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Regular paper
Tuning energy transfer in the peridinin–chlorophyll complex
by reconstitution with different chlorophylls
Toma
´s
ˇPolı
´vka
1,3,
*, Torbjo
¨rn Pascher
1
, Villy Sundstro
¨m
1
& Roger G. Hiller
2
1
Department of Chemical Physics, Lund University, Box 124, S-22100 Lund, Sweden;
2
Department of
Biological Sciences, Macquarie University, 2109 NSW, Australia;
3
Present address: Institute of Physical
Biology, University of South Bohemia, 373-33 Nove Hrady, Czech Republic; *Author for correspondence
(e-mail: polivka@ufb.jcu.cz; tomas.polivka@chemphys.lu.se; fax: +46-46-222 4119)
Received 5 December 2004; accepted in revised form 31 January 2005
Key words: carotenoids, energy transfer, excited state dynamics, PCP, peridinin, peridinin-chlorophyll
protein, reconstitution
Abstract
In vitro studies of the carotenoid peridinin, which is the primary pigment from the peridinin chlorophyll-a
protein (PCP) light harvesting complex, showed a strong dependence on the lifetime of the peridinin lowest
singlet excited state on solvent polarity. This dependence was attributed to the presence of an intramolecular
charge transfer (ICT) state in the peridinin excited state manifold. The ICT state was also suggested to be a
crucial factor in efficient peridinin to Chl-aenergy transfer in the PCP complex. Here we extend our studies
of peridinin dynamics to reconstituted PCP complexes, in which Chl-awas replaced by different chlorophyll
species (Chl-b, acetyl Chl-a, Chl-dand BChl-a). Reconstitution of PCP with different Chl species maintains
the energy transfer pathways within the complex, but the efficiency depends on the chlorophyll species. In
the native PCP complex, the peridinin S
1
/ICT state has a lifetime of 2.7 ps, whereas in reconstituted PCP
complexes it is 5.9 ps (Chl-b) 2.9 ps (Chl-a), 2.2 ps (acetyl Chl-a), 1.9 ps (Chl-d), and 0.45 ps (BChl-a).
Calculation of energy transfer rates using the Fo
¨rster equation explains the differences in energy transfer
efficiency in terms of changing spectral overlap between the peridinin emission and the absorption spectrum
of the acceptor. It is proposed that the lowest excited state of peridinin is a strongly coupled S
1
/ICT state,
which is the energy donor for the major energy transfer channel. The significant ICT character of the S
1
/ICT
state in PCP enhances the transition dipole moment of the S
1
/ICT state, facilitating energy transfer to
chlorophyll via the Fo
¨rster mechanism. In addition to energy transfer via the S
1
/ICT, there is also energy
transfer via the S
2
and hot S
1
/ICT states to chlorophyll in all reconstituted PCP complexes.
Abbreviations: aChl-a–acetyl chlorophyll-a; BChl-a–bacteriochlorophyll-a; CD – circular dichroism; Chl
– chlorophyll; EADS – evolution associated difference spectra; ESA – excited state absorption; ICT –
intramolecular charge transfer; LH2 – light harvesting complex 2 of purple bacteria; LHCII – light har-
vesting complex of Photosystem II; PCP – peridinin-chlorophyll-aprotein
Introduction
Excitation energy transfer in photosynthesis is a
vitally important process that has been a subject of
numerous studies in recent years (for reviews see
Sundstro
¨m et al. 1999; Ritz et al. 2000; van
Amerongen and van Grondelle 2001; Polı
´vka and
Sundstro
¨m 2004). Among the variety of light-
harvesting complexes, the water soluble peridinin-
chlorophyll-aprotein (PCP) isolated from the
dinoflagellate Amphidinium carterae, whose struc-
ture is known to 2.0 A
˚resolution (Hofmann et al.
Photosynthesis Research (2005) 86: 217–227 Springer 2005
1996), represents a special case. In contrast to
other light-harvesting complexes, a carotenoid,
peridinin is the principal light-harvesting pigment
as each PCP molecule contains 8 peridinins and 2
Chl-a. Efficient energy transfer between peridinin
and Chl-ais crucial to maintaining the high light-
harvesting capacity of PCP.
The highly substituted carotenoid peridinin
itself exhibits several unusual properties in its
excited states. In all carotenoids, the lowest excited
state (S
1
) is not directly accessible from the ground
state (S
0
) due to symmetry reasons and the second
excited state (S
2
) is responsible for the strong
absorption in the blue-green spectral region (Pol-
ı
´vka and Sundstro
¨m 2004). In peridinin the dra-
matic dependence of the S
1
lifetime on solvent
polarity was attributed to an intramolecular
charge transfer (ICT) state in the excited state
manifold (Bautista et al. 1999a; Frank et al. 2000;
Zigmantas et al. 2001). Although a number of
studies addressing the excited state dynamics of
peridinin have been carried out during past 5 years
(Bautista et al. 1999a; Frank et al. 2000; Damja-
novic et al. 2000; Zigmantas et al. 2001; Zimmer-
mann et al. 2002; Shima et al. 2003; Vaswani et al.
