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Acta Biochim Biophys
Sin 2006, 38: 310-317
doi:10.1111/j.1745-7270.2006.00171.x
Energy Transfer among
Chlorophylls in Trimeric Light-harvesting Complex II of Bryopsis corticulans
Su-Juan ZHANG1,2*,
Shui-Cai WANG2, Jun-Fang HE2,
and Hui CHEN3
1 Institute of
Photonics and Photo-Technology, Northwest University, Xi’an 710069, China;
2
State Key Laboratory of Transient Optics and Photonics,
Xi’an Institute of Optics and Precision Mechanics, Chinese Academy of Sciences,
Xi’an 710068, China;
3
Laboratory of Photosynthesis Basic Research,
Institute of Botany, Chinese Academy of Sciences, Beijing 100093, China
Received: October
25, 2005
Accepted: March 21,
2006
This work was
supported by a grant from the National Natural Science Foundation of China
(60308004)
*Corresponding
author: Tel, 86-29-88303281; Fax, 86-29-88303336; E-mail,
Abstract A study on energy transfer among chlorophylls (Chls) in the trimeric
unit of the major light-harvesting complex II (LHC II) from Bryopsis
corriculan, was carried out using time-correlated single photon counting.
In the chlorophyll Q region of LHC II, six molecules characterized as Chlb628, Chlb646, Chlb652654,657, Chla664666, Chla674677,680 and Chla682683 were discriminated according to their absorption spectrum and
fluorescence emission spectrum. Then, excited by pulsed light of 628 nm,
fluorescence kinetics spectra in the chlorophyll Q region were measured. In
accordance with the principles of fluorescence kinetics, these kinetics data
were analyzed with a multi-exponential model. Time constants on energy transfer
were obtained. An overwhelming percentage of energy transfer among chlorophylls
undergoes a process longer than 97 picoseconds (ps), which shows that, before
transferring energy to another Chl, the excited Chl might convert energy to
vibrations of a lower state with different multiplicity (intersystem crossing).
Energy transfer at the level of approximately 10 ps was also obtained, which
was interpreted as the excited Chls may go through internal conversion before
transferring energy to another Chl. Although with a higher standard deviation,
time constants at the femtosecond level can not be entirely excluded, which can
be attributed to the ultrafast process of direct energy transfer. Owing to the
arrangement and direction of the dipole moment of Chls in LHC II, the
probability of these processes is different. The fluorescence lifetimes of Chlb652654,657, Chla664666, Chla674677,680 and Chla682683 were determined to be 1.44 ns, 1.43 ns, 636 ps and 713 ps,
respectively. The percentages of energy dissipation in the pathway of
fluorescence emission were no more than 40% in the trimeric unit of LHC II.
These results are important for a better understanding of the relationship
between the structure and function of LHC II.
Key words energy transfer; LHC II; transient spectrum; fluorescence
kinetics
Bryopsis corticulans is a siphonous
green alga growing in intertidal areas. Owing to its adaptability to light, B.
corticulans can survive the periodic tide. The light-harvesting complex II
(LHC II) of photosystem II, which is the outermost and most abundant antenna
complex of chloroplasts, exists as a trimer and binds half of the thylakoid
chlorophyll molecules. It plays essential roles in harvesting solar energy and
transferring it in the process of photosynthesis. The structure of LHC II from
pea has been determined by electron crystallography at 3.4 ? resolution
parallel to the membrane plane, and at approximately 4.9 ? resolution
perpendicular to this plane [1]. This model revealed some basic structural
features of LHC II, including three transmembrane a-helices (helices A, B and
C), a short amphipathic helix (helix D), 12 chlorophyll tetrapyrroles with
roughly determined locations and orientations, and two carotenoids. A more
detailed structural picture of LHC II was reported by Liu et al., in
which the 14 chlorophylls (Chls) of each monomer can be unambiguously
distinguished as eight Chla and six Chlb molecules [2]. All Chlbs are located
around the interface between adjacent monomers, together with Chlas, they are
the basis for efficient light harvesting. Four carotenoid binding sites per
monomer have been observed [2]. Absorption kinetics in the LHC II of high plants has revealed the
Chl-Chl energy transfer in the LHC II complex. At
temperatures near room temperature the fastest Chlb
to Chla transfer seems to occur with a lifetime of approximately 150–200 femtosecond (fs) [3–6]. Further components have lifetimes of approximately 500–600 fs and 5–7 picoseconds
(ps) [3–6]. Energy transfer among the Chlas occurs on
a timescale of typically 1 ps and longer [4–7]. Given higher spectrum
sensitivity and temporal resolution, fluorescence kinetics spectra can provide
more detailed information on energy transfer. In the present work, we
investigated the pigment organization of trimeric LHC II by a combination of
transient absorption spectrum and transient fluorescence emission spectrum.
