PROS Ferdy S Rondonuwu, Y Koyama Excited state dynamics fulltext

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Excited state dynamics of carotenoids free and bound to

pigment-protein complexes

Ferdy S. Rondonuwua_{and Yasushi Koyama}b

a_{Faculty of Science and Mathematics, Satya Wacana Christian University,} Jl. Diponegoro 52 60, Salatiga 50711, Indonesia

b_{Faculty of Science and Technology, Kwansei Gakuin University,} 2-1 Gakuen, Sanda 669-1337, Japan

Abstract

Excited state dynamics and energetics of carotenoids having a different number of conjugated double bonds (_n), which include spheroidene (_n= 9) and lycopene (_n= 10) free and bound to LH2 complexes from _{Rhodobacter spaheroides} 2.4.1 and

Rhodospirillum moliscianum have been evaluated with the use of time-resolved absoprtion spectroscopy. A relaxation path, including a 11_B_u+₁1_B_u ₂1_A_g ₁1_A_g

singlet internal conversion and a 11_B_u _{T (triplet) has been identified for Car free in a}

solution. In antenna complexes, an additional two paths were identified as Car-to-BChl singlet-energy transfer channels; first starting from the 11_B_u+_{and/or 1}1_B_u _{states to the}

Qx state, and second starting from the 21Ag state to the Qystate. The second channel

may be deactivated depending on the number of conjugated double bonds (_n). The 21_A_g

(donor) state of spheroidene (_n= 9) promotoes the singlet-energy transfer to the Qy

(aceptor) state of BChl. In contrast to spheroidene, the 21_A_g _{state of lycopene (}_n_{= 10)}

prevents the energy transfer process.

Keywords spheroidene, lycopene, singlet-energy transfer, LH2 complexes

1. Introduction

Pigments are molecules that absorb light and give color to living matter. Carotenoids (Cars) are one of the most abundant pigments available in nature. Their presence can be easily recognized by bright yellow to red colors due to the lack of an absorption band in this region. Photosynthetic organisms, including green plants, photosynthetic bacteria, and algae sensitize Cars in nature. In green plants, Cars are mainly located in leaves, fruits, and roots. The strong orange color in carrots and yellowish red color in tomatoes, for example, basically originate from -carotene and lycopene, respectively. -carotene and lycopene are a few examples of more than 600 Cars that are naturally and chemically synthesized (Britton et al. 2004). In leaves, Cars are present in a relatively large amount but they are indistinct due to the strong green color given by chlorophylls. However, as the leaves become old and chlorophylls (Chls) degrade, the color gradually changes from green to yellow. Although only plants and microorganisms can synthesize Cars, they are also found accumulated in humans and animals through the nutrients they consume. Besides their color-giving ability, Cars perform various functions. In photosynthesis, Cars play two important roles: for light-harvesting (Magdoang et al., 2014) and photo-protection (Ruben et al., 2007; Staleva et al., 2015). As light-harvesters, Cars absorb light in the spectral region left over by (bacterio)chlorophylls ((B)Chls), thereby the unique configuration of carotenoids and BChl in photosystem antenna complexes, for example, enhance the absorption of sunlight energy

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from near ultraviolet to near infrared. As a photo-protector, Cars manage excessive light energy by quenching singlet and triplet states of Cars and (B)Chls in antenna complexes (Kirilovsky et al., 2015). Photon is absorbed by Cars in the form of excited-state energy. Later this excited-state energy is converted into redox energy, which is then used as fuel to drive the sophisticated photosynthetic "machines" in photosynthetic bacteria and green plants; those machines are immensely important to sustain life. Through a series of energy-transducing reactions, the photosynthetic machinery transforms light energy into a stable form of chemical energy of organic compounds, which can sometimes last as long as millions of years. The fossil fuels are burnt to support human activity, and the food we eat every day is the result of photosynthesis. Other than photosynthesis, Cars quench harmful singlet oxygen and free radicals by interrupting a sequence of oxidative reactions. The antioxidant ability of Cars makes them important molecules to prevent various diseases such as cancer or macular degeneration (Akufo et al., 2015). Although various functions of Cars have been well documented, a deeper understanding concerning the details mechanism of such functions at the molecular level is very limited.

