Excited state dynamics of carotenoids free and bound to pigment-protein complexes
SWUP
AZ.15
vibronic coupling is not allowed between those electronic states having different Pariser signs. Therefore, the direct 1
1
B
u +
2
1
A
g
internal conversion is symmetrically forbidden. Through the extrapolation of the low-lying singlet states of shorter polyenes that were
calculated on the basis of the Pariser-Parr-Pople multi-reference double-excitation configuration-interaction PPP MRD-CI method, it predicted that the 1
1
B
u
state should be present in-between the 1
1
B
u +
and the 2
1
A
g
states for polyenes having 11 n 6, while the
3
1
A
g
state should be present in-between the 1
1
B
u +
and 1
1
B
u
for polyenes having 9 n 10
Tavan Sculten, 1986, 1987. The optically-forbidden 1
1
B
u
state was first identified experimentally in crystalline spheroidene Sashima, 1999, lycopene, -carotene, and mini-9- -carotene Sashima et al.,
2000 by the use of resonance-Raman excitation profiles RREPs. Continuous efforts have been put into this work, leading to the very recent spectroscopic identification of the 3
1
A
g
state Furuichi et al., 2002. for a series of carotenoids, i.e., mini-9- -carotene, spheroidene, lycopene, anhydrorhodovibrin, and spirilloxanthin, and for further detection of the 1
1
B
u
state for anhydrorhodovibrin and spirilloxanthin Furuichi et al., 2002. The energy states determined by the measurements of the RREPs for carotenoids having the number of
conjugated C=C bonds, n = 9 13, are shown in Figure 2; the energy level of the Q
x
and the Q
y
states of BChls are also shown in the same figure for comparison.
Figure 2. An energy diagram, determined by the measurement of RREPs, for the 1
1
B
u +
, 3
1
A
g
, 1
1
B
u
, and 2
1
A
g
states of crystalline carotenoids including mini-9- -carotene n =
9, spheroidene n = 10, lycopene n = 11, anhydrorhodovibrin n = 12, and
spirilloxanthin n = 13. Broken lines indicate the Q
x
and Q
y
energy levels of BChls in the LH2 complexes. The carotenoids data in this figure was taken from Figure 5a of Furuichi
et al. 2002 .
3. Materials and methods 3.1 Materials
All- trans spheroidene was extracted from Rhodobacter Rhb. sphaerodes 2.4.1, while
lycopene was removed from tomato juice Rondonuwu et al., 2003. Spheroidene was dissolved in
n-hexane, and lycopene was dissolved in a benzene and n-hexane mixture 5:95
F.S. Rondonuwu, Y. Koyama
SWUP
AZ.16
vv. The preparation of LH2 complexes from Rhb. spaheroides 2.1.4 and Rhodospirillum
Rps. molischianum was carried out as described elsewhere Rondonuwu et al., 2004. Each LH2 complex was suspended in a 20 mM Tris-HCL pH 8 buffer and then adjusted to OD =
4.5 cm
-1
at the carotenoid absorption maximum.
3.2 Methods
Subpicosecond time-resolved absorption spectra in the region of 400-700 nm were recorded for carotenoids free in a solution, while time-resolved absorption spectra in the
region of 400-700 nm and 800 1000 nm were recorded for the LH2 complexes . Each
carotenoid solution in the amount of about 30 ml was circulated between a 1 mm optical path length of flow cell and a reservoir that was cooled in an ice-water mixture. Each sample
was excited by using a 120 fs laser pulse duration Spectra Physics OPA system at carotenoid 1
1
B
u +
v=0 using the following wavelengths: 467, 483, 511, and 529 nm for spheroidene, lycopene, LH2 from
Rhb. sphaeroides 2.1.4, and LH2 from Rps. molischianum, respectively. A temporal resolution of about 0.16 ps FWHM was obtained from cross correlation
measurements between excitation and probe pulses. The time resolved absorption spectra were measured with the use of a liquid nitrogen cooled CCD spectrometer Princeton
Instruments model 1152 EUV.
