modified with organic boron dye

Article abstract of DOI:10.1016/j.saa.2014.02.166

The plant light-harvesting complexes of photosystem II (LHC-II) play important roles in collecting solar energy and transferring the energy to the reaction centers of photosystems I and II. A two photon absorption compound, 4-(bromomethyl)-N-(4-(dimesitylboryl)phenyl)-N-phenylaniline (DMDP-CH ₂Br), was synthesized and covalently linked to the LHC-II in formation of a LHC-II-dye complex, which still maintained the biological activity of LHC-II system. Under irradiation with femtosecond laser pulses at 754 nm, the LHC-II-dye complex can absorb two photons of the laser light effectively compared with the wild type LHC-II. The absorbed excitation energy is then transferred to chlorophyll a with an obvious fluorescence enhancement. The results may be interesting and give potentials for developing hybrid photosystems.

Full text of DOI:10.1016/j.saa.2014.02.166

Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 128 (2014) 295–299
Contents lists available at ScienceDirect
Spectrochimica Acta Part A: Molecular and
Biomolecular Spectroscopy
journal homepage: www.elsevier.com/locate/saa
Two photon absorption energy transfer in the light-harvesting complex
of photosystem II (LHC-II) modified with organic boron dye
a
b,
Li Chen a,1, Cheng Liu b,1, Rui Hu a, Jiao Feng a, Shuangqing Wang a, , Shayu Li , Chunhong Yang
⇑
⇑
,
Guoqiang Yang a,
⇑
a Beijing National Laboratory for Molecular Sciences, CAS Key Laboratory of Photochemistry, Institute of Chemistry, Chinese Academy of Sciences, Beijing 100190, China
b Key Laboratory of Photobiology, Institute of Botany, Chinese Academy of Sciences, Beijing 100093, China
h i g h l i g h t s
g r a p h i c a l a b s t r a c t
A two photon absorption compound, 4-(bromomethyl)-N-(4-(dimesitylboryl)phenyl)-N-phenyl-aniline
(DMDP-CH2Br), was synthesized and covalently linked to the LHC-II in formation of a LHC-II-dye com-
plex. The LHC-II-dye complex can absorb two photons of the laser light effectively. The absorbed excita-
tion energy is then transferred to chlorophyll a with an obvious fluorescence enhancement. The work
may be interesting and give potentials for developing hybrid photosystems.
ꢀ
ꢀ
ꢀ
A two-photon absorption compound,
DMDP-CH2Br, was synthesized and
linked to LHC-II.
The LHC-II-dye complex can absorb
two photons of the laser light
effectively.
The absorbed energy in the LHC-II-
dye complex can transfer to
chlorophyll a.
a r t i c l e i n f o
a b s t r a c t
Article history:
The plant light-harvesting complexes of photosystem II (LHC-II) play important roles in collecting solar
energy and transferring the energy to the reaction centers of photosystems I and II. A two photon absorp-
tion compound, 4-(bromomethyl)-N-(4-(dimesitylboryl)phenyl)-N-phenylaniline (DMDP-CH2Br), was
synthesized and covalently linked to the LHC-II in formation of a LHC-II-dye complex, which still main-
tained the biological activity of LHC-II system. Under irradiation with femtosecond laser pulses at
754 nm, the LHC-II-dye complex can absorb two photons of the laser light effectively compared with
the wild type LHC-II. The absorbed excitation energy is then transferred to chlorophyll a with an obvious
fluorescence enhancement. The results may be interesting and give potentials for developing hybrid
photosystems.
Received 27 December 2013
Received in revised form 19 February 2014
Accepted 24 February 2014
Available online 12 March 2014
Keywords:
Light-harvesting complex of photosystem II
LHC-II
Two photon absorption
Energy transfer
Ó 2014 Elsevier B.V. All rights reserved.
Organic boron dye
Introduction
⇑
Corresponding authors. Tel.: +86 10 82617263; fax: +86 10 82617315.
E-mail addresses: g1704@iccas.ac.cn (S. Wang), yangch@ibcas.ac.cn (C. Yang),
gqyang@iccas.ac.cn (G. Yang).
Collecting the solar energy and transferring the energy to the
reaction centers of photosystems I and II are the most important
1
Co-first author.
http://dx.doi.org/10.1016/j.saa.2014.02.166
1386-1425/Ó 2014 Elsevier B.V. All rights reserved.