Recently Cars researches focused on structures and dynamics to widen their applications not only for natural colorants but also for medicine and artificial antenna such as biosolar cells (Xiang et al., 2005; Hug et al., 2015). This paper outlines early events in photosynthesis; how Cars capture sunlight energy and transfer it to neighboring (B)Chl through an S1-state will be discussed. Advancements of time-resolved absorption spectroscopies up to femtosecond and subpicosecond time-resolution bring up the opportunity to explore excited state dynamics of low-lying singlet excited states including 11_B

u+, 11Bu , and 21Ag of Cars.

2. Carotenoid structure

The basic structure of carotenoids consists of the C40 hydrocarbon skeleton

constructed from eight isoprenoid units in a sequence forming a conjugated chain (Britton et al., 2004). The optical properties of Cars are determined by the number of conjugated C=C bonds (_n). The left-hand side of Figure 1 depicts the molecular structures of five different kinds of Cars with_n= 9 13 that are often found in bacterial photosynthetic.

Figure 1. Molecular structures of carotenoids (left) and their electronic absorption spectra in_n-hexane solution (right).

The conjugated chain of Cars has approximately C2hsymmetry (see the structure of all

-trans-polyene, for example), which gives rise to the singlet states classified as k1_A

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m1_A

g+, and n1Bu+ groups. Here, the superscript 1 signifies the singlet states; + and are

Pariser signs showing the symmetry of electronic configuration (Pariser, 1956); g (gerade) and u (ungerade) represent the even and odd parity of the total wave function; and k, l, m, and n are labels of a series of electronic states having the same symmetry from lower to higher energy (note, n is different from the symbol_nthat is being used to specify the number of conjugated C=C bonds). Concerning the one-photon optical transition from the 11_A

g state,

the m1_A

g+and n1Bu+states can be classified as the optically-allowed states, whereas the k1Ag

and l1_B

u states can be classified as the optically-forbidden states. The singlet states of

carotenoids in the order: 11_B

u+> 21Ag> 11Ag (the ground state), have been well-documented.

In all-_trans-carotenoids, the optically-allowed 11_B

u+11Ag transitions can be clearly

observed in the near-ultraviolet to the visible spectral regions. Fig. 1 (right panel) shows the absorption spectra due to this transition for the above-mentioned five different kinds of carotenoids in _n-hexane solution. The spectral pattern of each 11_B

u+ 11Ag absorption

consists of a pair of vibrational progressions due to the C=C and C C stretching vibrations with frequencies of ~1520 cm 1 _{and ~1150 cm}1 _{(Sashima et al., 1999). The pairs of}

progressions are overlapped with each other to form the apparent vibrational progressions. The 11_B

u+  11Ag transitions strongly depend on intramolecular interactions with the

surroundings. For a nonpolar carotenoid in a nonpolar solvent, a dispersion interaction (Shulten et al., 1980) becomes predominant, which gives rise to the red shift in the transition. The solvent effect is particularly important to comprehend how the 11_B

u+energy is modified

when the carotenoid molecule is embedded in proteins such as the antenna complexes and the reaction centers.

Unlike the energies of the optically-allowed 11_B

u+ state, which can be readily

determined by the use of a conventional spectrometer, the energy of the optically-forbidden 21_A

g state, for example, needs to be determined by high-sensitivity fluorescence

spectroscopy overcoming the low quantum yield of the 21_A

g  11Ag fluorescence

originating from a state mixed with the strongly optically-allowed 11_B

u+state (Christensen,

1999). As the length of the conjugated chain increases, the energy gap between the 21_A g and

the 11_B

u+ states widens, and therefore, reduces the mixed state, leading to weaker

fluorescence. It implies that an accurate determination of the 21_A

g-state energy of

carotenoids is extremely difficult. Since knowledge of a 21_A

g state is important in

understanding the mechanisms of singlet-energy transfer in photosynthesis, substantial efforts have been made to determine the 21_A

g-state experimentally. The methods include:

