全 文 ：第 35卷第 2期
2006 年 2 月 　　　　　　　　　　　　 光　子　学　报ACTA PHO TON ICA S INICA
Vo l. 35 No. 2
Februa ry 2006
*Suppo r ted by Na tional Natural Science Fund of China
(60308004)
**Tel:029 88472107 8612 Email:sujuan_ zhang@yahoo. com.cn
Received date:2005 07 12
Study on Energy Transfer in LHC Ⅱ of Bryopsis Corticulans by
Femtosecond Spectra*
Zhang Sujuan1 , ** ,Wang Shuicai1 , He Junfang 1 ,Chen Hui2
1 State Key Laboratory o f Transient Optics and photonics , X i′an Institute of Optics and Precision Mechanics ,
Chinese Academy of S ciences , Xi′an 710068
2 Laboratory of Photosynthesis B asic Research , I nstituteo f Botany , Chinese Academy of S ciences , Bei jing 100093
Abstract　Based on the technique of T ime Co rrela ted Sing le Photon Counting (TCSPC), energy
t ransfer kinetics among chlorophylls in a monomeroic unit of major light-harvesting complex
(LHC Ⅱ) f rom bryopsis corriculan w ere studied. In the chlorophyll Q region , six characte ristic
molecules marked as Chlb630 , Chlb642 , Chla653652 , Chla667664 , Chla676 , 680675 , Chla683682 were discriminated.
Fluorescence kinetics measured in the chlo rophyl l Q region w as exci ted by pulsed light of 630nm
(70 fs). Af ter analy zing , results of energ y t ransfer among chls w ere obtained as fo llow ing :only ～
20% of the energy absorbed by Chlb630 is ini tially t ransferred directly to o ther Chls w ith t ime
constants sho rte r than 150 fs. Overw helming par t of the energy t ransfer among chlorophylls occur
wi th time constants longer than 76 ps. Energy t ransfe r pathw ay w ith time constants o f several
hundred femo to seconds and tens of pico seconds were also obtained. The f luorescence lifet imes of
Chlb
653
652 , Chla667664 , Chla676 , 680675 , Chla683682 were determined to be 1. 41 ns , 1. 39 ns , 676 ps , 709 ps
respectively . T he pe rcentages of energy dissipation in the pathw ay of fluorescence emission are no
mo re than 40% in the monomeric uni t of LHC Ⅱ.
Keywords　Energy t ransfer;LHC Ⅱ;Fem tosecond spectra;Fluo rescence kinet ics
CLCN　　Q615　　　　Document Code　　A
0　Introduction
Bryopsis co rticulans is a siphonous green alga
grow ing in intert idal areas. It can be survival
during the periodic tide. LHC Ⅱ of photosy stem
II , the outermost and most abundant antenna
complex o f chlo roplasts , exists as a t rimer and
binds half of the thy lakoid chlorophy ll molecules ,
which has an essential ro le in harvest ing solar
energy and transfer in the process o f
photosynthesis.
Measured by the abso rption kinetics in the
LHC Ⅱ of high plants , there now exists a gene ral
ag reement in the f ield about the Chl-Chl ene rg y
t ransfer in the LHC Ⅱ complex. A t temperatures
near room temperature the fastest Chlb to Chla
t ransfer seems to occur w ith a lifet ime of ～ 150 ～
200 fs[ 1 ～ 4] . Further components have lifet imes o f
～ 500 ～ 600 fs and 5 ～ 7 ps[ 1 ～ 4] . Energ y t ransfer
among the Chlas occur on a time scale typically 1 ps
and longer[ 2 ～ 5] . Given higher spect ra sensitivity and
tempo ral resolution , measured by the fluorescence
kinetics , more detailed info rmation on energy t ransfer
can be obtained. In the present w ork , the pigment
organization of monomeric LHC Ⅱ by combination of
t ransient absorption spectrum and transient
f luo rescence emission spect rum was investig ated. Then
the energy transfer betw een Chls of monomeric LHC Ⅱ
was studied by the fluorescence kinetics. All these
results are important for a further understanding of the
relation between structure and function of LHC Ⅱ.
