**3.2 Peridinin and chlorophyll triplet state in solvent**

It is important to firmly establish our spectral assignment of Per and Chl-*a* modes in the 3Per and 3Chl-*a* states using an artificial system where Per and Chl-*a* are not strongly coupled. To this end, we performed step-scan time resolved FTIR measurements on Per mixed with Chl*a* in organic solvent to observe TEET on the µs timescale and extract their individual spectra using global and target analysis. The two chromophores were mixed in THF with a stoichiometric ratio Per: Chl-*a* of 1:8 with a Chl-*a* OD (670 nm) of about 100 cm-1. Timeresolved FTIR data has been obtained by direct excitation of Chl-*a* at 625 nm and the ensuing spectral evolution was analyzed using global and target analysis. Global analysis in terms of a sequential kinetic scheme shows that three components are required to fit the data. In order to determine the origin of the third component, we used target analysis. The best fit was obtained with a three level scheme, in which the first component decays in 3.5 µs in parallel to the second and the third component, which decay to the ground state in 7 µs and 3 ms, respectively. The kinetic model is displayed in Fig.3. The first component decays into the second and third components with an estimated yield of 60% and 12% respectively. About 28% of the first component amplitude is lost *via* triplet-triplet annihilation and decays to the ground state. Experiments with Chl-*a* only in THF confirmed that the first component decays into a component having similar lifetime and spectral features as compared to the third component observed in the mixed Chl-*a*/Per sample. We note that Per is unable to quench the third component which suggests that it can be assigned to a radical state of Chl-*a*. The three SADS that result from the target analysis of the mixed Chl-*a*/Per data are shown

Time-Resolved FTIR Difference Spectroscopy Reveals the Structure and Dynamics

follows from a spurious contribution from SADS1 (3Chl-*a*).

1671

1733

1747

1761

1698


Delta Absorption,

*mOD*

relatively homogenous and non H-bonded with a main frequency at 1761 cm-1.

of Carotenoid and Chlorophyll Triplets in Photosynthetic Light-Harvesting Complexes 239

Per. Thus, C=O lactone conformers in THF, which is moderately polar and aprotic, are

In SADS2, the small amplitude bandshift observed around 1697(-)/1666(+) cm-1 arises from a contribution by SADS1 (i.e. 3Chl-*a*) that the target analysis fails to fully remove due to our limited time resolution, S/N ratio and baseline fluctuations. A Chl-*a* – Per sample at a 1:1 stoichiometric ratio (OD670 = 150 cm-1) exhibits a shorter 3Chl-*a* lifetime of about 1 µs. Target analysis resulted in a 3Per SADS (SADS-Per) almost free of 3Chl-*a* contributions. By comparing this latter SADS-Per with SADS2 (Figure 4, light gray line) we can confirm that in SADS2 (Figure 4) the small amplitude bandshift observed around 1697(-)/1666(+) cm-1

> SADS1 (3.5 µs) SADS2 (7 µs) SADS3 (3 ms) (x 2) SADS-Per (7 µs)

1461

1450 1306 1367 1336

radical pair

1800 1700 1600 1500 1400 1300

W avenumbers, *cm-1*

Fig. 4. SADS resulting from target analysis applied to data obtained upon direct Chl-*a* excitation at 625 nm of Per and Chl-*a* mixed in THF with a stoichiometric ratio Per: Chl-*a* of 1:8 with a Chl-*a* OD at 670 nm of about 100 cm-1. The black spectrum is assigned to 3[Chl-*a*]\*, it decays in 3.5 µs into the dash-dot gray and light gray SADS. The dash-dot gray SADS is assigned to 3[Per]\* and decays to the ground state in 7 µs. The light gray SADS contains signature of Chl-*a* cation and THF radical anion; it decays in 3 ms. The gray spectrum (SADS-Per) is obtained upon direct Chl-*a* excitation at 625 nm of Per and Chl-*a* mixed in THF with a

stoichiometric ratio Per: Chl-*a* of 1:1 with a Chl-*a* OD at 670 nm of about 150 cm-1.