(i) measurements of resonanceRaman excitation profiles (RREPs) for crystalline lycopene, -carotene, and mini-9- -carotene (Sashima et al. 2000); (ii) fluorescence spectroscopy for all -trans-neurosporene and spheroidene (Fujii et al., 1998), -carotene (Onaka et al., 1999), violaxanthin, and zeaxanthin (Frank et al., 2000) in _n-hexane solution; and (iii) transient absorption spectroscopy using the 11_B

u+21Ag absorption in the infrared region for

zeaxanthin and violaxanthin in methanol (Polívka et al., 1999), spheroidene bound to the pigment-protein (LH2) complexes from _{Rhodobacter sphaeroides} 2.4.1, and rhodophin glucoside bound to the LH2 complex from_{Rhodospseudomonas acidophila}(Polívka, 2002).

It took almost three decades to completely understand the dynamics of the Car singlet states in terms of the state ordering of 11_B

u+> 21Ag > 11Ag. Based on this particular scheme,

the Car-to-BChl singlet-energy transfer reactions were explained in terms of channels starting from these two low-lying singlet states, i.e., 11_B

u+ and 21Ag. However, the pseudoparity

selection rule (Callis et al., 1983) using Pariser s label (Pariser, 1956), which is defined within the framework of the Pariser-Parr-Pople (PPP) approximation (Pople, 1953), suggests that

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vibronic coupling is not allowed between those electronic states having different Pariser signs. Therefore, the direct 11_B

u+21Ag 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 11_B

u state should be

present in-between the 11_B

u+ and the 21Ag states for polyenes having 11n6, while the

31_A

g state should be present in-between the 11Bu+and 11Bu for polyenes having 9n10

(Tavan & Sculten, 1986, 1987). The optically-forbidden 11_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 31_A

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 11_B

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 Qxand the Qy

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 11_B_u+_,

31_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 Qxand Qyenergy 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-_transspheroidene 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

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v/v). The preparation of LH2 complexes from _{Rhb. spaheroides} 2.1.4 and _{Rhodospirillum} (Rps.) molischianumwas 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 11_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 11_B

u+, 11Bu, and the 21Ag

state to the Snstate. It is important to note that the Snstate in the diagram represents only a

simplified state of the corresponding transisition. The Sn state should have a different

symmetry depending on the origin of the transition. The 11_B

u and 21Ag states are populated

through an internal conversion from the 11_B

u+ by photoexcitation from the ground (11Ag)

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 11_B

u+to the 2, 1, and 0 vibrational levels of

the ground 11_A

g state accompanied by excited-state absorptions (ESAs) from the 11Bu+state

that appear in longer wavelengths. After the stimulated emission disappears, broad ESAs appear acompanied by ground state bleaching of the 11_A

g state. Based on the sequential

appearance, these ESAs can be ascribed to the 11_B

u state. Finally, typical sharp ESAs from

the 21_A

g state appear together with the ground state bleaching from the 11Ag state. Almost

parallel with the ESAs from the 21_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 11_B

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Figure 3.An electronic transition diagram for carotenoids. Transient signals from low-lying singlet excited states are measured following photoexcitation (thick_). The Snstate

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 11_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 21_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 21_A

g state for spherodende and

lycopene. The time profiles feature stimulated emissions at time-zero followed by the rise and decay of the 21_A

g state. Although the rise time constants of the 21Ag state of both Cars

are similar, their decay time constants are substantially different, i.e., 9.1 ps (decay rate constant,_k= 0.11 ps1_{) in spheroidene and 3.85 ps (}_k_{= 0.26 ps}1_{) in lycopene.}

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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. molischianumis 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 Qxabsorption. Double peaks

at 800 nm and 848 nm can be assigned to the Qyabsorptions. Multiple peaks in the range of

400 550 nm can be ascribed to the lycopene absorption due to the transition from the ground (11_A

g ) state to the 11Bu+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 Qystate. The presence of BChl QyESAs 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 21_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 21_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 Qybleaching of BChl is also shown for a

comparison. The population of the Qystate in the LH2 complex fromRps. 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 21_A

g states of Cars free

and bound to the LH2 complexes and rise rate constants of the Qystates of Bchls in the LH2

complexes. The results can be explained as follows: Since the rate constant of the Qy

formation in the LH2 complex from_{Rps. molischianum}is much faster than the decay of the 21_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 21_A

g state does not contribute to Car-to-Bchl

singlet-energy transfer. The rapid formation of the Qystate (4.76 ps1) must then come from

either an internal conversion within BChl (QxQy) and/or a singlet energy transfer from a

higher singlet excited state of Cars directly to the Qy state of BChl (11Bu+/11Bu  Qy).