Then the energy transfer among Chls of trimeric LHC II was studied by the
fluorescence kinetics spectra. These results are important for a better
understanding of the relationship between the structure and function of LHC II.
Materials and Methods
Isolation of trimeric LHC II
A light-harvesting chlorophyll a/b-protein complex was isolated
directly from thylakoid membranes of marine green alga, B. corticulans,
through two consecutive runs of liquid chromatography. The trimeric form of the
light-harvesting complex was obtained by sucrose gradient ultra-centrifugation
in the Institute of Botany, Chinese Academy of Sciences (Beijing, China). All
detailed procedures were described previously [8].
Experimental apparatus
The measurement of femtosecond transient absorption and fluorescence
emission were carried out using a titanium-sapphire laser system (Spectra-Physics,
California, USA). The master oscillator was a Ti:sapphire laser
(Spectra-Physics) excited by a CW diode pumped intracavity doubled Nd:YVO4 (Spectra-Physics). The laser provided a train of approximately 60
fs at an 82 MHz repetition rate with 0.4 W of average power at the central
wavelength of 800 nm. To get a higher energy/pulse, the pulse amplification was
obtained by injecting the pulses in a Ti:sapphire regenerative amplifier
(Spitfire/Hurricane, Spectra-Physics) pumped at 1 kHz by a Q-switched,
intracavity doubled Nd:YLF running at the second harmonic wavelength, 527 nm
(model 527 DP-H; Evolution, Spectra-Physics). After amplification, pulsed light
of 800 nm at 1 kHz was injected into optical parametric amplification (OPA)
(OPA-800CF; Spectra-Physics) to generate white light continuum pulses and
tunable light. The white light used in OPA as a seed was created by focusing a
few mJ of energy (800 nm) into sapphire. With a broad spectral coverage,
white light continuum provides an ideal seed source for OPA. As the visible
light range (<800 nm) in seed light is of no use in parametric amplification, this range was split with a dichroic mirror and used for the detection of transient absorption spectroscopy. The
OPA converted the wavelength to approximately 628 nm with a pulse width of
approximately 150 fs and a spectral width of approximately 3 nm. Pulse energy
of approximately 0.5 mJ in a 1 mm diameter spot was used. The FLS920 (Edinburgh
Instruments, Livingston, UK) was chosen for measuring spectra and the kinetics
of fluorescence emission. The measurements were based on time-correlated single
photon counting, using the ultrafast photodetectors of the microchannel plate
photomultiplier R3809U-50 (Hamamatsu, Chiba, Japan) with a C4878 cooling system (Hamamatsu). After numerical
reconvolution, the lower limit of the lifetime range could be estimated to be
2.5 ps.
