全 文 :第43卷第8期
2014年8月
光 子 学 报
ACTA PHOTONICA SINICA
Vol.43No.8
August 2014
Foundation item:The National Natural Science Foundation of China(No.60978038)
First author:HE Jun-fang(1971-),female,research felow,Ph.D.degree,mainly focuses on ultrafast phenomena.Email:amilyhjf@163.com
Received:Jan.1,2014;Accepted:Apr.1,2014
http:∥www.photon.ac.cn
doi:10.3788/gzxb20144308.0832001
假根羽藻外周天线LHC II寡聚体的瞬态吸收光谱
贺俊芳1,朱长军2,赵小侠1,李院院1
(1西安文理学院 物理与机械电子工程学院,西安710065)
(2西安工程大学 理学院物理系,西安710048)
摘 要:采用时间分辨瞬态吸收光谱技术研究了假根羽藻外周天线寡聚体的光保护机制.分别以
667nm飞秒激光脉冲和白光脉冲作为泵浦光和探测光,探测光与泵浦光之间的延时范围和准确度分别
为340ps和134fs.实验结果表明在泵浦光激发之后外周天线对探测光的吸收是动态变化的.对瞬态吸
收光谱进行多指数拟合,并结合外周天线的荧光发射谱和激发谱进行分析,结果表明:500~600nm的
瞬态吸收谱主要来源于类胡萝卜素分子,外周天线寡聚体至少包含四种具有光保护作用的类胡萝卜素
分子,对应的S0→Sn 跃迁光谱为511nm和554nm,522nm和541nm,530nm和563nm(对应管藻黄
素),536nm和575nm;类胡萝卜素分子以两种方式参与到光保护过程中:一种是直接方式,在几皮秒范
围内猝灭叶绿素三重态;另一种是间接方式,在几百皮秒范围内猝灭从叶绿素分子获得能量的单线
态氧.
关键词:瞬态吸收光谱;光合作用;泵浦探测;外周天线;光保护;类胡萝卜素;叶绿素;单线态氧
中图分类号:Q631;Q632 文献标识码:A 文章编号:1004-4213(2014)08-0832001-6
Transient Absorption Spectra of Aggregated LHC II in Broypsis Corticulans
HE Jun-fang1,ZHU Chang-jun2,ZHAO Xiao-xia1,LI Yuan-yuan1
(1 School of Physics and Mechatronics Engineering,Xi′an University of Arts and Science,Xi′an,710065,China)
(2 Department of Physics,School of Science,Xi′an Polytechnic University,Xi′an,710048,China)
Abstract:A time resolved transient absorption spectroscopy was performed to investigate the
photoprotection mechanism in light harvesting complex II of broypsis corticulans. A 667nm
femotosenond laser and white light pulse were used as pump and probe lights.The range and accuracy of
the delay line between probe and pump were 340ps and 134fs,respectively.The experiment results
show that the absorption of the probe is dynamicaly changing after pump light exciting.The results of
the multi-exponential fitting,fluorescence emission and excitation spectra show that the transient
absorption spectra from 500nm to 600nm mainly originate from carotenoids and the aggregated light
harvesting complex II contains at least four types of carotenoids functioning photoprotection which have
S0→Sntransition spectral properties 511nm and 554nm,522nm and 541nm,530nm and 563nm
(corresponding to siphonaxanthin),536nm and 575nm.Carotenoids play the photoprotection role not
only in a direct manner by quenching the triplet state of chlorophyl ain several picoseconds,but also in
an indirect manner by quenching the singlet state of oxygen in several hundreds of picoseconds.
Key words:Transient absorption spectrum;Photosynthesis;Pump-probe;Light harvesting complex II;
Photoprotection;Carotenoids;Chlorophyl;Singlet state oxygen
OCIS Codes:320.7120;320.7100;170.1420;170.7160
1-1002380
光 子 学 报
0 Introduction
Photosynthesis is of fundamental significance in
the conversion of solar energy to energies utilized by
living things which are adapted to different
environments and may be modulated in response to
changing light environments. Light Harvesting
Complex(LHC II)in green plant and algapossesses
the most abundant light harvesting pigment-protein
complex.In essence,it is believed to be organized in
trimeric units and it binds about 50%of the chlorophyl
present in the chloroplast.Besides chlorophyls,LHC
II contains carotenoids which are in close contact with
chlorophyl molecules[1-5].Carotenoids can absorb light
in the blue-green and yelow regions and,then,
transfer the energy to chlorophyl,which increases the
efficiency of colection of solar energy in the wavelength
region where chlorophyl does not absorb light[6].On
the other hand,the reverse energy transfer process
may occur,that is,carotenoids can quench both triplet
excited states in chlorophyl and singlet state in
oxygen,protecting the photosynthetic apparatus from
photodamage.