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was reported to be 4.7 ps1_{(Rondonuwu et al., 2004), which is very similar with the formation}

rate of the Qystate inRps. moliscianum. Thus, the Qyformation in this complex is mainly

determind by the Qx Qy internal conversion; therefore, the presumed singlet-energy

transfer from the higher singlet states of Car can be omitted. In addition, the Qystate is

energetically higher than the 21_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 Qy state to the 21Ag 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 11_B_u+_,

11_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 21_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 21_A_g-_{state absorption maxima. The Q}_y_{time profiles}

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Table 1.The rise and decay rate constants (_k) of the 21_A_g _{state of Cars free in a solution}

and bound to the LH2 complexes are revealed. The rise time constants of the Qystate 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 Qytime profiles. The decay rate constant of the 21_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 21_A

g to the Qx

state. This is almost in agreement with the 0.53 ps 1_{formation rate of Q}

y. Thus, the 21Ag

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 (11_B

u+/11Bu) of lycopene to a Qxstate 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 21_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 11_B

u+and 11Bu states.

5. Conclusion and remarks

Excited state dynamics and energetics of Cars free and bound to the LH2 complexes have been evaluated. A relaxation scheme, including a 11_B

u+11Bu 21Ag 11Ag singlet

internal conversion and a 11_B

u triplet state has been identified for Cars free in a solution.

In antenna complexes, two additional paths were obtained as Car-to-BChl singlet-energy transfer channels, namely 11_B

u+/ 11Bu Qx, and 21Ag Qy. Depending on the energetics

of the donor and acceptor states, singlet-energy transfer channels may be shut down when donor states are lower than that of acceptor states. In the LH2 complexes, the energy of the 21_A

g (donor) state of spheroidene (n= 9) is higher than the energy of the Qy(acceptor) state

of BChl; thus, it promotes a singlet-energy transfer. In contrast to spheroidene, the energy of the 21_A

g state of lycopene (n= 10) is lower than the Qystate; hence, it prevents the energy

transfer process.

These donor and acceptor energy levels are important for Car applications, such as for antioxidative agents or artificial antenna. Apart from a Car-to-BChl singlet energy transfer, an ultrafast singlet to triplet conversion is also observed within Cars from the 11_B

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Those singlet to triplet conversions are predominant for Cars bound to pigment protein complexes. The enhancement of singlet to triplet conversions within Cars in pigment protein complexes is not clear yet, but it is most probably because Cars are twisted when they are bound to protein complexes to facilitate one spin flip for a triplet formation. The triplet state may function as an energy outlet when Cars absorb excesive photon energy.

It should be mentioned that the 31_A

g state must also play a role in both Cars internal

conversions and the Car-to-Bchl singlet-energy transfer in an LH2 complex from _Rsp. Moliscianum, as this hidden state is present in-between the optically active 11_B

u+and newly

identified 11_B

u states for lycopene (n=11). However, a shorter pulse duration (less than 60

fs) is necessary to be employed, since the lifetime of the 11_B

u+and 31Ag and 11Bu states are

extremely short (less than 100 fs) for Cars have a number of conjugated double bonds greater than 10. Knowledge about Cars excited state dynamics and energetics is particularly important not only for ultrafast electron injection in a Cars-semiconductor interface study but also to understand the molecular mechanisms of Cars antioxidant applications.

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13_B_u_{state in carotenoids: dependence on the conjugation length.} _{Chem. Phys. Lett.}_,₃₇₆_{, 292}

301.

Rondonuwu, F.S., Fujii, R., Yokoyama, K., Koyama, Y., Cogdell, R.J., & Watanabe, Y. (2004). The role of the 11_B_u _{state in carotenoids-to-bacterioclirophyll singlet-energy transfer in the LH2 antenna}

complexes from _{Rhodobacter sphaeroides} G1C, _{Rhodobacter sphaeroides} 2.4.1. and Rhodospirillum molischianum and _{Rhodopseudomonas achidophylla}. _{Chem. Phys. Lett.}, ₃₉₀, 314 322.