The third SADS has an overall low amplitude. It is assigned to the Chl-*a*+/THF-

which is formed with a relatively low yield from the 3Chl-*a* and decays with an estimated lifetime of 3 ms. In the carbonyl region, the third SADS exhibits up-shifted ester and keto C=O stretch frequencies at 1738(-)/1751(+) and 1692(-)/1712(+), 1657(-)/1680(+) cm-1, characteristic for radical cation formation (Breton, Nabedryk et al. 1999). The presence of

1550

<sup>1599</sup> <sup>1657</sup>

(a) Triplet-triplet annihilation proceeds with a yield of 28% (3[Chl-*a*]\* + 3[Chl-*a*]\* = 1[Chl-*a*]\* + 1[Chl-*a*]). (b) Triplet excitation energy transfer from Chl-*a* to Per takes place in 3.5 µs with a yield of 60%. (c) Chl-*a* radical formation have a yield of 12%.

Fig. 3. Target analysis kinetic model applied to the time-resolved data of Per mixed with Chl-*a* in THF (excitation at 625 nm).

in Figure 4. The first component (SADS1), shown in black in Fig. 4, is assigned to 3Chl-*a* and has a lifetime of 3.5 µs. Its short lifetime indicates that the 3Chl-*a* is strongly quenched in the presence of Per. This SADS matches the FTIR triplet minus singlet (T-S) spectrum of Chl-*a* in THF at 90K (Breton, Nabedryk et al. 1999). Bands at 1747(-)/1733(+) and 1698(-)/1671(+) cm-1 are assigned to the Chl-*a 10a*-ester and *9*-keto carbonyl, respectively. The *9*-keto carbonyl stretch of Chl-*a* in THF at 1698 cm-1 is non H-bonded and in a moderately polar environment.

In SADS1 (Fig. 4), the band at 1599 (-) cm-1 is typical of a 6-coordinated Chl-*a* Mg-atom (Fujiwara 1986; Fujiwara and Tasumi 1986; Groot 2004). If Chl-*a* is monomeric, then Chl-*a* will be 6-coordinated with two THF molecules ligated to the central Mg atom forming the complex [Chl-*a*]-[THF]2. However the presence of a dip in the ESA at 1657 cm-1, which is not present in the FTIR T-S spectra in ref.(Breton, Nabedryk et al. 1999), may indicate that the observed triplet state actually resides on aggregated Chl*-a*. Chl-*a* at high concentration in an apolar solvent, in the absence of water, is known to aggregate with a typical IR band near 1657 cm-1 for Chl-*a* dimers (Katz, Ballschmiter et al. 1968). The aggregation most likely follows from the high concentration used for the FTIR experiments of about 1 mM (OD at 670 nm of about 100 cm-1) in THF. Assuming that Chl-*a* is dimeric in our sample, the 1657 cm-1 band most likely indicates a second Chl-*a* (Chl-*a*2) with its 9-keto bound to the Mg atom of the first Chl-*a* (Chl-*a*1) (Katz, Ballschmiter et al. 1968; Fujiwara 1986). As SADS1 is dominated by modes of Chl-*a*1, this suggest that the triplet state is mainly localized on this Chl-*a* for which the ester and keto are free of (H-) bonding.

The second SADS rises in 3.5 µs and decays in 7 µs. It is assigned to 3Per (dash-dot gray SADS in Fig. 3) according to its characteristic decay time. 3Per is formed *via* TEET from 3Chl*a*, note that this component is not observed in Chl-*a* only samples in THF. The 3Per SADS is dominated by a band-shift at 1761(-)/1733(+) cm-1, assigned to the C=O lactone stretch of

3[Chl-a]\* <sup>3</sup> (a) [Chl-*a*]\*

(c) (c) (a)

Long Living (ms time scale)

(a) Triplet-triplet annihilation proceeds with a yield of 28% (3[Chl-*a*]\* + 3[Chl-*a*]\* = 1[Chl-*a*]\* + 1[Chl-*a*]). (b) Triplet excitation energy transfer from Chl-*a* to Per takes place in 3.5 µs with a yield of 60%. (c) Chl-*a*