So far,intensive researches have been conducted
on the energy transfer from carotenoids to
chlorophyl[7-8].Solar energy can be transferred from
carotenoids to chlorophyl S1or S2state on picosecond
time scale,or even femtoseconds time scale[9-10].Much
attention has also been paid to the photoprotection
mechanism caused by carotenoids in photosynthesis
when the photosystem is under high light
ilumination[11-12].There were currently four models
suggesting how to quench excess excited state Chl
a[13-16]:1)via energy transfer to the carotenoid S1
state,2)via electron transfer,resulting in a Chl-a
anion and a carotenoid cation that recombine on a
picosecond time scale,3)it is achieved via carotenoid-
Chl exciton coupling that provides a pathway for
deactivation of excess excited Chl via the rapidly
decaying S1state of the carotenoid,and 4)it results
from formation of Chl/Chl exciton pairs which undergo
charge transfer as the pathway for Chl excited state
deactivation.Except the forth model,the first three
models involve carotenoids directly. Some
researches[11,17-19]showed that xanthophyl cycle play an
important role in photoprotection.Zeaxanthin can
accept energy from Chl Qy states in al possible
orientations,whereas violaxanthin can do so only at
very close distances by charge transfer.
Little effort,however,has been dedicated to the
dynamics of the photoprotection process.This paper
focuses on the revelation of the dynamics of
photoprotection using a time resolved transient
absorption spectroscopy.
1 Experiment
1.1 Materials
Bryopsis corticulans is a siphonous green alga
growing in intertidal areas,where periodic changes of
light accepted by the green alga during a cycle of tides
may demand that LHC Ⅱ of the alga operate with
some mechanisms to adapt the drasticaly changing
light.Thus it is a suitable material for investigating the
photoprotection information.The LHC II aggregates
were obtained from bryopsis corticulans.The complex
contains a specific carotenoid,siphonaxanthin,as wel
as Chl a, Chl b,neoxanthin and violaxanthin.
Siphonaxanthin which is present in the light-harvesting
siphonaxanthin-chlorophyl a/b-protein complex is
responsible for enhanced absorption in blue-green
region(530nm).The light harvesting chlorophyl a/b-
protein complex with a Chl a/Chl b ratio of 1.1is
similar to that of higher plants.
1.2 Experiment setup
Experiment setup for transient absorption
difference measurements in visible spectral range using
a femtosecond pump-probe scheme was described
previously[20].Briefly,a Ti:Sapphire laser acting as
master oscilator was pumped by the intracavity
frequency doubled cw Nd∶ YVO4 laser and emits
ultrashort pulses of about 80fs at 800nm.The laser
pulses were subsequently stretched,amplified and then
recompressed at 1kHz repetition rate by a regenerative
amplifier(Spectra Physics,Spitfire).The amplified
pulses have high average power(about 640mW)and
short pulse duration (about 100fs).The amplified
pulses were used to drive an Optical Parametric
Amplifier (OPA),tunable in UV-Vis-IR and to
generate a white light continuum in a 1mm glass plate
used as the probe.The output of OPA was used as
pump light.The probe light traveled through sample in
a separate time with respect to the pump light,which
was controled by a high precision optical delay line.
The range and accuracy of the delay line were 340ps
and 134fs.The two lights overlapped spatialy inside
the sample.The probe light was polarized under magic
angle(54.7°)with respect to the pump light.In order
to explore the photoprotection mechanisms related to
carotenoids,the wavelength of pump light was tuned
around 667nm.The pump light intensity was about 15
nJ/pulse.The probe intensity was typicaly 10times
lower.Absorption difference spectra were recorded by
a high sensitivity micro-channel plate photo-multiplier
tube and time correlation single photon count
technology.