4. Results and discussion Dynamics of carotenoids free in solution
Figure 3 shows a schematic diagram of the low-lying singlet excited state of Cars, including the transition corresponding to the transient signal of the 1
1
B
u +
, 1
1
B
u
, and the 2
1
A
g
state to the S
n
state. It is important to note that the S
n
state in the diagram represents only a simplified state of the corresponding transisition. The S
n
state should have a different symmetry depending on the origin of the transition. The 1
1
B
u
and 2
1
A
g
states are populated through an internal conversion from the 1
1
B
u +
by photoexcitation from the ground 1
1
A
g
state. The transient signals were measured following the photoexcitation. Figure 4 upper panels depicts the time-resolved excited state absorption of
spheroidene n=10 and lycopene n=11. Time-dependent spectral changes are similar
among the two carotenoids. They can be characterized based on the results of neurosporene in
n-hexane Zhang et al., 2000 as follows: first, a stimulated emission appears immediately after photo-excitation with vibrational progression. The resulting stimulated emission
originates from the lowest 0 vibrational level 1
1
B
u +
to the 2, 1, and 0 vibrational levels of the ground 1
1
A
g
state accompanied by excited-state absorptions ESAs from the 1
1
B
u +
state that appear in longer wavelengths. After the stimulated emission disappears, broad ESAs
appear acompanied by ground state bleaching of the 1
1
A
g
state. Based on the sequential appearance, these ESAs can be ascribed to the 1
1
B
u
state. Finally, typical sharp ESAs from the 2
1
A
g
state appear together with the ground state bleaching from the 1
1
A
g
state. Almost parallel with the ESAs from the 2
1
A
g
state, aditional ESAs appear on their blue side and persist up to 50 ps for spheroidene and lycopene. Based on their spectral patterns and life
times, they can be ascribed to the triplet state of carotenoids. This state is presumably generated from the optically forbidden 1
1
B
u
state.
Excited state dynamics of carotenoids free and bound to pigment-protein complexes
SWUP
AZ.17
Figure 3. An electronic transition diagram for carotenoids. Transient signals from low-
lying singlet excited states are measured following photoexcitation thick . The S
n
state in this diagram only represents a simplified final excited state with three different
symmetries, depending on the transition origins. Broken arrows represent internal
conversions.
Figure 4. Excited state absorptions and bleachings upper panels and time profiles of
lower panels of a spheroidene and b lycopene free in a solution measured at a selected delay time. Based on their sequential appearance these spectra were assigned
to the 1
1
B
u +
, 1
1
B
u
, 2
1
A
g
state absorptions, respectively. Spectra shown at the bottom of the upper panels can be assigned to the triplet state absorptions. The time profiles
shown in the lower panels were plotted at the 2
1
A
g
state absorptions maxima. Each of the fitting curves smooth solid lines, which almost perfectly overlap with the time
profile data, are also shown for comparisons.
Figure 4 lower panels shows the time profiles of the 2
1
A
g
state for spherodende and lycopene. The time profiles feature stimulated emissions at time-zero followed by the rise
and decay of the 2
1
A
g
state. Although the rise time constants of the 2
1
A
g
state of both Cars are similar, their decay time constants are substantially different, i.e., 9.1 ps decay rate
constant, k = 0.11 ps
1
in spheroidene and 3.85 ps k = 0.26 ps
1
in lycopene.
F.S. Rondonuwu, Y. Koyama
SWUP
AZ.18
Figure 5. An electronic absorption spectrum of the LH2 complex from Rsp.
molischianum.
Carotenoid energy transfer in LH2 complexes
The electronic absorption spectra spectrum of the LH2 complex from Rsp.
molischianum is is presented in Figure 5. The peak at 372 nm can be assigned to the Soret absorption, whereas the peak at 590 nm can be assigned to the Q
x
absorption. Double peaks at 800 nm and 848 nm can be assigned to the Q
y
absorptions. Multiple peaks in the range of 400 550 nm can be ascribed to the lycopene absorption due to the transition from the
ground 1
1
A
g
state to the 1
1
B
u +
state. Car absorptions in LH2 complexes shift to the red side when compared to those free in a solution.
Figure 6 shows ESAs in the visible and near infrared region of spheroidene and lycopene bound to the LH2 complexes from
Rhb. sphareoides 2.4.1 and Rps. molischianum, respectively. In the visible region, the spectral features are similar to those free in a solution.
Rapid formations of triplet states in these complexes are apparently more efficient. In the near infrared region, strong bleaching appears at around 850 nm accompanied by ESAs in
almost overlapping positions, although the tails are still clearly seen on the blue side. Those ESAs and bleachings can be ascribed to the BChls Q
y
state. The presence of BChl Q
y
ESAs and bleaching immediately after Car photoexcitation indicates that Car-to-BChl energy-transfer
takes place in the LH2 complexes. Figure 7 compares the time profile of the 2
1
A
g
state for Cars free in a solution and bound to LH2 complexes. For spheroidene bound to the LH2 complex from
Rhb. sphaeroides 2.4.1, the 2
1
A
g
state decays much faster than that free in a solution, while lycopene bound to the LH2 complex from
Rps. molischianum decays almost in the same phase when compared to that free in a solution. Time dependent Q
y
bleaching of BChl is also shown for a comparison. The population of the Q
y
state in the LH2 complex from Rps. molischianum rises
within a very short time, whereas in Rhb. sphaeroides 2.4.1 it rises with two components.