2003; Zigmantas et al. 2003; Linden et al. 2004;
Papagiannakis et al. 2004), a consistent picture is
still missing. Various models have been suggested
to explain experimental data: (1) the ICT state
being identical with the S
1
state (Shima et al.
2003), (2) a strong S
1
-ICT coupling forming a
common S
1
/ICT state with a complicated potential
surface (Zigmantas et al. 2003), (3) the S
1
and ICT
viewed as two distinct electronic states that equil-
ibrate on the time scale of a few picoseconds
(Papagiannakis et al. 2004). Although models 2
and 3 are quite similar, the crucial difference is that
while Model 2 assumes one lifetime for the
strongly coupled S
1
/ICT state, in model 3 the S
1
and ICT states have distinct lifetimes. To date,
none of these models have been able to consis-
tently explain all the experimental data.
Efficient peridinin-chlorophyll energy transfer
in PCP was demonstrated prior to structural
knowledge of the complex (Song et al. 1976;
Akimoto et al. 1996), but availability of the high-
resolution structure (Hofmann et al. 1996) became
a landmark for systematic time-resolved studies.
The energy transfer between peridinin and chlo-
rophyll in the PCP complex utilizes both S
2
and S
1
pathways, resulting in total energy transfer effi-
ciency of 88% (Bautista et al. 1999b). Although
calculations did not predict the S
2
-mediated energy
transfer (Damjanovic et al. 2000), experiments
clearly showed that the S
2
state transfers energy
with efficiency ranging from 25% to 50% (Krueger
et al. 2001; Zigmantas et al. 2002; Linden et al.
2004). The rest of the energy transfer proceeds via
the S
1
and/or ICT channel. Efficiency of the S
1
channel is not easy to estimate, because the
intrinsic (without energy transfer) S
1
lifetime of
peridinin in PCP is not known. The measured S
1
/
ICT lifetime of peridinin in PCP ranges from 2.7–
3.5 ps (Krueger et al. 2001; Zigmantas et al. 2002;
Linden et al. 2004), and is significantly shorter
than that obtained in the most polar solvents
(Frank et al. 2000; Zigmantas et al. 2003). Since
peridinin in PCP is in a mildly polar environment,
it was concluded that the S
1
-mediated energy
transfer pathway must work with efficiency around
80% (Krueger et al. 2001; Zigmantas et al. 2002;
Linden et al. 2004).
The peridinin ICT state has a characteristic
stimulated emission in the near-infrared region
(Zigmantas et al. 2001). Time resolved absorption
changes showed that the ICT state is involved in
the peridinin-chlorophyll energy transfer, since the
decay of the ICT emission at 930 nm matched the
rise of Chl-ableaching (Zigmantas et al. 2002).
This observation raises an important question
concerning the mechanism of peridinin to chloro-
phyll energy transfer, because the ICT state has
apparently too low energy to allow energy transfer
to the Q
y
band of Chl-alying at 14800 cm
)1
in
PCP. To explain this discrepancy, Zigmantas et al.
(2003) pointed out that in the case of a strongly
coupled S
1
/ICT state, energy transfer may still
occur from the S
1
-like part of the potential surface.
The role of the ICT state is to enhance the dipole
moment of the S
1
/ICT state, allowing efficient
energy transfer via a Fo
¨rster mechanism (Zig-
mantas et al. 2002).
Reconstitution of light-harvesting complexes
with different pigments is a promising method for
studying mechanism of energy transfer (Herek
et al. 2000). Here we make use of recent successful
reconstitution of the PCP complex with a variety
of chlorophyll species to provide further insight
into mechanisms of peridinin-chlorophyll energy
transfer in the PCP complex (Miller et al. 2005
[accompanying paper]). The reconstituted PCP
complexes have the chlorophyll Q
y
bands spanning
218
the spectral region from 650 nm (Chl-b) to 790 nm
(BChl-a), leading to significant changes in spectral
overlap between peridinin emission and chloro-
phyll Q
y
absorption. In this paper, we present
results of time-resolved experiments on energy
transfer in the reconstituted PCP complexes. The
experimental results combined with calculations
show that peridinin to chlorophyll energy transfer
can be explained by the Fo
¨rster mechanism.