1　Materials and methods
1. 1　Isolation of monomeric LHC Ⅱ
A light-harvesting chlorophyll a /b-protein
complex w as iso lated direct ly f rom thy lakoid
membranes of marine green alga , Bryopsis
cort iculans , by tw o consecut ive runs o f liquid
chromatog raphy . T he monomeric fo rm of LHC Ⅱ
was obtained by the procedure as described in the
Reference[ 6] .
1. 2　Experimental apparatus
Fem to second transient abso rpt ion and
f luorescence emission measurements w ere
perfo rmed by using a ti tanium-sapphire laser
sy stem. The master o sci llato r is a T i ∶sapphire
laser ex cited by a CW diode pumped int racavity
doubled Nd ∶YVO. The lase r provides a t rain of
fem tosecond pulses (56 fs) at 82 MHz repeti tion
rate w ith 0. 4W of average pow er at the central
w aveleng th of 800 nm. Seed pulses w ere amplif ied
by a Ti∶sapphire reg enerative amplifier(Spi tf ire /
Hurricane) pumped at 1 kHz by a Q-swi tched ,
2 期 Zhang Sujuan , e t al. S tudy on Ene rgy T ransfer in LHC Ⅱ of Bryopsis Co rticulans by Fem to second Spectr a
int racavity doubled Nd ∶ YLF running at the
second ha rmonic w avelength , 527 nm (model 527
DP-H , Evolution). The w hi te light used in OPA
sy stems (OPA-800CF)(Spect ra-Physics) as a seed
was created by focusing a few μJ of energy(8 00 nm)
into sapphire. Thus the broad spect ral cove rage o f
w hite light continuum provides an ideal seed source
fo r an OPA. A s the visible light range (<800 nm)
in the range of seed light is no w orthy in the
pro cess of parametric amplication , this range w as
spli t o ff w ith dichroic mir ror and utilized for
detection for t ransient abso rption spect roscopy .
The OPA stage converted the w avelength to ～ 630 nm
with a pulse width of ～ 70 fs. Pulse energy of ～ 0. 1μJ
in a 1mm diameter spot w as used. The Edinburgh
Inst ruments FLS920 w as chosen for measuring
spect ra and the kinetic of f luorescence emission.
Based on time co rrelated single photon counting
technique (TCSPC) equipped wi th the ult rafast
photo-detecto rs of micro-channel plate
photomul tiplier MCP-PMT ( HAMAMA TSU
R3809U-50 wi th the cooling sy stem C4878), the
measuring temporal resolution of this sy stem
fluo rescence emit ting can be obtained to ～ 20 ps.
1. 3　Data analysis
As exciting pulse is not a real Dirac function in
the experiments , the measured t ime resolved
fluo rescence emission spect rum h (t) is the
convo lution integ ral of real f luo rescence em ission
spect rum g(t) w ith instrument response f (t).
S uch situation w as represented by the follow ing
integral:h(t)=∫g(t -τ)f(τ)dτ=∫g(τ)f(t - τ)dτ.
S o the numerical procedure required the use o f the
convo lution integ ral to ex t ract the lifetime
paramete rs. The model o f numerical f it w as
expre ssed in mathemat ical term s as fo llow s:Y =y 0
+A i ∑n
i =1exp ( - x / ti), w ith pre-exponential facto rs
A i , the characteristic lifetime ti and an addit iona
backg round y0 . The pre-exponential factors can be
either positive or negative. A positive A i value
represented a decay process (energy dissipation),while
negative Ai value was characteristic for grow th process
(accepting energy). The numerical routine to ex tract
the parameters Ai and ti was following in a matlab
procedure based on marquardt-levenberg algo rithm
compiled by ourselves. The reduced chi-square of
fitting result w as calculated to evaluate the quality of
the fit results.
2　Results
2. 1 　Pigment composition and its Spectroscopic
characteristic in the chlorophyll Q region
Unde rstanding o f the energy transfe r of
monomeric LHC Ⅱ required recognit ion of the
pigment composition and i ts spect roscopic
characteristic. The pigment composition o f LHC Ⅱ
was analy zed by using reversed-phase High
Pe rfo rmance Liquid Chromatog raphy (HPLC )
method
[ 7]
. The ratios of Chla / Chlb trimer was 1 . 2 ,
which were similar to that of the native LHC Ⅱ
reported in higher plants befo re
[ 8]
. The know ledge
of spect roscopic characterist ic has been obtained
from transient absorption spect rum (Fig . 1) and
transient f luorescence emission spect rum (Fig. 2).