Fig. 3. Target analysis kinetic model applied to the time-resolved data of Per mixed with

in Figure 4. The first component (SADS1), shown in black in Fig. 4, is assigned to 3Chl-*a* and has a lifetime of 3.5 µs. Its short lifetime indicates that the 3Chl-*a* is strongly quenched in the presence of Per. This SADS matches the FTIR triplet minus singlet (T-S) spectrum of Chl-*a* in THF at 90K (Breton, Nabedryk et al. 1999). Bands at 1747(-)/1733(+) and 1698(-)/1671(+) cm-1 are assigned to the Chl-*a 10a*-ester and *9*-keto carbonyl, respectively. The *9*-keto carbonyl stretch of Chl-*a* in THF at 1698 cm-1 is non H-bonded and in a moderately polar

In SADS1 (Fig. 4), the band at 1599 (-) cm-1 is typical of a 6-coordinated Chl-*a* Mg-atom (Fujiwara 1986; Fujiwara and Tasumi 1986; Groot 2004). If Chl-*a* is monomeric, then Chl-*a* will be 6-coordinated with two THF molecules ligated to the central Mg atom forming the complex [Chl-*a*]-[THF]2. However the presence of a dip in the ESA at 1657 cm-1, which is not present in the FTIR T-S spectra in ref.(Breton, Nabedryk et al. 1999), may indicate that the observed triplet state actually resides on aggregated Chl*-a*. Chl-*a* at high concentration in an apolar solvent, in the absence of water, is known to aggregate with a typical IR band near 1657 cm-1 for Chl-*a* dimers (Katz, Ballschmiter et al. 1968). The aggregation most likely follows from the high concentration used for the FTIR experiments of about 1 mM (OD at 670 nm of about 100 cm-1) in THF. Assuming that Chl-*a* is dimeric in our sample, the 1657 cm-1 band most likely indicates a second Chl-*a* (Chl-*a*2) with its 9-keto bound to the Mg atom of the first Chl-*a* (Chl-*a*1) (Katz, Ballschmiter et al. 1968; Fujiwara 1986). As SADS1 is dominated by modes of Chl-*a*1, this suggest that the triplet state is mainly localized on this

The second SADS rises in 3.5 µs and decays in 7 µs. It is assigned to 3Per (dash-dot gray SADS in Fig. 3) according to its characteristic decay time. 3Per is formed *via* TEET from 3Chl*a*, note that this component is not observed in Chl-*a* only samples in THF. The 3Per SADS is dominated by a band-shift at 1761(-)/1733(+) cm-1, assigned to the C=O lactone stretch of

[Chl-*a*] +/ THF And/or [Chl-*a*] 2

+/ [THF]

ISC

(b)

60% 12%

1[Chl-a]

Chl-*a* for which the ester and keto are free of (H-) bonding.

radical formation have a yield of 12%.

Chl-*a* in THF (excitation at 625 nm).

environment.

**Excitation 625 nm**

3[Per]\*

(c)

1[Chl-a]\*

28%

Per. Thus, C=O lactone conformers in THF, which is moderately polar and aprotic, are relatively homogenous and non H-bonded with a main frequency at 1761 cm-1.

In SADS2, the small amplitude bandshift observed around 1697(-)/1666(+) cm-1 arises from a contribution by SADS1 (i.e. 3Chl-*a*) that the target analysis fails to fully remove due to our limited time resolution, S/N ratio and baseline fluctuations. A Chl-*a* – Per sample at a 1:1 stoichiometric ratio (OD670 = 150 cm-1) exhibits a shorter 3Chl-*a* lifetime of about 1 µs. Target analysis resulted in a 3Per SADS (SADS-Per) almost free of 3Chl-*a* contributions. By comparing this latter SADS-Per with SADS2 (Figure 4, light gray line) we can confirm that in SADS2 (Figure 4) the small amplitude bandshift observed around 1697(-)/1666(+) cm-1 follows from a spurious contribution from SADS1 (3Chl-*a*).

Fig. 4. SADS resulting from target analysis applied to data obtained upon direct Chl-*a* excitation at 625 nm of Per and Chl-*a* mixed in THF with a stoichiometric ratio Per: Chl-*a* of 1:8 with a Chl-*a* OD at 670 nm of about 100 cm-1. The black spectrum is assigned to 3[Chl-*a*]\*, it decays in 3.5 µs into the dash-dot gray and light gray SADS. The dash-dot gray SADS is assigned to 3[Per]\* and decays to the ground state in 7 µs. The light gray SADS contains signature of Chl-*a* cation and THF radical anion; it decays in 3 ms. The gray spectrum (SADS-Per) is obtained upon direct Chl-*a* excitation at 625 nm of Per and Chl-*a* mixed in THF with a stoichiometric ratio Per: Chl-*a* of 1:1 with a Chl-*a* OD at 670 nm of about 150 cm-1.