Materials and methods
Reconstitution of PCP
PCP was reconstituted from heterologously
expressed N-domain apoprotein and purified pig-
ments as described in the preceding paper (Miller
et al. 2005)
Spectroscopic measurements
Femtosecond pulses were obtained from a
Ti:Sapphire oscillator pumped by the 5 W output
of a CW frequency-doubled, diode-pumped
Nd:YVO
4
laser. The oscillator, operating at a
repetition rate of 82 MHz, was amplified by are-
generative Ti:Sapphire amplifier pumped by a
Nd:YLF laser (1 kHz), producing 130 fs pulses
with an average energy of 0.9 mJ/pulse and a
central wavelength at 800 nm. The amplified pul-
ses were divided into two paths: one to pump an
optical parametric amplifier for generation of
excitation pulses, and the other to produce white-
light continuum probe pulses in a 0.5 cm sapphire
plate. The mutual polarization of the pump and
probe beams was set to the magic angle (54.7 )
using a polarization rotator placed in the pump
beam. For signal detection, the probe beam and an
identical reference beam (that had no overlap with
the pump beam) were focused onto the entrance
slit of a spectrograph, which than dispersed both
beams onto a home-built dual photodiode array
detection system. Each array contained 512 pho-
todiodes and allowed a spectral range of 270 nm
to be measured in each laser shot. The spectral
resolution of the detection system was 80 cm
)1
and the energy of excitation was attenuated by
neutral density filters to 5·10
14
photons
pulse
)1
cm
)2
. Absorption spectra were measured
before and after measurements to ensure that no
permanent photochemical changes occurred over
the duration of experiment.
Analysis of time resolved spectra
To gain deeper insight into the excited state
dynamics, all kinetic traces collected by the diode-
array detection system were fitted globally. This
approach allows more precise determination of the
time constants of the excited state processes and,
more importantly, assignment of spectral profiles
of the intermediate excited state species (van
Stokkum et al. 2004). The data were fitted to a sum
of exponentials, including numerical deconvolu-
tion of the FWHM of the response function, and a
fourth degree polynomial describing the chirp. The
fitting procedure used general linear regression for
the amplitudes of the exponentials and the Nelder-
Mead simplex method for the rate constants, the
FWHM and the chirp polynomial. To visualize the
excited state dynamics, we assume that the excited
PCP complexes evolve according to a sequential,
irreversible scheme A fiB, B fiC, C fiD... The
arrows represent increasingly slower monoexpo-
nential processes and the time constants of these
processes correspond to lifetimes of the species A,
B, C, D... The spectral profiles of the species are
called evolution-associated difference spectra
(EADS), and although they do not correspond to
the pure spectra of the excited state species, they
provide valuable information about the time evo-
lution of the whole system (van Stokkum et al.
2004).
Results
Absorption spectra of the reconstituted PCP
complexes normalized to the peridinin absorption
at 535 nm are depicted in Figure 1. The reconsti-
tuted complexes exhibit a single, narrow chloro-
phyll Q
y
band located at 650 nm (Chl-b), 672 nm
(Chl-a), 687 nm (acetyl Chl-a), 700 nm (Chl-d)
and 790 nm (BChl-a). The protein-induced shift of
the Q
y
bands, as compared with that measured in
acetone solution, is comparable with the shift
observed in native PCP, demonstrating successful
incorporation of different chlorophyll species. In
the PCP complex containing BChl-a, an additional
weak band at 690 nm signals a mild contamination
by acetyl-Chl-a(aChl-a). The distinct Q
x
band of
219
BChl-aat 585 nm is also clearly visible. Based on
the absorption spectra, the excitation wavelength
was chosen to be at 530 nm for all complexes,
because only peridinin is excited at this wavelength.
Transient absorption spectra shown in Figure 2
demonstrate energy transfer between peridinin
excited at 530 nm and chlorophyll. At 300 fs, the
transient absorption spectra are dominated by the
characteristic broad peridinin excited state
absorption (ESA) peaking around 630 nm
(Papagiannakis et al. 2004), accompanied by
ground state bleaching below 550 nm. Even at
300 fs after excitation, the onset of the chlorophyll
bleaching is apparent in all PCP complexes, sug-
gesting that a fraction of energy is transferred via
the peridinin S
2
state. After 30 ps, the peridinin
signal has decayed and the spectra show only the
chlorophyll Q
y
bleaching and a weak featureless
ESA, confirming that peridinin to chlorophyll
energy transfer takes place in all reconstituted
complexes. Energy transfer from peridinin moni-
tored by the rise of chlorophyll bleaching is shown
in Figure 3. The rise is slowest for Chl-b, while for
aChl-a, Chl-dand BChl-athe energy transfer
becomes faster than that for Chl-ain the native
PCP complex.
The global fitting results for the PCP complex
reconstituted with Chl-aare shown in Figure 4.