The t ransient f luo rescence emission spectrum was
excited by 630 nm in case the excita tion of
carotenoids. The abso rpt ion and f luo rescence
spectra of the samples w ere measured befo re and
af ter the fem to second li fetime measurements ,
showing no changes due to photochemical o r o ther
damage. From the analy sis of on the spect roscopic
charactistic (Fig1 , Fig2. ) combined wi th the
abso rption spectral of individual pigments[ 9 , 10]
there w ere six characteristic molecules marked as
Chlb630 , Chlb642 , Chla653652 , Chla667664 , Chla676 , 680675 , Chla683682
(the subscript deno tes the cente r w aveleng th of
abso rption , the superscript deno tes the center
Fig. 1　T ransient abso rption spectrum o f LHC Ⅱ
monomer at r oom tempera ture
Fig . 2　The fourth de riva tive o f fluore scence Emission
spectrum of o f LHC Ⅱ trimer at r oom tempera tur e
excited by 630 nm with a pulse w idth of ～ 70 fs
167
光　子　学　报 35 卷
wavelength of f luo rescence emission.) were
discriminated in the chlorophy ll Q region.
2. 2　Energy transfer between Chls
There appeared many peaks in the
fluo rescence emission spect rum (Fig . 2), but only
during the range o f 655 nm ～ 695 nm , fluorescence
emission can be detected in the time resolved
experiments. Considering the co rrespondence w ith
the t ransient abso rpt ion spectrum (Fig. 1), four
representat ive time-resolved fluo rescence spect ra
characterized w ith the pigments w ere analy zed
fo llow ing the routine as describing in the data
analysis. Af ter reconvolution simulation , the most
appropriate fi t ting curve (Fig. 3. ) and results log
(not show n) were created. The reduced chi-square
of fi tt ing results w ere all in the level of 10- 4 , and
the quali ty of fit ting results w ere seem to be w ell
accepted. A s the f it ting curve is also a combination
of g row th pro cess (accepting ene rg y) and decay
process (dissipation energy). Therefo re the fi tt ing
lifetimes in the results log w ere also divided into
tw o major processes:accepting energy pro cess and
energ y dissipation process. Among each process ,
considering pigment composition of LHC Ⅱ and i ts
spectro scopic characteristic in the chlorophy ll Q
region ,which allow ed us to specifically assign these
lifetimes and dif ferentiate in detail the energy
t ransfe r paths betw een Chls. The co rresponding
percentage o f each subproce ss w as calculated as
A i
∑A i (pre-exponential factor s A i w ith the same
sign).
Fig. 3　T ime reso lved fluo rescence spectra o f LHC Ⅱ excited by 630nm measured a t diffe rent w aveband
　　All the results of lifetimes and percentage o f
energy t ransfer betw een Chls w ere summarized
clearly in Table. 1. Resul ts can be reduced to five
points:1)Only ～ 20% of the energy abso rbed by
Chlb628 was ini tially t ransfer red direct ly to other
Chls wi th time constants shorter than 150 fs:
Chlb630
90 fs
Chlb
653
652 , Chlb630 153 fs Chla667664 , Chlb630
124 fs
Chla676 , 680675 , Chlb630 127 fs Chla683682 ;2 )
Ove rw helming part of the energy t ransfer among
chlo rophylls o ccur wi th time constants longer than
76 ps:Chlb630 …→Chlb642 … →Chla653652(137 ps),
Chlb630 …→Chlb642 →Chla667664(148 ps), Chlb630 …→
Chlb642 …→Chla676 , 680675 (106 ps), Chlb630 …→Chlb642
…→Chla683682 (76 ps), which denotes that befo re
t ransfe r energy to another Chls , the excited Chl
must exchange energy to vibrat ions of a lowe r state
w ith dif ferent mult iplici ty (intersy stem crossing);
3) Energy transfer di rectly f rom Chla653652 to
Chla676 , 680675 and Chla66664 to Chla6 77 , 6806 74 were t rif ling (<
0. 02 %), yet the t ransfer time w ere determined to
be 197 fs and 93 fs. So energy t ransfer s f rom
Chlb628 to Chla677 , 680674 via Chla654 , 657652 with t ime
constant of 83 fs w ere also subt le. How ever , energy
t ransfe rs f rom Chlb628 to Chla
666
664 via Chlb
654 , 657
652 w ith
lifetime o f 174 fs ,Chlb628 to Chla683682 via Chlb654 , 657 652
and Chla666664 with lifetime of 217 fs w ere
considerable (>10 %). The dif ference can be owed
to the dif ferences in the arrangement and direction
of dipole moment of Chls in LHC Ⅱ;4) Some
168
2 期 Zhang Sujuan , e t al. S tudy on Ene rgy T ransfer in LHC Ⅱ of Bryopsis Co rticulans by Fem to second Spectr a
slow er energ y t ransfer such as Chlb653 652
13. 8 ps
Chla683682 , Chla667664 8. 4 ps Chla683682 , Chla676 , 680674 9. 1 ps
Chla
683
682 were also obtained , which can be at tributed
to internal conversion befo re t ransferring energy to
ano ther Chls;5) The f luo rescence lifet imes o f
Chla
653
652 ,Chla667664 ,Chla676 , 680675 , Chla683682 were de termined
to be 1. 41 ns ,1. 39 ns , 676 ps ,709 ps respectively.
