Original Paper |
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Acta Biochim Biophys |
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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,
[email protected]
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 Å resolution
parallel to the membrane plane, and at approximately 4.9 Å 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).
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