To reproduce the experimental data, at least four
time components (0.1, 0.6, 2.9 and 25 ps) are
needed. An additional non-decaying component,
which represents excitations being trapped in the
lowest excited state of Chl-a(lifetime 4.5 ns,
Koka and Song 1977), is needed to reproduce the
final state of the system. The first EADS depicted
by the black line is created directly upon excita-
tion. Below 575 nm it consists of peridinin
ground state bleaching and the S
2
stimulated
emission. The positive signal above 700 nm is
assigned to an ESA from the S
2
state. Interest-
ingly, the Chl-ableaching around 670 nm is
clearly visible immediately after excitation, sig-
naling that a fraction of energy is transferred on
a time scale beyond our time resolution. The first
EADS is replaced within 0.1 ps by the second
EADS (red), which shows that all signals associ-
p
Figure 1. Absorption spectra of the PCP complexes
reconstituted with Chl-b(solid), Chl-a(dash), aChl-a
(dot), Chl-d(dash-dot), and BChl-a(dash-dot-dot). The
spectra are normalized to peridinin absorption at 535 nm.
Inset shows enlargement of the Q
y
region of the chloro-
phylls.
Figure 2. Transient absorption spectra of the reconstituted
PCP complexes recorded after 530 nm excitation at 300 fs (top)
and 30 ps (bottom).
220
ated with the S
2
state of peridinin are gone, and
the S
1
/ICT ESA peaking at 630 nm dominates
the EADS. The Chl-ableaching is more pro-
nounced in the second EADS, but it is also
slightly blue-shifted as compared with the first
EADS, suggesting that S
2
-mediated energy
transfer occurs on two different time scales. The
faster time scale is beyond our time resolution
and represents S
2
transfer to the Chl-ahaving
lower energy. The slower time scale is character-
ized by the 0.1 ps component and corresponds to
S
2
transfer from peridinin to the Chl-awith Q
y
band located at higher energy. Further evolution
of the system proceeds with the 0.6 ps time con-
stant leading to EADS shown as a green line in
Figure 4. This evolution is characterized by a
decrease of both the S
1
/ICT ESA and the perid-
inin ground state bleaching, suggesting a loss of
excited state population on peridinin. Since it is
also accompanied by an increase of the Chl-a
bleaching, it strongly supports previous assign-
ments of this component to a peridinin to Chl-a
energy transfer channel that proceeds via the hot
S
1
/ICT state of peridinin (Zigmantas et al. 2002;
Linden et al. 2004). The third EADS (green)
decays with a 2.9 ps time constant to form the
EADS shown in blue. This step is characterized
by a further loss of both the S
1
/ICT ESA and
ground state bleaching of peridinin, accompanied
by a significant increase of the Chl-ableaching.
Accordingly, the 2.9 ps time evolution is assigned
to the peridinin to Chl-aenergy transfer pro-
ceeding via the relaxed S
1
/ICT state. Further
dynamics, occurring with a 25 ps time constant,
are observed even after the energy transfer is
completed. The observed changes are small (from
blue to cyan EADS in Figure 4) and again
include a loss of peridinin S
1
/ICT ESA and
ground state bleaching, but the Chl-ableaching
decreases in this step. Consequently, the process
characterized by the 25 ps component cannot be
due to energy transfer from peridinin to Chl-a.A
similar time constant has previously been found
for native PCP and assigned to S
1
/ICT-S
0
internal
conversion of a peridinin that does not transfer
energy to Chl-a(Krueger et al. 2001). Alterna-
tively, equilibration and/or annihilation between
Figure 3. Rise of the chlorophyll bleaching after peridinin
excitation at 530 nm. Kinetics were measured at probing
wavelengths corresponding to the maximum of the Q
y
bleach-
ing: 650 nm (Chl-b), 672 nm (Chl-a), 689 nm (aChl-a), 700 nm
(Chl-d) and 790 nm (BChl-a). The inset shows the time evolu-
tion in the first 3 ps.
Figure 4. EADS resulting from global fitting analysis of data
collected after peridinin excitation at 530 nm for the PCP
complex reconstituted with Chl-a(top) and BChl-a(bottom).
n.d. stands for the long-living component that is non-decaying
at the time scale of experiments. See text for details.
221
the two Chl-amolecules in the PCP complex have
been proposed (Zigmantas et al. 2002), because
Chl–Chl energy transfer in PCP occurs on a
comparable time scale (Kleima et al. 2000a; Ila-
gan et al. 2004). Loss of the S
1
/ICT ESA sup-
ports the former explanation, while the decrease
of the Chl-ableaching rather points to the latter.
It is likely that both processes take place in the
PCP complex, and the 25 ps evolution observed
here represents a mixture of them. The final, non-
decaying EADS (cyan) is due to the equilibrated
Chl-a, and shows a strong bleaching at 670 nm
and a weak ESA spanning most of the visible
spectral region.