The percentages of energy dissipation in the
pathw ay o f fluo rescence emission are no mo re than
40%in the monomeric unit of LHC Ⅱ.
Table. 1　Lifetimes and percentage of energy transfer between Chls
C653652(Chlb) C667664(Chla) C676 , 680675 (Chla) C683682(Chla)
C630 →C653652
90 fs(22%)
C630 …→C642 …→C653652
149 ps(78%)
C653652 →C667664
89 fs(27%)
C653652 →C676 , 680675
197 fs(0. 02%)
C653652 …→C683682
13. 8 ps(48%)
C630→C667664
153 f s(16%)
C630 …→C642 …→C667664
148 ps(69%)
C630 →C653652 →C667664
125 f s(27%)
C667664 →C676 ,680675
93 fs(0. 02%)
C667664 …→C683682
8. 4 ps(60. 9%)
C630 →C676 , 680675
124 fs(19%)
C630 …→C642 …→C676 , 680675
106 ps(69%)
C630 →C653652 →C676 ,680675
68 f s(27%)
C630 →C667664 →C676 ,680675
174 fs(10. 3%)
C676 ,680675 …→C683682
9. 1 ps(48%)
C630 →C683682
127 fs(18%)
C630 →C653652 →C667664 →C683682
217 fs(10%)
C630 …→C642 …→C683682
76 ps(71%)
C683682→C
128 fs(0. 002%)
C683682 …→C
9. 55 ps(58. 9%)
C653652 →S0
1. 41 ns(25%)
C667*664 →S0
1. 39 ns(37%)
C676 , 680*675 →S 0
676 ps(39%)
C683*682 →S 0
709 ps(41%)
3　Discussion
The consistent under standing of the LHC-II
absorption in an overall subst ructure model ,
including po ssible excitonic effects , was thus
complicated by the unknown o rigin o f the observed
spect ral hete rogeneity of at least 10 subbands[ 11] .
In the process of detecting the abso rpt ion and
emission spect ra of LHC-II exicted by steady
exci tation sources , considering the nonconservative
contribut ions to the biolo gical sample induced by
consistent irradiation , the spect ra w ould be
disto rted by the final bleaching and excited state
absorption signal in Chls range[ 1] . Therefore , in
this article , a new approach to assess the
spect roscopic characterization o f monomeric LHC
Ⅱ by probing the absorption peaks and em ission
peaks direct ly by femtosecond pulses w as
presented. All the measured spect ras w ere exci ted
by the laser w ith a pulse w idth o f ～ 70 fs at 1
kHz ,which ensured the restoration o f sample w hen
exci ted by utrafast lig ht. How eve r , there also w as
somewhat smaller pa rt of inv olved subbands
hindered in t ransient spect ra. For
complementarity , some derivative spect ras w ere
made to explore the hindered subbands (Fig. 2).
There are at least six characteristic molecules in
the Q range of chls marked as Chlb628 , Chlb646 ,
Chlb654 , 657652 , Chla666664 , Chla677 , 680674 , Chla683682 , which w ere
recognized by t ransient spect roscopic technique.