Data analysis
The interaction of visible light with a molecular system generally produces
a vibrationally hot electronic state. The primary event following excitation is
therefore vibrational relaxation. The way in which this occurs depends on the
vibrational modes involved, the coupling between them and the shape of the
multidimensional potential surface of the excited state [9–12]. When one
kind of chlorophyll molecule (M1) absorbs the excited light during a femtosecond period of time, it
would reach the state S1*. Then S1* would dissipate this excited energy and come back to ground state S0 (Fig. 1). According to quantum statistics, there are three
ways for energy to dissipate. First, some part of S1* transfers its energy directly to another kind of chlorophyll
molecule M2 and goes back to ground
state S0. Second, some part of S1* might convert energy to vibrations of a lower state with the same
multiplicity (internal conversion) or different multiplicity (intersystem
crossing), then transfer its energy to M2 before it gets to the lowest vibrational state of S1. Finally, some part of S1* comes through vibrational relaxation to the lowest vibrational
state of S1, then emits fluorescence
and goes back to ground state S0. These three existing reactive channels compete with each other and
speed up the decay of the excited state [10,11]. That is, during the process of
fluorescence emitting (e.g., l3), apart from receiving
the energy from M1, M2 also transfers the excited energy to M3 at the same time. In actual examples, there are many kinds of M1 that can transfer energy to M2. M2 also transfers
energy to many kinds of M3. Therefore, the
time resolved fluorescence emission spectrum measured is a combination of the
growth process (accepting energy) and the decay process (dissipating energy). The interaction of visible light with a molecular system generally produces
a vibrationally hot electronic state. The primary event following excitation is
therefore vibrational relaxation. The way in which this occurs depends on the
vibrational modes involved, the coupling between them and the shape of the
multidimensional potential surface of the excited state [9–12]. When one
kind of chlorophyll molecule (M1) absorbs the excited light during a femtosecond period of time, it
would reach the state S1*. Then S1* would dissipate this excited energy and come back to ground state S0 (Fig. 1). According to quantum statistics, there are three
ways for energy to dissipate. First, some part of S1* transfers its energy directly to another kind of chlorophyll
molecule M2 and goes back to ground
state S0. Second, some part of S1* might convert energy to vibrations of a lower state with the same
multiplicity (internal conversion) or different multiplicity (intersystem
crossing), then transfer its energy to M2 before it gets to the lowest vibrational state of S1. Finally, some part of S1* comes through vibrational relaxation to the lowest vibrational
state of S1, then emits fluorescence
and goes back to ground state S0. These three existing reactive channels compete with each other and
speed up the decay of the excited state [10,11]. That is, during the process of
fluorescence emitting (e.g., l3), apart from receiving
the energy from M1, M2 also transfers the excited energy to M3 at the same time. In actual examples, there are many kinds of M1 that can transfer energy to M2. M2 also transfers
energy to many kinds of M3. Therefore, the
time resolved fluorescence emission spectrum measured is a combination of the
growth process (accepting energy) and the decay process (dissipating energy). As the exciting pulse is not a real Dirac function in these
experiments, the measured fluorescence kinetics spectrum h(t) is
the convolution integral of the real fluorescence emission spectrum g(t)
with instrument response f(t). Their relationship is represented
in Equation 1.
Eq. 1
So the numerical procedure requires the use of the convolution
integral to extract the lifetime parameters. The model of numerical fit is
expressed in mathematical terms as Equation 2,
Eq. 2
with pre-exponential factors as Aj, the characteristic lifetime as tj and an additional background as y0. The pre-exponential factors can be either positive or negative. A
positive Aj value
represents a decay process (energy dissipation), whereas a negative Aj value is characteristic for a growth process (accepting energy).
The numerical routine to extract the parameters Aj and tj was made in a
Matlab procedure based on the Marquardt-Levenberg algorithm compiled by us. The
reduced c2 of the fitting result was calculated to evaluate the quality of the
fit results.
Results
Protein and pigment
composition of LHC II and its spectroscopic characteristics in the chlorophyll Q
region
The pigment composition of the trimeric LHC II has already been analyzed
using reversed-phase high performance liquid chromatography. The construction
of protein in the trimeric LHC II was analyzed by sodium
dodecylsulfate-polyacrylamide gel electrophoresis [13]. The protein map and
typical chromatograms of the pigment extracts from trimeric subcomplexes of LHC
II were described previously [13]. According to the results of sodium
dodecylsulfate-polyacrylamide gel electrophoresis, the trimeric LHC II used in
this experiment is heterogeneous. The ratio of the Chla/Chlb trimer is 1.2,
which is similar to that of the native LHC II reported in higher plants [14].