The EADS resulting from the global fitting of
the PCP complex reconstituted with BChl-aare
shown in the bottom panel of Figure 4. The overall
evolution pattern is similar to that described above
for the PCP complex containing Chl-a, but one
difference that is specific for the Bchl-aPCP
complex must be mentioned. Contrary to other
PCP complexes, the BChl-aPCP also contains a
trace of aChl-a(see Figure 1) and the presence of
the second chlorophyll species is reflected in the
excited state dynamics. As for the other PCP
complexes, the S
2
route operates as evidenced by
appearance of the BChl-ableaching at 790 nm
when going from the first to the second EADS
(black to red in Figure 4). A hint of the BChl-a
bleaching is already in the first EADS (black), but
it is weaker than for the PCP complex containing
Chl-a(Figure 4, top). The majority of the peridi-
nin to BChl-aenergy transfer takes place with a
0.45 ps time constant. The transition from the
second to the third EADS (red to green in
Figure 4b) that monitors this process exhibits a
major loss of the S
1
/ICT ESA and the ground state
bleaching of peridinin, accompanied by simulta-
neous increase of the BChl-ableaching. On the
other hand, the process occurring with the 1.9 ps
time constant (transition from green to blue
EADS) does not involve BChl-a. Instead, the loss
of the peridinin excited state population goes
along with an increase of a bleaching signal at
685 nm that is characteristic of aChl-a(Figure 1).
Therefore, the 1.9 ps component represents the S
1
/
ICT energy transfer for the fraction of the PCP
complexes containing aChl-a. This assignment is
further supported by the fact that nearly the same
time constant is found in the PCP complexes
reconstituted with aChl-a(Table 1). The time
evolution of the PCP complexes reconstituted with
other chlorophyll species is similar to that
observed for the PCP complex reconstituted with
Chl-a, except the time constant characterizing
the energy transfer via the S
1
/ICT channel (see
supplementary material for EADS). The time
constants extracted from the global fitting
are summarized in Table 1 for all PCP complexes.
The S
1
/ICT state of peridinin in native PCP has
a significant ICT character, with a characteristic
near-infrared emission peaking around 930 nm
(Zigmantas et al. 2002). We have measured
kinetics at 930 nm for all reconstituted PCP com-
plexes and the resulting traces are shown in
Figure 5. The negative signal due to ICT stimu-
lated emission is found in all reconstituted com-
plexes, signaling involvement of the ICT state. The
930 nm kinetics were fitted with the time constants
obtained from the global fitting of kinetics in the
visible spectral region, and fits are shown along
with the kinetics in Figure 5.
Table 1. Time constants extracted from global fitting analysis
a
s
1
(ps) s
2
(ps) s
3
(ps) s
4
(ps)
Chl-b0.1 0.55 5.9 18
Chl-a0.1 0.6 2.9 25
aChl-a0.09 0.65 2.2 16
Chl-d0.08 0.5 1.7 15
Bchl-a0.1 0.45
b
1.9
b
15
native PCP 0.08 0.65 2.7 30
a
The error margins are 25% for s
1
, 15% for s
2
, 10% for s
3
and 25% for s
4
. In addition, a non-decaying component with s> 1000 ps
due to the chlorophyll decay is present for all complexes.
b
The 0.45 ps lifetime represents the main component for the PCP complex reconstituted with BChl-a; the 1.9 ps component is due to
the S
1
/ICT lifetime of aChl-athat is in small amount present in this complex (see text for details).
222
Discussion
Comparison with native PCP complexes
Prior to discussing mechanisms of energy transfer in
the reconstituted PCP complexes, we demonstrate
success of the reconstitution by comparing the
results obtained for the reconstituted PCP complex
with those known from previous measurements on
the native PCP complex. Besides similarity in the
absorption spectra, CD and fluorescence excitation
spectra discussed in the accompanying paper
(Miller et al. 2005), the time-resolved studies pro-
vide further evidence of successful reconstitution.
The S
1
/ICT lifetime in the PCP complex reconsti-
tuted with Chl-a(2.9 ps) matches the values 2.7–
3.3 ps obtained from different measurements of the
S
1
/ICT lifetime in the native PCP complex (Akim-
oto et al. 1996; Bautista et al. 1999a, b; Krueger et al.
2001; Zigmantas et al. 2002; Linden et al. 2004).
Since, regardless the exact mechanism, energy
transfer is very sensitive to distances and mutual
orientation of donor and acceptor molecules, simi-
lar S
1
/ICT lifetimes in the native and reconstituted
PCP complexes provide strong evidence that
reconstituted PCP complexes must be structurally
very close to the native ones. This is underscored by
the presence of the S
2
channel in reconstituted PCP
complexes. Because of the short S
2
lifetime of
peridinin (Zigmantas et al. 2003; Linden et al.