They w ere mostly consistent w ith the results
studied by o ther g roups wi th different techniques
that there exits absorption peak at 64 8 nm 、66 0 nm 、
669 nm 、678 nm 、684 nm 、695 nm[ 1 , 3 , 12 ～ 14] .
In Table. 1 , it w as not iced that w hen energy
t ransfe r betw een Chls via C642 , the transfer t ime
w ould last up to 100 ps , and this pathw ay w as the
main channel of energy t ransfer between Chls fo r
it s higher percentage. It implicated that Chlb642
must be assembled in a special posi tion in the
st ructure of monomeric LHC Ⅱ;o r Chlb642 maybe
alw ay s w ent through the intersystem crossing and
exists in a t riplet state befo re exchange ene rgy to
o thers. Some slowe r energy transfer time of 10 ps ,
assigned to the accept ing ene rg y time of Chla
683
682
f rom o ther Chls di rect ly , and it occupied most ly in
the energy t ransfer percentag e. It also implicated
Chla
683
682 was the main acceptor in LHC Ⅱ. With the
longe r of w aveleng th , the mo re i t dissipated energy
in the mode o f fluorescence. That is to say , the
eff iciency o f ene rg y transfer w as decreasing w i th
the turn of Chlb
654
657 652 , Chla666664 ,Chla677 , 680674 , Chla683682 .
4　Conclusion
This study w as the fi rst demonst ration to
discriminate the pigments composition in
monomeric LHC Ⅱ by i ts transient spect rum
characteristic. In the lifet ime data analy sis , as the
f luorescence emission curve including the grow th
process (accepting energy ) and decay pro cess
(dissipate energy);a 6-exponential fi t w as ve rif ied
to be appropriated to fi t these data. This al lowed us
169
光　子　学　报 35 卷
to specifically assign and different iate in detail the
energy t ransfer paths among Chls. And all the
lifetime w as assigned to dif ferent pathw ay o f
energy t ransfer reasonably. The long-term
objective fo r this pro ject is to provide answ ers to
key que stions on the st ructure-function
relationships of the apparatus enabling eff icient
utilisat ion of light for the synthesis o f metabolic
energy in oxygenic photosynthesis.
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假根羽藻外周天线内能量传递的飞秒光谱研究
张苏娟1 　王水才1 　贺俊芳1 　陈　晖2
(1 中国科学院西安光学精密机械研究所瞬态室 ,西安 710068)
(2 中国科学院植物所 , 北京 100093)
收稿日期:2005 07 12
摘　要　在时间相关单光子技术的基础上 ,对假根羽藻外周天线内叶绿素分子间的能量传递进行研究. 采
用瞬态吸收与荧光发射谱识别样品内的具有特征光谱组分的分子 ,得到在叶绿素分子的 Q带区主要存在以
下六个特征分子:Chlb630 ,Chlb642 ,Chla653652 ,Chla667664 ,Chla676 , 680675 ,Chla683682. 在 630 nm 的飞秒脉冲光的激发下 ,通
过对不同特征发射峰出的荧光动力学进行解析得到:1)Chlb628分子所吸收的能量仅有大约 20%被直接传递
给其他叶绿素分子 ,传能时间小于 150 fs;2)叶绿素间大部分的能量传递发生在长于 76 ps的时间范围内;3)
传能时间常量在几百 f s以及 10 ps左右的间接传能可能与具有不同光谱组分特征的叶绿素分子在外周天线
内的排列方式以及偶极距的取向有关;4)Chlb654 , 657 652 ,Chla666664 ,Chla677 , 680674 和 Chla683682以荧光形势耗散能量的时
间常量分别为 1. 41 ns , 1. 39 ns , 676 ps , 709 ps ,这部分在整个能量耗散中占的比例不超过 40%.
关键词　能量传递;外周天线;飞秒光谱;荧光动力学
Zhang Sujuan　was bo rn in Chengcheng , in 1975. She received her B. S. degree in 1996 and
M . S. deg ree in 1999 from No rthw estUniversity and her Ph. D. deg ree f rom Xi′an Jiao tong
Unive rsity in 2004. He r resea rch invo lves the development of a technique fo r the
measurement of ult rafast events and its application in biolo gy and medicine.
170