296
L. Chen et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 128 (2014) 295–299
role in plant light-harvesting complex of photosystem II (LHC-II)
[1–3]. Isolated from different plants, such as pea, spinach and bam-
boo, each LHC-II polypeptide bound with chlorophyll a, chlorophyll
b and carotenoid still remains the light-harvesting function [4,5].
Actually, the chlorophyll a and chlorophyll b are the main chemical
pigments for light-harvesting. The absorption coefficients of LHC-II
in blue portion of the sunlight spectrum are high enough, followed
by the red portion. However, the absorption coefficient is poor in
the green and near-infrared portions, which means that the
LHC-II can not sufficiently capture the green and near-infrared
light from the sun light [6]. Because of the solar energy is partly ab-
sorbed by the atmosphere, IR light of solar radiation reaching the
Earth may have about 60% energy fractions. In order to absorb
the solar energy efficiently, organic dye-modified complexes of
LHC-II or its peptide fragments, such as LHCb1, LHCb2 and LHCb3
proteins (LHC-II-dye complex) had been prepared [7]. The excita-
tion energy flow in the LHC-II system had been studied in different
techniques for the targets of interpreting high energy efficacy of
LHC-II as the major plant antenna and fabricating the potential
artificial photosynthesis [8–10]. For efficient conversion of solar
energy to chemical energy, the artificial photosynthetic systems
had been studied for several decades [11]. The studies of artificial
photosynthesis may involve introduction of different organic dyes
to the photosystems I and II as a model to mimic the process and
mechanism of photosynthesis, which will be a useful contribution
to better understanding the photosynthesis and solar energy
conversion.
region. The organic dye, DMDP-CH2Br, can react with the
sulphydryl group on the peptide fragment of LHC-II. After protein
recombinant in vitro, a new light-harvesting photosystem II com-
plex modified with organic boron dye (LHC-II-dye complex) was
obtained, which retains its biological activity, and does not
change the basic function of the LHC-II polypeptide. Under irradi-
ation with femtosecond laser pulse at 754 nm, the modified LHC-
II can absorb two photons of the laser light more effectively than
the wild LHC-II. The energy is then transferred to chlorophyll a,
resulting in an obvious fluorescence enhancement compared with
the wild type LHC-II.
This work concerns the collection of the solar energy and con-
version of the energy to the reaction centers of the organic boron
dye-modified photosystems II, which may be a potential candidate
for artificial photosynthesis. The corresponding research gives an
approach to improve the energy transfer yield in artificial photo-
synthesis by two photo techniques, which contributes a better
understanding of photosynthesis and also gives potentials in devel-
oping new hybrid photosystems.
Materials and methods
Synthesis of the organic boron dye, DMDP-CH2Br
4-(Dimesitylboryl)-N,N-diphenylaniline (DMDP) was synthe-
sized from 4-bromo-N,N-diphenylaniline as starting material in
the temperature at -78 °C in 85% yield [14,15]. The DMDP was
transferred to corresponding benzaldehyde and phenylmethanol
derivatives in usual methods with the yields of 82% and 99%,
respectively [16,17]. And then, the corresponding phenylmethanol
derivative was bromized with PBr3 to get the target organic boron
compound [18], 4-(bromomethyl)-N-(4-(dimesitylboryl)phenyl)-
N-phenylaniline (DMDP-CH2Br), with the yield of 95%. The syn-
thetic route is shown in Scheme 1.
In the polypeptide sequences of the LHC-II [5], a sulphydryl
group on cysteine residue could be modified by reaction with
some organic dyes. Triarylboron compounds have attracted much
attention due to their high fluorescence quantum yields and two
photon absorption properties [12,13]. Recently, we synthesized a
series of triarylboron compounds, i.e. 4-(dimesitylboryl)-N,N-
diphenylaniline (DMDP) and its bromomethyl-linked derivative,
4-(bromomethyl)-N-(4-(dimesitylboryl)phenyl)-N-phenylaniline
(DMDP-CH2Br), as two photon fluorescence dyes in near-IR or IR
Scheme 1. The synthesis scheme of the organic boron compound, DMDP-CH2Br.