2004), proper arrangement of donor and acceptor is
crucial to achieve S
2
-mediated energy transfer. As
the efficiency of this channel is comparable with that
of the native PCP complex for all reconstituted PCP
complexes (Table 2), it is a clear proof of successful
reconstitution. Besides energy transfer, the spectral
features in the transient absorption spectra of the
reconstituted PCP complexes are very similar to
those observed for native PCP. The broad, fea-
tureless S
1
/ICT ESA peaking at 630 nm together
with the presence of the ICT stimulated emission
around 930 nm (Zigmantas et al. 2001) shows that
peridinins in the reconstituted PCP complexes
experience the same protein environment as the
native ones. Further evidence of successful recon-
stitution is the presence of a weak negative band
around 535 nm in the 30-ps transient absorption
spectra of all reconstituted PCP complexes
(Figure 2). At this delay, the transient absorption
features correspond solely to Chl signal. Moreover,
this feature is present also after direct excitation of
Chl-aat 670 nm (Salverda 2003). This band was not
observed for other light-harvesting complexes and
is thus specific for PCP. The origin of this band is not
completely clear. Salverda et al. (2003) suggested
that it could be due to weak interaction between
peridinin and chlorophyll, caused, for example, by
Figure 5. Kinetics of ICT stimulated emission measured at
930 nm after excitation of peridinin at 530 nm. The kinetics are
normalized to the maximum of the stimulated emission. The
solid lines represent fits using the time constants obtained from
the global fitting analysis.
Table 2. Parameters of the S
1
/ICT and S
2
energy transfer
a
sET;S1=ICTaUS1=ICT bUS2c
Chl-b9.4 0.63 0.45
Chl-a3.55 0.82 0.35
acChl-a2.55 0.86 0.35
Chl-d1.9 0.89 0.28
Bchl-a0.46 0.97 0.35
Native PCP 3.2
d
0.84
d
0.25–0.50
e
a
The energy transfer times s
ET,S1/ICT
were calculated from
equation s1
S1=ICT ¼s1
ET;S1=ICT þs1
I, where s
S1/ICT
and s
I
are the
experimentally obtained S
1
/ICT lifetime and intrinsic peridinin
S
1
/ICT lifetime in the PCP complex, respectively. The value
s
I
=16 ps was used for calculations (Zigmantas et al. 2002).
b
Efficiency is calculated from equation U¼k=ðkET þkI),
where k
ET
and k
I
are S
1
/ICT energy transfer rate and rate of S
1
/
ICT-S
0
internal conversion, respectively (k¼s1
ET;S1=ICT and
kI¼s1
I)
c
The values are obtained from estimation of the chlorophyll
bleaching in the EADS corresponding to early times after
excitations (see text for details). The error caused by this esti-
mation is around 20%.
d
From Zigmantas et al. (2002)
e
From Krueger et al. (2001); Zigmantas et al. (2002); Linden
et al. (2004).
223
excited state mixing. It might also be due to an
electrochromic response of peridinin to the electric
field generated by the excited chlorophyll nearby.
Such a carotenoid response has been found in the
LH2 complex of purple bacteria (Herek et al. 1998,
2004) and similar effects were found also for LHCII
(Gradinaru et al. 2003). The fact that the position of
the band is not shifted upon change of chlorophyll
species supports the hypothesis of an electrochro-
mic response of peridinin. As the electrochromic
response is again very sensitive to distances and
orientation (Herek et al. 2004), the presence of the
535 nm band in all reconstituted complexes further
underlines the successful reconstitution. Therefore,
based on the arguments above, we conclude that
both structural arrangement of the pigments and
pigment-protein interaction in the reconstituted
PCP complexes are very similar to those in the
native PCP complex.
Peridinin-chlorophyll energy transfer
The results show that there is a clear correlation
between the position of the Q
y
band of the particular
chlorophyll species and efficiency of the S
1
/ICT
energy transfer. The energy transfer rates and effi-
ciencies calculated under assumption that the
intrinsic peridinin S
1
/ICT lifetime in PCP is 16 ps
(Zigmantas et al. 2002) are summarized in Table 2.
It is apparent that the efficiency of the S
1
/ICT
pathway increases with shifting the Chl Q
y
band
towards lower energies. For the chlorophyll species
having the Q
y
energy below that of Chl-a, the effi-
ciency is even higher than for the native PCP com-
plex. The obvious correlation between the position
of the Q
y
band of the acceptor and energy transfer
efficiency suggests that spectral overlap between
donor emission and acceptor absorption is a key
factor for determining the energy transfer efficiency.