Ruben, A.V., Berera, R., Ilioaia, C., van Stokkum, I.H.M., Kennis, J.T.M., Pascal, A.A., van Amerongen, H., Robert, B., Horton, P., & van Grondelle, R. (2007). Identification of a mechanism of photoprotective energy dissipation in higher plants._Nature, 450, 575 578.

Sashima, T., Nagae, H., Kuki, M., & Koyama, Y. (1999). A new singlet-excited state of all-trans-spheroidene as detected by resonance-Raman excitation profiles. _{Chem. Phys. Lett.},₂₉₉, 187 194.

Sashima, T., Koyama, Y., Yamada, Y., & Hashimoto, H. (2000). The 1Bu+, 1Bu, and 2Ag energies of

crystalline lycopene, -carotene, and mini-9- -carotene as determined by resonance-Raman excitation profiles: Dependence of the 1Bu-state energy on the conjugation length.J. Phys. Chem. B,₁₀₄, 5011 5019.

Shulten, K., Dinur, U., & Honig, B.J. (1980). The spectra of carbonium ions, cyanine dyes, and protonated Schiff base polyenes._{J. Chem. Phys.},₇₃, 3927 3935.

Staleva, H., Komenda, J., Shukla, M.H., Slouf, V., Karna, R., Polivka, T., & Sobotka, R. (2015). Mechanism of photoprotection in the cyanobacterial ancestorof plant antenna proteins. _{Nature Chemical}

Biology,₁₁, 287 291.

Tavan, P., & Schulten, K. (1986). The low-lying electronic excitations in long polyenes: A PPP-MRD-CI study._{J. Chem. Phys.},₈₅, 6602 6609.

Tavan, P., & Schulten, K. (1987). Electronic excitations in finite and infinite polyenes. _{Phys. Rev. B},

8(36), 4337 4357.

Xiang, J., Rondonuwu, F.S., Kakitani, Y., Fujii, R., Watanabe, Y., & Koyama, Y. (2005). Mechanisms of electron injection from retinoic acid and carotenoic acids to TiO2 nanoparticles and charged

recombination via the T1 state as determined by subpicosecond to microsecond time-resolved absorption spectroscopy: Dependence on the conjugation length._{J. Phys. Chem. B},₁₀₉, 17066 17077.

Zhang, J.-P., Inaba, T., Watanabe, Y., & Koyama, Y. (2000). Excited-state dynamics among the 1Bu+, 1Bu

-and 2Ag-states of all-trans-neurosporene as revealed by near-infrared time-resolved absorption

(1)

Figure 3.An electronic transition diagram for carotenoids. Transient signals from low-lying singlet excited states are measured following photoexcitation (thick_). The Snstate

in this diagram only represents a simplified final excited state with three different symmetries, depending on the transition origins. Broken arrows (_) represent internal conversions.

of the upper panels can be assigned to the triplet state absorptions. The time profiles shown in the lower panels were plotted at the 21_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

_A

state for spherodende and

lycopene. The time profiles feature stimulated emissions at time-zero followed by the rise

and decay of the 2

_A

state. Although the rise time constants of the 2

A

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

_{) in spheroidene and 3.85 ps (}

_k

_{= 0.26 ps}

_{) in lycopene.}

(2)

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

absorption. Double peaks

at 800 nm and 848 nm can be assigned to the Q

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

_A

) state to the 1

B

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

state. The presence of BChl Q

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

_A

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

_A

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

bleaching of BChl is also shown for a

comparison. The population of the Q

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

_A

states of Cars free

and bound to the LH2 complexes and rise rate constants of the Q

states of Bchls in the LH2

complexes. The results can be explained as follows: Since the rate constant of the Q

formation in the LH2 complex from

_{Rps. molischianum}

is much faster than the decay of the

2 _A

state and their rate constants are practically the same for those free and bound to the

LH2 complexes, one can conclude that the 2

_A

state does not contribute to Car-to-Bchl

singlet-energy transfer. The rapid formation of the Q

state (4.76 ps

) must then come from

either an internal conversion within BChl (Q



Q

) and/or a singlet energy transfer from a

higher singlet excited state of Cars directly to the Q

state of BChl (1

B

/1

B



Q

).