L. Chen et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 128 (2014) 295–299
297
Preparation of light-harvesting complex of photosystem II modified
with organic boron dye, LHC-II-dye complex
The LHCb1 apoprotein was isolated from pea using our published
method [19], the sulphydryl group of cysteine residue in the
sequence of LHCb1 acts as the reaction site to connect the organic
boron dye at benzyl bromide active group. The reaction is illumi-
nated in Scheme 2. LHCb1 60 mg was dissolved in 5 mL 0.5% sodium
dodecyl sulfate (SDS) solution, to this aqueous solution DMDP-
CH2Br 4.2 mg in 0.3 mL THF was added in dropwise at room temper-
ature. After addition of 0.3 mg NaHCO3, the mixture was stirred for
3 days. The LHCb1-dye was precipitated by adding 6 mL ethanol,
purified by centrifuging and washing with ethanol and water
[20–22]. And then the LHCb1-dye was reconstituted to get the
LHC-II-dye complex using published method [22,23]. Fig. 1 gives
the results of sucrose density gradient ultracentrifugation of the
wild type LHC-II and LHC-II-dye complexes. The clear difference in
the results indicates that the organic boron dye has been connected
to the LHC-II protein. The further experiments show that the artifi-
cial mutation of LHC-II with a covalently bonded organic dye has
the similar biological activities with the wild type LHC-II protein.
The chemical modified apoprotein LHCb1 does not interfere with
LHC-II assembly, function and stability. The structure of the LHC-
II-dye complex is shown in Fig. 2 for interpretation of the complex.
Fig. 2. The structure of the LHC-II-dye complex.
new formed LHCb1-dye has maximum absorption at 382 nm
(Fig. 3), which is homologous with the absorption of the organic
dye, DMDP-CH2Br. The result indicates that the organic boron
dye is connected to the LHCb1 apoprotein.
The LHCb1 and LHCb1-dye were reconstituted respectively in
the same conditions, resulting in formation of wild type LHC-II
and LHC-II-dye complex. Fig. 4 shows the absorption spectra of
the wild type LHC-II and the LHC-II-dye complex in 0.5% SDS solu-
tions. The absorption spectra of the two proteins are similar, which
means that the LHC-II-dye complex may have similar optical
absorption property with wild type LHC-II. As consistent with
other published results, the absorptions are mainly contributed
from the chlorophyll a and chlorophyll b [6,22]. The contribution
from organic boron dye to the absorption is not obvious due to
its low molar ratio in the LHC-II polypeptide system. This result
indicated that the LHC-II-dye complex could retain the biological
activity of normal wild LHC-II polypeptide in efficient capture of
light energy without affect from the labeled dye.
Absorption and fluorescence measurements
The absorption spectra were recorded using Hitachi U-3010
spectrophotometer, the fluorescence emission spectra were re-
corded with a Hitachi F-2500 fluorescence spectrophotometer.
The two photon absorption induced emission spectra were carried
using Coherent Chameleon XR Ti-Sapphire femtosecond laser
(78 MHz) as excited light source and detected using Edinburgh
FLS 900 spectrophotometer [24,25].
Results and discussion
Fluorescence spectra
Absorption spectra
The fluorescence spectra of LHCb1 and LHCb1-dye in SDS solu-
tions were measured and shown in Fig. 5. The apoprotein LHCb1
has not obvious fluorescence emission under excitation at
377 nm, while the LHCb1-dye has a fluorescence maximum at
446 nm, which is the typical fluorescence of the organic boron dye.
The fluorescence spectra of the recombined wild type LHC-II
and LHC-II-dye complex showed in Fig. 6, which were obtained
at the excitation wavelengths of 377, 438 and 471 nm, near the
absorption maximum of organic boron dye, chlorophyll a and
LHCb1 apoprotein in 0.5% sodium dodecyl sulfate (SDS) aqueous
solution does not show obvious absorption in visible range. The
Scheme 2. Reaction of the organic boron dye and the LHCb1 apoprotein.
0.12
DMDP-CH2Br in methanol
0.10
LHCb1-dye in SDS solution
0.08
0.06
0.04
0.02
0.00
-0.02
350
400
450
500
Wavelength/ nm
Fig. 1. Sucrose density gradient ultracentrifugation of wild type and LHC-II-dye
Fig. 3. The absorption spectra of LHCb1-dye in 0.1% dodecyl b-D-maltoside solution
complex.
and DMDP-CH2Br in methanol.