The spectral overlap for all chlorophyll species with
peridinin emission measured in methanol is depic-
ted in Figure 6. The spectral overlap Hwas calcu-
lated according to the equation
H¼ZFðmÞAðmÞ
m4dm
For the calculation, the absorption spectra of
all chlorophyll species were measured in acetone
solution to avoid problems with the high energy
tail of chlorophyll absorption overlapping with
peridinin S
2
absorption. The resulting spectra were
then shifted to match the Q
y
maximum in the
reconstituted PCP complexes. The peridinin
emission spectrum F(m) and absorption spectra of
chlorophyll species A(m) were converted to the
energy scale and normalized to unit area. Knowl-
edge of the spectral overlap allows calculation of
the energy transfer rate in terms of the Fo
¨rster
mechanism according to the following equation
(Pullerits et al. 1997; Scholes 2003).
kET ¼1:18V2H
where Vis the interaction term equal to
V¼5:04lAlDj
R3
where l
A
and l
D
are transition dipole moments of
the acceptor and donor, respectively, jis the ori-
entation factor and Ris the distance between the
donor and acceptor. It must be noted that for
carotenoids, which have negligible dipole moment
of the S
1
state, calculation of absolute values for
the energy transfer rate requires more than the
Fo
¨rster dipole approximation; full Coulombic
coupling between the transition densities of the
donor and acceptor states must be calculated
(Krueger et al. 1998; Damjanovic
´et al. 1999).
Consequently, use of the above equations cannot
give the absolute values for the energy transfer
Figure 6. Spectral overlap between peridinin emission and Q
y
absorption of the PCP complexes reconstituted with different
chlorophyll species. Absorption spectra are normalized to the Q
y
maximum. Peridinin emission measured in methanol was taken
from Zigmantas et al. (2001) and normalized to maximum.
224
rates. Nevertheless, their use permits exploration
of relative changes in energy transfer rate occur-
ring upon change of chlorophyll species. Assuming
that all reconstituted PCP complexes are struc-
turally identical, the only parameters that vary
with different chlorophyll species are the overlap
integral and transition dipole moment of the
acceptor. The transition dipole moments for the
Q
y
transitions for all the chlorophylls were calcu-
lated from absorption spectra using the known
molar extinction coefficients. It is worth mention-
ing that the transition dipole moments should be
corrected for the effects of the surrounding med-
ium (Kleima et al. 2000b), but since this effect
should be the same for all PCP complexes, it is
omitted. To compare the energy transfer rates
obtained from a Fo
¨rster calculation and experi-
ment, the rates are normalized to the values
obtained for the PCP complex reconstituted with
Chl-a. The results are plotted in Figure 7. The
trend of the change of the energy transfer rate
obtained from experimental data is well repro-
duced by the Fo
¨rster calculations, except for the
PCP complex reconstituted with BChl-a. A possi-
ble origin of this behavior can be related to
enhanced energy transfer via the hot peridinin
S
1
/ICT state (see below).
The S
2
energy transfer pathway is active in all
reconstituted PCP complexes. Efficiency of the S
2
channel, as estimated from the contribution of the
chlorophyll signal in the first and second EADS
(Figure 4 and supplementary material), falls into
the 25–45% range (Table 2). This is in agreement
with values obtained for the native PCP complex
(Krueger et al. 2001; Zigmantas et al. 2002; Linden
et al. 2004). The values shown in Table 2 demon-
strate that for the S
2
pathway the trend is opposite
to that for the S
1
/ICT-mediated energy transfer
and can be explained by a better spectral overlap
between the peridinin S
2
emission and Q
x
bands of
the chlorophyll species absorbing at higher ener-
gies. This trend is again broken for the PCP
complex reconstituted with BChl-a, but the reason
may be a strong Q
x
transition of Bchl-alocated at
590 nm (Figure 1). Thus, as for the S
1
/ICT path-
way, energy transfer via the S
2
state seems to be
controlled by the spectral overlap.
Besides the S
2
and S
1
/ICT routes, a minor
energy transfer channel from peridinin to chloro-
phyll characterized by a 0.5–0.7 ps component, is
present in all reconstituted PCP complexes. This
channel was also found in the native PCP complex
and assigned to energy transfer via a ‘hot’ peridi-
nin S
1
/ICT state (Zigmantas et al. 2002; Linden
et al. 2004). In contrast to the time constants for
the S
2
and S
1
/ICT channels, the 0.5–0.7 ps com-
ponent is insensitive to the chlorophyll species in
reconstituted PCP. However, the amplitude of this
component does vary with different chlorophyll
species, as can be judged from the increase of the
chlorophyll bleaching (see the change from red to
green EADS in Figure 4a and supplementary
material). These facts exclude a possibility of this
channel being due to energy transfer via the
S
1
/ICT state of just one of the four peridinins,
which might have a better coupling to chlorophyll.