Experiment was carried out at room temperature
2-1002380
HE Jun-fang,et al:Transient Absorption Spectra of Aggregated LHC II in Broypsis Corticulans
and atmospheric pressure.The sample was prepared in
the near natural state,without treatments of deoxidize
or saturated oxygen.
2 Results and discussions
Fig.1shows the transient absorption spectrum of
aggregated LHC II in bryopsis corticulans. A
femtosecond continuum light was used to iluminate the
LHC II aggregates.To avoid photo-saturation of LHC
II,the femtoseconds light was very weak,with pulse
energy of 1nJ.The absorption spectrum obtained in
our experiments shows more detailed information than
traditional absorption spectra.In the spectrum,almost
twenty absorption peaks were identified at 457,463,
469,478,482,489,493,511,522,530,536,541,
548,554,563,569,575and 592nm,respectively.
The carotenoid absorption wavelength ranges from
400nm to 600nm[21].Therefore,the peaks shown in
Fig.1correspond to the absorption lines of carotenoids
and chlorophyls of the aggregated LHC II.
Fig.1 White light spectrum and transient absorption
spectrum of aggregated LHC II
When chlorophyl molecules are iluminated,they
are excited to a high singlet state,and then
intercrossed to their first excited triplet state.The
triplet-excited chlorophyls can react with molecular
oxygen to produce singlet oxygen,which is a powerful
oxidising agent and rapidly kils those cels exposed to
it.Carotenoids are able to overcome this effect in two
ways,as shown in Fig.2.The first way is to quench
Fig.2 Direct and indirect quenching channels
the triplet state of chlorophyl adirectly,which can be
expressed as
3 Chl a*+Car→Chl a+3 Car* (1)
The second way is to quench singlet oxygen,
preventing the production of singlet oxygen,which can
be written as
O2 1Δ( )g +Car→O2 3Δ-( )g +3 Car* (2)
In order to investigate the protecting role of
carotenoids,the samples were excited at 667nm,
corresponding to the absorption of chlorophyl Qyband.
Time varying characteristics of 4 representative
absorption peaks were analysed using the absorption
difference spectra shown in Fig.3.
The time varying characteristics of absorption
peaks can be analysed by
y=y0+a1exp(-t/τ1)+a2exp(-t/τ2)+
a3exp(-t/τ3) (3)
where y0,ai,τi are background,pre-exponential
factors and lifetimes, respectively. The multi-
exponential model is often used on energy transferring
between pigments of photosynthesis[22-23].In this fitting
model,the pre-exponential factors,ai,can be either
positive or negative.A positive airepresents a decay
process,while negative aivalues are characteristic for
growth processes.The multiple exponential fittings
reveal that al the kinetics curves have a fast rise
component with several hundred of fs,corresponding to
the excitation of chlorophyl a.Meanwhile,two delay
components,one fast process of several ps and one
slow process of hundreds of ps,correspond to the two
energy transfer processes displayed in Eqs.(1)and
(2).It can be seen from Fig.3that the time durations
of direct energy transfer process and indirect energy
transfer process are on several ps and hundreds of ps
time scales.Pumped at 667 nm,chlorophyl a
molecules were first excited and,then,transfer energy
in an optimized way to a reaction center where the
energy was transformed in the form of chemistry.But
in the LHC II aggregates,there exists no acceptor
transforming energy in the form of chemistry.Thus,
the energy must be dissipated in other manners.The
first manner is emitting fluorescence directly from
chlorophyl a.The second is transferring energy in
direct and indirect manners to other pigments,e.g.,
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光 子 学 报
Fig.3 Time dependence of absorption difference spectra of LHC II pumped at 667nm
carotenoid and chlorophyl b.The energy transferred
directly from chlorophyl ato carotenoid,or indirectly
from chlorophyl athough oxygen to carotenoid,was
eventualydissipated thermaly,leading to photoprotection.
In the case of excitation with 436 nm,the
emission spectrum was detected and was fitted by six
Gaussian bands,i.e.,658nm,680nm,686nm,
696nm,726nm,736nm(dot lines),as displayed in
Fig.4.Thereinto,the band 658nm is originated from
chlorophyl b absorbing at 650 nm,whereas,the
436nm light is likely to excite chlorophyl a,implying
that energy can be transferred from chlorophyl ato
chlorophyl b.To confirm this process,the excitation
Fig.4 Fluorescence emission and Gauss fitting,and
excitation spectra of aggregated LHC II
spectrum at emission 658nm was also detected in the
red region,as shown in Fig.4.It is clear that the light
from 665nm to 690nm,corresponds to the chlorophyl
aQyabsorption,which induces chlorophyl b emitting
fluorescence.