298
L. Chen et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 128 (2014) 295–299
1.0
0.8
0.6
0.4
0.2
0.0
280
260
240
Ex: 377nm
Ex: 471nm
Ex: 438nm
220
200
180
160
140
120
100
80
LHC-II-dye complex
Wt
Wt
60
40
20
0
-20
350 400 450 500 550 600 650 700 750 800
Wavelength /nm
300
400
500
600
700
800
Wavelength /nm
Ex: 377
Ex: 471
Ex: 438
240
220
200
180
160
140
120
100
80
Fig. 4. Absorption spectra of wild type LHC-II and LHC-II-dye complex in SDS
solutions (at same concentration).
LHC-II-dye complex
chlorophyll b, respectively. In all the fluorescence spectra with dif-
ferent excitation wavelengths, the fluorescence maximums of the
wild type LHC-II and LHC-II-dye complex are the same at
685 1 nm as the emission from chlorophyll a [22]. The result
indicates that the introduction of organic boron dye in the LHC-II
protein will not change the energy transfer path in the light har-
vesting process (from chlorophyll b to chlorophyll a). Meanwhile,
the excitation of the organic boron dye with 377 nm gave only
the emission of chlorophyll a. It indicated clearly that the energy
transfer is from the boron dye to the chlorophyll a or chlorophyll b.
60
40
20
0
-20
350 400 450 500 550 600 650 700 750 800
Wavelength /nm
Fig. 6. Fluorescence spectra of wild type LHC-II (top) and LHC-II-dye complex
(bottom) (at same concentration).
Two photon excitated fluorescence spectra
In order to investigate the energy transfer in the organic boron
dye-labeled LHC-II-complex, femtosecond laser at 754 nm was
used to excite the organic boron fragment for evaluating the
change of the two photon excitated fluorescence spectra (Fig. 7).
Under excitation of the femtosecond laser, the relative fluores-
cence intensity of LHC-II-dye complex is obviously enhanced with
1.44 times higher than the wild type LHC-II, which presumes that
the LHC-II-dye complex can absorb two photons of the laser more
effectively compared with the wild type LHC-II, and then the en-
ergy is transferred to chlorophyll a. The results also suggest that
chemical modified LHC-II system such as introduction of an organ-
ic dye to the LHC-II system could improve the light harvesting
properties and the solar energy absorption efficiency.
500000
LHC-II-dye complex Ex:754nm
400000
300000
200000
100000
0
Wt Ex:754nm
400
450
500
550
600
650
700
Wavelength /nm
1600
1400
Fig. 7. Two photon excitated fluorescence spectra of wild type LHC-II and LHC-II-
dye complex (at same concentration).
LHCb1-dye
1200
1000
Conclusions
Ex: 377nm
800
600
400
200
We have synthesized a two photo absorption compound,
4-(bromomethyl)-N-(4-(dimesitylboryl)phenyl)-N-phenylaniline
(DMDP-CH2Br). It is clearly shown that the organic boron com-
pound can be covalently linked with the LHC-II to get LHC-II-
dye complex. The LHC-II-dye complex maintains the biological
activity of the LHC-II system, and the organic boron dye does
not change the basic function of the LHC-II polypeptide. Under
irradiation with 754 nm femtosecond laser, the LHC-II-dye com-
plex can absorb two photons of the laser more effectively
compared with the wild type LHC-II. The absorbed excitation
energy is then transferred to chlorophyll a with an obvious
0
LHCb1
-200
350 400 450 500 550 600 650 700 750 800
Wavelength /nm
Fig. 5. Fluorescence spectra of LHCb1 and LHCb1-dye in SDS solutions (at same
concentration).

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Financial support from the National Basic Research Program of
China (2011CBA00905, 2011CBA00904), Chinese Academy of
Sciences (KSZD-EW-Z-018) and the National Natural Science
Foundation of China (21373240, 21233011, 21205122,
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