In such a case, the energy transfer should be sen-
sitive to the spectral overlap and its amplitude
should be approximately 25% regardless the
chlorophyll species. On the other hand, if the hot
peridinin S
1
/ICT state is an energy donor, the
hypothetical hot S
1
/ICT emission will have a good
spectral overlap with the higher vibrational states
of the Q
y
band. Since these higher vibrational
states form a broad weak absorption extending to
600 nm for all chlorophyll species except BChl-a,
it is not surprising that this component is insensi-
tive to the change of chlorophyll. Thus, the
hypothesis of the 0.5–0.7 ps component being due
to the energy transfer via the hot peridinin S
1
/ICT
state is consistent with the data presented here.
The PCP complex reconstituted with BChl-a
Figure 7. Comparison of the energy transfer time constants
obtained from experiment and calculated using the Fo
¨rster
equation. The calculated values were normalized to the exper-
imentally obtained value for the PCP complex reconstituted
with Chl-a.
225
represents a special case, because no separate S
1
/
ICT and hot S
1
/ICT channels were found. Perhaps
for Bchl-athe time components of these two pro-
cesses are too close to be separated. For Bchl-athe
time constant of 0.45 ps is significantly faster than
for PCP reconstituted with other chlorophyll spe-
cies, and the channel from peridinin to BChl-avia
the hot S
1
/ICT state likely becomes dominant.
Role of the ICT state
Involvement of the peridinin ICT state in peridi-
nin-chlorophyll energy transfer in native PCP is
shown by the matching decay of the characteristic
near-infrared ICT stimulated emission with rise of
the chlorophyll bleaching (Zigmantas et al. 2002).
The same situation occurs in the reconstituted
PCP complexes and again questions the precise
role of the ICT state. Femtosecond pump-dump-
probe spectroscopy on peridinin in methanol
solution provided evidence that the S
1
and ICT
states are separate (Papagiannakis et al. 2004). In
their model, Papagiannakis et al. (2004) suggested
that at early times, predominantly the ICT state is
populated and that in PCP ICT-S
1
equilibration is
slower than energy transfer. Consequently, the
ICT state must be the energy donor in the PCP
complex, and an energy transfer mechanism
insensitive to spectral overlap was proposed to
explain the discrepancy between ICT emission in
the near-infrared and the Chl-aabsorption around
670 nm (Papagiannakis et al. 2004). However, the
results presented here clearly show that the energy
transfer depends on the spectral overlap and that
the Fo
¨rster mechanism can explain the observed
data, challenging the proposal of Papagiannakis
et al. (2004). Since the ICT emission peaks at
930 nm in PCP (Zigmantas et al. 2002), it is highly
unlikely that the ICT state could transfer energy
directly to Chl-bwhose Q
y
band lies 4500 cm
)1
higher in energy. Yet, the peridinin-Chl-btransfer
occurs with 63% efficiency (Table 2). One expla-
nation could be that the ICT emission extends
much more toward higher energies, and the peak
at 930 nm in the transient absorption spectra is a
result of overlapping ICT ESA and stimulated
emission. However, both steady-state emission
(Bautista et al. 1999a, b) and time-resolved fluo-
rescence data (Zigmantas et al. 2001) on peridinin
in solution suggest that below 750 nm the S
1
emission dominates. The results obtained here can
then be explained in terms of a strongly coupled
S
1
/ICT state (Zigmantas et al. 2003). According to
this model, the energy transfer still occurs from
the S
1
-like part of the S
1
/ICT potential surface,
which is above the chlorophyll Q
y
band. Both the
S
1
and ICT parts of the potential surface have the
same lifetime due to the strong coupling, leading
to the observed match in the ICT emission decay
(Figure 5). Then, as suggested by Zigmantas et al.
(2002), the role of the ICT state is to enhance the
dipole moment of the S
1
/ICT state, facilitating the
peridinin to chlorophyll energy transfer. It is
worth noting, however, that direct ICT-mediated
energy transfer may take place in the PCP com-
plex reconstituted with BChl-a,sinceitsQ
y
tran-
sition partly overlaps the ICT emission. In that
case, the energy transfer may be further enhanced,
leading to the very fast S
1
/ICT energy transfer
which, as shown in Figure 7, does not match the
rate expected from Fo
¨rster calculations based on
the peridinin S
1
emission spectrum.
Acknowledgements
We thank David Miller and Julian Catmull for
some of the N-domain PCP apoprotein used for
the reconstitutions and Frank Sharples for assis-
tance with pigment purification. RGH was sup-
ported by the Australian Research Council
(A0000264). TPo thanks Tonu Pullerits for useful
discussions, Ozlem Ipek for technical assistance,
and the Swedish Energy Agency for financial
support. The work at Lund University was sup-
ported by grants from the Swedish Research
Council, the Wallenberg Foundation and the
Crafoord Foundation.
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