(3)

was reported to be 4.7 ps

_{(Rondonuwu et al., 2004), which is very similar with the formation}

rate of the Q

state in

Rps. moliscianum

. Thus, the Q

formation in this complex is mainly

determind by the Q



Q

internal conversion; therefore, the presumed singlet-energy

transfer from the higher singlet states of Car can be omitted. In addition, the Q

state is

energetically higher than the 2

_A

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

state to the 2

A

state probably takes place (Staleva, 2015), but the sequential

appearance is difficult to be distinguished.

11_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 21_A_g _{excited state absorptions of (a) spheroidene free}

(4)

Table 1.The rise and decay rate constants (_k) of the 21_A_g _{state of Cars free in a solution}

and bound to the LH2 complexes are revealed. The rise time constants of the Qystate 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 Qytime profiles.

The decay rate constant of the 2

_A

state of spheroidene bound to the LH2 complexes

from

_{Rhb. sphaeroides}

2.4.1 were substantially enhanced by 0.38 ps

_{when compared to the}

free in a solution, indicating that singlet energy transfer took place from the 2

_A

to the Q

state. This is almost in agreement with the 0.53 ps

_{formation rate of Q}

. Thus, the 2

A

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

_B

/1

B

) of lycopene to a Q

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

_A

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

_B

and 1

B

states.

5. Conclusion and remarks

Excited state dynamics and energetics of Cars free and bound to the LH2 complexes

have been evaluated. A relaxation scheme, including a 1

_B



1 B



2 A



1 A

singlet

internal conversion and a 1

_B



triplet state has been identified for Cars free in a solution.

In antenna complexes, two additional paths were obtained as Car-to-BChl singlet-energy

transfer channels, namely 1

_B

/ 1

B



Q

, and 2

A



Q

. Depending on the energetics

of the donor and acceptor states, singlet-energy transfer channels may be shut down when

donor states are lower than that of acceptor states. In the LH2 complexes, the energy of the

2 _A

(donor) state of spheroidene (

n

= 9) is higher than the energy of the Q

(acceptor) state

of BChl; thus, it promotes a singlet-energy transfer. In contrast to spheroidene, the energy of

the 2

_A

state of lycopene (

n

= 10) is lower than the Q

state; hence, it prevents the energy

transfer process.

These donor and acceptor energy levels are important for Car applications, such as for

antioxidative agents or artificial antenna. Apart from a Car-to-BChl singlet energy transfer,

an ultrafast singlet to triplet conversion is also observed within Cars from the 1

_B

(5)

Those singlet to triplet conversions are predominant for Cars bound to pigment protein

complexes. The enhancement of singlet to triplet conversions within Cars in pigment protein

complexes is not clear yet, but it is most probably because Cars are twisted when they are

bound to protein complexes to facilitate one spin flip for a triplet formation. The triplet state

may function as an energy outlet when Cars absorb excesive photon energy.

It should be mentioned that the 3

_A

state must also play a role in both Cars internal

conversions and the Car-to-Bchl singlet-energy transfer in an LH2 complex from

_Rsp.

Moliscianum

, as this hidden state is present in-between the optically active 1

_B

and newly

identified 1

_B

states for lycopene (

n

=11). However, a shorter pulse duration (less than 60

fs) is necessary to be employed, since the lifetime of the 1

_B

and 3

A

and 1

B

states are

extremely short (less than 100 fs) for Cars have a number of conjugated double bonds greater

than 10. Knowledge about Cars excited state dynamics and energetics is particularly

important not only for ultrafast electron injection in a Cars-semiconductor interface study

but also to understand the molecular mechanisms of Cars antioxidant applications.

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recombination via the T1 state as determined by subpicosecond to microsecond time-resolved absorption spectroscopy: Dependence on the conjugation length._{J. Phys. Chem. B},₁₀₉, 17066 17077.

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