Table 1 summarizes the rise and decay rate constants of the 2
1
A
g
states of Cars free and bound to the LH2 complexes and rise rate constants of the Q
y
states of Bchls in the LH2 complexes. The results can be explained as follows: Since the rate constant of the Q
y
formation in the LH2 complex from Rps. molischianum is much faster than the decay of the
2
1
A
g
state and their rate constants are practically the same for those free and bound to the LH2 complexes, one can conclude that the 2
1
A
g
state does not contribute to Car-to-Bchl singlet-energy transfer. The rapid formation of the Q
y
state 4.76 ps
1
must then come from either an internal conversion within BChl Q
x
Q
y
andor a singlet energy transfer from a higher singlet excited state of Cars directly to the Q
y
state of BChl 1
1
B
u +
1
1
B
u
Q
y
. However, decay rate constant of Q
x
Q
y
internal conversion within BChl a in a THF solution
Excited state dynamics of carotenoids free and bound to pigment-protein complexes
SWUP
AZ.19
was reported to be 4.7 ps
1
Rondonuwu et al., 2004, which is very similar with the formation rate of the Q
y
state in Rps. moliscianum. Thus, the Q
y
formation in this complex is mainly determind by the Q
x
Q
y
internal conversion; therefore, the presumed singlet-energy transfer from the higher singlet states of Car can be omitted. In addition, the Q
y
state is energetically higher than the 2
1
A
g
state of lycopene see Figure 1. These energetics force an uphill energy transfer which is practically unfavorable. A back energy transfer from the BChl
Q
y
state to the 2
1
A
g
state probably takes place Staleva, 2015, but the sequential appearance is difficult to be distinguished.
Figure 6. a Excited state absorptions and bleachings of spheroidene upper panels
and lycopene lower panels bound to the LH2 complexes measured at a selected delay time. Based on their sequential appearance, these spectra were assigned to the 1
1
B
u +
, 1
1
B
u
, 2
1
A
g
state absorptions, respectively. b Excited state absorptions and Q
y
bleachings of BChl in LH2 complexes.
Figure 7. The time profiles of the 2
1
A
g
excited state absorptions of a spheroidene free in a solution and bound to the LH2 complexes from
Rhb. sphaeroides 2.4.1 and b lycopene free and bound to the LH2 complexes from
Rps. molischianum. Each of the Cars time profiles was plotted at the 2
1
A
g -
state absorption maxima. The Q
y
time profiles of BChl in the corresponding LH2 complexes are also shown for comparisons.
F.S. Rondonuwu, Y. Koyama
SWUP
AZ.20
Table 1. The rise and decay rate constants k of the 2
1
A
g
state of Cars free in a solution and bound to the LH2 complexes are revealed. The rise time constants of the Q
y
state of BChl in the LH2 complexes from
Rhb. sphaeroides 2.4.1 and Rps. molischianum are also shown for comparisons.
Molecule Rise ps
-1
Decay ps
-1
Free Bound
Free Bound
Spheroidene 2.63
7.69 0.11
0.49 Lycopene
3.13 6.25
0.26 0.28
Bchl in LH2 from Rhb
4.76, 0.53 Bchl in LH2 from
Rps 4.35
The value in parentheses was used as a fixed rate when fit to the BChl Q
y
time profiles.
The decay rate constant of the 2
1
A
g
state of spheroidene bound to the LH2 complexes from
Rhb. sphaeroides 2.4.1 were substantially enhanced by 0.38 ps
1
when compared to the free in a solution, indicating that singlet energy transfer took place from the 2
1
A
g
to the Q
x
state. This is almost in agreement with the 0.53 ps
1
formation rate of Q
y
. Thus, the 2
1
A
g
state of spheroidene in LH2 complex from Rhb sphaerodes 2.4.1 promotes Car-to-BChl singlet
energy transfer. Overall, the efficiency of Cars-to-BChl singlet-energy transfer in the LH2 complexes
from Rhb. sphaeroides 2.4.1 and Rps. Molischianum as determined by comparing the
absorption and fluorescence-excitation spectra Rondonuwu et al., 2004 were reported to be 89 and 53 respectively. The above mentioned 53 overall energy transfer efficiency
for lycopene bound to an LH2 complex from Rps. moliscianum was very likely promoted by a
higher excited state 1
1
B
u +
1
1
B
u
of lycopene to a Q
x
state of BChl. Unlike lycopene, an 89 overall efficiency transfer of spheroidene in an LH2 complex from
Rhb. sphaeroides 2.4.1 must be promoted by two chanels, one from the higher excited state with an efficiency of at
least 53 and the other one from the 2
1
A
g
state with an efficiency of at most 36. However, more detailed studies are needed concerning energy transfer mechanisms in higher excited
states that require shorter pulse durations in order to probe the very short-life components of the 1
1
B
u +
and 1
1
B
u
states.
5. Conclusion and remarks