The knowledge of spectroscopic characteristics was obtained from transient
fluorescence emission spectrum (Fig. 2) and transient absorption
spectrum (Fig. 3). To avoid the excitation of carotenoids, the transient
fluorescence emission spectrum was excited at 628 nm. The absorption and
fluorescence spectra of the samples were measured before and after the femtosecond lifetime measurement, which showed no changes due to photochemical or other damage.
From the analysis of the spectroscopic characteristics combined with the
absorption spectra of individual pigments [15,16], there were six
characteristic molecules, marked as Chlb628, Chlb646, Chlb652654,657
Energy transfer between Chls
Though there are many peaks in the fluorescence emission shown in Fig.
2, fluorescence emission could only be detected in the time-resolved
experiments during the range 660–695 nm. Eight time-resolved fluorescence
spectra were recorded at 660, 665, 670, 675, 680, 685, 690 and 695 nm.
Considering the correspondence with the transient absorption spectrum of Fig.
3, four representative time-resolved fluorescence spectra characterized
with the pigments were analyzed following the routine described in “Data
analysis”. After reconvolution simulation, the most appropriate fitting
curve was created and the results in log are shown in Fig. 4. The
reduced c2 of the fitting results were all in the level of 10–4, which shows the quality
of the fitting results was well accepted. The fitting curve is also a
combination of the growth process (accepting energy) and the decay process
(dissipating energy). Therefore the fitting lifetimes in the results in log are
also divided into two major processes: the energy acceptance process and the
energy dissipation process. In each process, the pigment composition of LHC II
and its spectroscopic characteristics in the chlorophyll Q region allow us to specifically assign these lifetimes and
differentiate the energy transfer paths in detail among Chls. The corresponding
percentage of each subprocess was calculated as (Aj with the same
sign). All the fitting results of lifetimes and the percentage of energy
transfer among Chls are summarized clearly in Table 1. An overwhelming percentage
of the energy transfer among chlorophylls undergoes a process longer than 97
ps:Chlb628®Chlb646®Chlb652654,657 (149 ps)Chlb628®Chlb646®Chla664666 (157 ps)Chlb628®Chlb646®Chla674677,680 (103 ps)Chlb628®Chlb646®Chla682683 (97 ps)which indicates that, before
transferring energy to another Chl, the excited Chl might convert energy to
vibrations of a lower state with different multiplicity (intersystem crossing).
Some slower processes of energy transfer were also obtained, such as Chlb652654,657®Chla682683 (14.5 ps)Chla664666®Chla682683 (8.1 ps)Chla674677,680® Chla682683 (9.3 ps)These pathways could be described in terms of the excited Chl
undergoing internal conversion before transferring energy to another Chl. With
a higher standard deviation, time constants at the femtosecond level can be
attributed to the process of direct energy transfer, such as C628®C652654,657C628®C664666C628®C674677,680C628®C682683C628®C652654,657®C664666C628®C652654,657®C674677,680C652654,657®C664666and the difference in the probability is owing to the difference in
the arrangement and direction of the dipole moment of Chls in LHC II. The
fluorescence lifetimes of Chlb652654,657, Chla664666, Chla674677,680 and Chla682683 were determined to be 1.44 ns, 1.43 ns, 636 ps and 713 ps,
respectively. The percentages of energy dissipation in the pathway of
fluorescence emission were no more than 40% in the trimeric unit of LHC II. All
of these results are important for a further understanding of the relationship
between the structure and function of LHC II.