The third SADS has an overall low amplitude. It is assigned to the Chl-*a*+/THF radical pair which is formed with a relatively low yield from the 3Chl-*a* and decays with an estimated lifetime of 3 ms. In the carbonyl region, the third SADS exhibits up-shifted ester and keto C=O stretch frequencies at 1738(-)/1751(+) and 1692(-)/1712(+), 1657(-)/1680(+) cm-1, characteristic for radical cation formation (Breton, Nabedryk et al. 1999). The presence of

Time-Resolved FTIR Difference Spectroscopy Reveals the Structure and Dynamics

of Carotenoid and Chlorophyll Triplets in Photosynthetic Light-Harvesting Complexes 241

Fig. 6. (A) Time slices of raw data at different excitations. (B) DADS of PCP obtained using a parallel model after different excitations. DADS1 (dotted line) and DADS2 (black line) are

experiments on PCP in the visible spectral region reported one Per triplet lifetime of 10 µs at RT (Bautista, Hiller et al. 1999; Kleima, Wendling et al. 2000) and two lifetimes of 13 and 40 µs at 77 K (Kleima, Wendling et al. 2000) and 13 and 58 µs up to 200 K (Carbonera, Giacometti et al. 1999), respectively. In Fig. 6 the positive DADS signals originate from excited state absorption (ESA) of the triplet states, and negative DADS signals from the

In our spectral analysis, we distinguish four regions of interest: The carbonyl region (1800- 1630 cm-1); the C=C stretch region, characteristic of the polyene backbone of carotenoids and chlorophylls giving rise to bands between 1610 and 1525 cm-1; the CH-deformations and possible lactone-ring modes in the region of 1450-1380 cm-1 and the fingerprint region below

As we can see in Fig. 6B, the two DADS show many spectral similarities. The weak bands at 1623,1633 cm-1 (bleach) and strong bands at around 1555/1530 cm-1 (bleach/ESA) are Per C=C stretching modes. Typically, carotenoid C=C stretching modes are around 1520 cm-1 – e.g. spheroidene, ß-carotene (Noguchi, Hayashi et al. 1990; Hashimoto, Koyama et al. 1991) – and down-shifted by 20 cm-1 to around 1500 cm-1 in the T1 state (Hashimoto, Koyama et al. 1991). In Per we observe slightly higher frequencies, indicating a decrease in bond-order, as observed normally for peripheral C=C stretches (Nagae, Kuki et al. 2000). Moreover, the broad band extending from ~1480 cm-1 to lower frequencies (bleach) due to CH-deformation

1380 cm-1 with e.g. CH-out-of-plane, C-C and C-O stretches and their combinations.

associated with a 13 and 42 µs lifetime, respectively.

bleach of the ground state.

**3.3.1 Spectral analysis** 

two different 9-keto stretches confirms our earlier hypothesis that a Chl-*a* dimer (Chl*a*1/Chl-*a*2) is present under these conditions. In contrast to the triplet state, which seemed to be mainly localized on Chl-*a*1 (SADS1), the radical appears to be delocalized over the two Chl-*a* molecules of the dimer, as its infrared signature is in fact similar to P700+ in the Photosystem I RC (Breton, Nabedryk et al. 1999). Below 1500 cm-1 new bands have appeared in SADS3. A strong negative band with a double peak character is observed centered at 1450 and 1461 cm-1 in addition to bands at 1367, 1336 and 1306 cm-1. These bands are typical for THF (data not shown) and up-shifted after Chl-*a* excitation to 1630-1515, 1382, 1354 and 1321 cm-1, respectively. Such up-shifts indicate radical formation of THF, probably the radical anionic ([THF]●-) state, forming the [Chl-*a*]●+/[THF]●- radical pair.