Previous research results show that there is a large
range of overlapping region between chlorophyls and
carotenoids′absorption spectra in the region below 500
nm[24].Therefore,it is possible that energy transfer
from chlorophyl ato chlorophyl b and,thus,the
difference absorption dynamics curves below 500nm
are originated not only from carotenoid but also from
chlorophyl b molecules,causing the faster delay
lifetimes below 500 nm.Consequently,only the
absorption kinetic curves in the region 500~600nm are
originated from carotenoids.
An interesting phenomenon appears,namely,
511nm and 554 nm have similar lifetimes and
percentages in the decay process,as shown in Table 1.
These peaks corresponds to the same molecule.Similar
situation can be found 522nm and 541nm,530nm and
563nm,536nm and 575nm.Since the S0 →S1
transition is opticaly forbidden,these wavelengths
might be due to the S0→Sntransition.According to
previous report[3],the 530nm and 563nm are due to
siphonaxanthin.In the calculation model,A+B/(N+
C),511nm and 554nm,522nm and 541nm,536nm
4-1002380
HE Jun-fang,et al:Transient Absorption Spectra of Aggregated LHC II in Broypsis Corticulans
and 575nm originate from three carotenoids with 10or
11carbon-carbon double bonds.Herein,the three
carotenoids are named as Car511,Car522and Car536.
The two delay lifetimes of the curves correspond to the
two photoprotection ways described in Eqs.(1)and
(2).Fig.5shows the transient absorption difference
spectra developing with time increasing after pump
light exciting.At about 1.3 ps,the absorption
difference spectra from 500nm to 600nm reach the
maximum. Then with the time increasing,the
transient absorption difference spectra are decreasing.
Table 1 Lifetime parameters of the kinetic curves
Wavelength
/nm
τ1(fs)
with
negative a1
τ2(ps)with a2
(percentage in
delay processes)
τ3(ps)with a3
(percentage in
delay processes)
478 670 1.06(12%) 44.2(88%)
482 676 1.24(6%) 60.8(94%)
489 361 1.44(5%) 38.7(95%)
493 328 1.18(3%) 51.6(97%)
511 354 8.22(4%) 302(96%)
522 706 5.83(12%) 88.4(88%)
530 640 4.77(1%) 487(99%)
536 488 5.62(3%) 320(97%)
541 843 5.64(15%) 91.9(85%)
548 1 871 3.01(24%) 117(76%)
554 353 8.73(5%) 386(95%)
563 649 4.52(1%) 634(99%)
569 1 683 2.85(3%) 541(97%)
575 546 7.46(3%) 717(97%)
592 549 6.15 /
Fig.5 The transient absorption difference spectra of
aggregated LHC II at four time constant.
(The inset is for about 667nm)
Correspondingly,the transient absorption difference at
about 667nm(exciting wavelength)has little change in
several picoseconds,then increase sharply at several
hundred picoseconds(in the inset).The results show
that the quenching process of excited state Chl a mostly
occur in or over several hundred picoseconds.So
combined with the lifetimes parameters of the kinetic
curves,it is clear that the four carotenoids quench the
triplet state of chlorophyl ain several picoseconds with
low percentages,while they mostly quench the singlet
oxygen state on several hundreds picoseconds
timescale. Car522 seems to faster protect
photosynthetic apparatus than the other three
carotenoids,and has a larger percentage in quenching
triplet state of chlorophyl a.
3 Conclusion
Photoprotection mechanism in LHC II of broypsis
corticulans was studied using time resolved transient
absorption spectroscopy. Transient absorption
spectrum of LHC II was obtained using femtoseconds
continuum light and,furthermore,transient absorption
difference spectra of aggregate LHC II were obtained
using pump-probe scheme with the pump light centered
at 667nm.The results indicate that carotenoids play
an important role in the photoprotection process in two
fashions,in a direct manner by quenching the triplet
state of chlorophyl ain several picoseconds and,in the
meantime,in an indirect manner by quenching the
singlet state of oxygen in several hundreds of
picoseconds.
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