Discussion
Given the recent progress in the molecular structure determination
of the LHC II complexes [1,2], it is now possible to obtain very detailed descriptions
of the functional and optical properties of these systems by different spectral
and kinetic information on energy transfer [3–7,17,18]. In this study,
after recognition of the protein and pigment composition of LHC II and its
spectroscopic characteristics in the chlorophyll Q
region, the lifetimes obtained in the fluorescence kinetics were assigned to
different energy transfer pathways.A thorough understanding of the LHC II absorption in an overall substructure model, including possible excitonic effects, is complicated by the unknown origin of the observed spectral heterogeneity of at least 10 sub-bands [19]. In the process
of detecting the absorption and emission spectra of LHC II excited by steady excitation
sources, considering the nonconservative contributions to
the biological sample induced by consistent irradiation, the spectra would be
distorted by the final bleaching and excited state
absorption signal in the Chls range [3,20–24]. Therefore, in this
article, we present a new approach to assessing the spectroscopic
characteristics of trimeric LHC II by probing the absorption peaks and emission
peaks directly by ps. All the spectra measured were excited by laser with a
pulse width of approximately 150 fs at 1 kHz, which ensured the restoration of
the sample when excited by ultrafast light. However, there are also somewhat
smaller involved sub-bands hindered in transient spectra. To solve this
problem, some derivative spectra were made to explore the hindered sub-bands (Fig.
2). There are at least six characteristic molecules in the Q range of Chls,
marked as Chlb628, Chlb646, ,, and , recognized by the transient spectroscopic technique. Our
results are mostly consistent with those of other groups using different
techniques. Absorption peaks exist at 648 nm, 660 nm, 669 nm, 678 nm, 684 nm
and 695 nm [25–29].According to the measurement principle of time-correlated single
photon counting, a spectrometer equipped with the fastest detection system combines
ultimate sensitivity [Dark Count Rate (–25 ?C)<5 cps] with high spectral resolution (0.1 nm) and excellent stray light rejection (1:1010). It offers the highest dynamic range (count rates of up to 100
MHz) and temporal resolution (Multi Channel Scaling mode cards are available
with a minimum time window down to 2.5 ns with 4096 channels). Furthermore, the
technique is digital, making it insensitive to background noise from detectors
and electronics (a signal to noise ratio of more than 6000:1 for a measurement
can be guaranteed). In the lifetime data analysis, we started from one single
exponential model to six exponential models in turn. A six-exponential fit
would be the most appropriate by evaluating the fit results using the reduced c2.When the fluorescence kinetics spectra measured at different
wavebands were superposed at one window, some energy transfer among Chls marked
with the characteristic fluorescence bands could also be obtained according to
the shift of emission peaks on the timescale. The maximum was first observed at
675 nm and 685 nm after the excitation (approximately 440 ps after the excited
pulse). The maximum shifted to 665 nm in the end within 640 ps. So the transfer
time among Chls in the Q range should be no more than 200 ps, which is
consistent with the analysis in Table 1. The lifetime of the
characteristic fluorescence band fitted by the fluorescence time fitting
software FLS 920 (Edinburgh Instruments, Livingston, UK) (data not shown) is
consistent with the analysis in Table 1 (600 ps–1.44 ns).In Table 1, we noticed that when energy was transferred among
Chls through Chlb646, the percentage
is higher and the transfer time lasted up to 100 ps, so this pathway is the
main channel of energy transfer among Chls. which implies that Chlb646 must be assembled in a special position in the structure of
trimeric LHC II, or that Chlb646 might always go through the intersystem crossing and exist in a
triplet state before transferring energy to others. An energy transfer time of
approximately 10 ps was assigned to the direct energy accepting process of from
other Chls, which occupied the highest percentage in the energy transfer. This
indicates that was the main acceptor in LHC II. With the longer wavelength,
more energy is dissipated in the mode of fluorescence. That is to say, from Chlb652654,657, to Chla664666, to Chla674677,680, to Chla682683, the efficiency of energy transfer is gradually decreasing.
This study is the first effort to
discriminate the pigment composition of trimeric LHC II by its transient
spectrum characteristics. In the lifetime data analysis, as the fluorescence
emission curve includes the growth process (accepting energy) and the decay
process (dissipating energy), a six-exponential fit was verified to be
appropriate. This allowed us to specifically assign and differentiate in detail
the energy transfer paths among the Chls. The lifetime was assigned to
different pathways of energy transfer. The long-term objective for this project
is to solve key issues in the relationship between the LHC II’s structure and
function for the efficient use of light in oxygenic photosynthesis.
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