#### **3.3 A PCP triplet state dynamic**

In this section we use microsecond time-resolved FTIR spectroscopy to measure triplet formation in PCP by monitoring excitation-induced variations in the vibrational modes of the PCP chromophores. In the following paragraphs, we present the estimated lifetimes, the decay associated difference spectra (DADS) and the vibrational mode assignment of three sets of time-resolved IR data resulting from excitation of Chl-*a* at 670 nm (Qy), and Per at 480 nm (maximum of Per absorption) and 530 nm (red edge of Per absorption, Fig.5). Excitation at 550 nm gave essentially the same result as 530 nm (data not shown).

Fig. 5. Differential absorbance signals of PCP obtained by step-scan Fourier-transform infrared spectroscopy. Raw data with 5 µs time resolution for 530 nm excitation; intensity 2 mJ/cm2.

Time-resolved mid-IR spectra were collected (Fig. 6A) and globally analyzed at frequencies between 1100 and 1800 cm-1, and the resulting decay associated difference spectra (DADS) are shown in Fig. 6B. To describe the time resolved data, we used a parallel model, which required three components: two fast lifetimes, of the order of tens of microseconds and a longer one that we do not consider further We find the triplet state dynamics of PCP at RT to be described by lifetimes of 13 and 42 µs which are typical of carotenoid triplets. Previous

Fig. 6. (A) Time slices of raw data at different excitations. (B) DADS of PCP obtained using a parallel model after different excitations. DADS1 (dotted line) and DADS2 (black line) are associated with a 13 and 42 µs lifetime, respectively.

experiments on PCP in the visible spectral region reported one Per triplet lifetime of 10 µs at RT (Bautista, Hiller et al. 1999; Kleima, Wendling et al. 2000) and two lifetimes of 13 and 40 µs at 77 K (Kleima, Wendling et al. 2000) and 13 and 58 µs up to 200 K (Carbonera, Giacometti et al. 1999), respectively. In Fig. 6 the positive DADS signals originate from excited state absorption (ESA) of the triplet states, and negative DADS signals from the bleach of the ground state.

#### **3.3.1 Spectral analysis**

240 Infrared Spectroscopy – Life and Biomedical Sciences

two different 9-keto stretches confirms our earlier hypothesis that a Chl-*a* dimer (Chl*a*1/Chl-*a*2) is present under these conditions. In contrast to the triplet state, which seemed to be mainly localized on Chl-*a*1 (SADS1), the radical appears to be delocalized over the two Chl-*a* molecules of the dimer, as its infrared signature is in fact similar to P700+ in the Photosystem I RC (Breton, Nabedryk et al. 1999). Below 1500 cm-1 new bands have appeared in SADS3. A strong negative band with a double peak character is observed centered at 1450 and 1461 cm-1 in addition to bands at 1367, 1336 and 1306 cm-1. These bands are typical for THF (data not shown) and up-shifted after Chl-*a* excitation to 1630-1515, 1382, 1354 and 1321 cm-1, respectively. Such up-shifts indicate radical formation of THF, probably the radical

In this section we use microsecond time-resolved FTIR spectroscopy to measure triplet formation in PCP by monitoring excitation-induced variations in the vibrational modes of the PCP chromophores. In the following paragraphs, we present the estimated lifetimes, the decay associated difference spectra (DADS) and the vibrational mode assignment of three sets of time-resolved IR data resulting from excitation of Chl-*a* at 670 nm (Qy), and Per at 480 nm (maximum of Per absorption) and 530 nm (red edge of Per absorption, Fig.5). Excitation

Fig. 5. Differential absorbance signals of PCP obtained by step-scan Fourier-transform infrared spectroscopy. Raw data with 5 µs time resolution for 530 nm excitation; intensity 2

Time-resolved mid-IR spectra were collected (Fig. 6A) and globally analyzed at frequencies between 1100 and 1800 cm-1, and the resulting decay associated difference spectra (DADS) are shown in Fig. 6B. To describe the time resolved data, we used a parallel model, which required three components: two fast lifetimes, of the order of tens of microseconds and a longer one that we do not consider further We find the triplet state dynamics of PCP at RT to be described by lifetimes of 13 and 42 µs which are typical of carotenoid triplets. Previous

anionic ([THF]●-) state, forming the [Chl-*a*]●+/[THF]●- radical pair.

at 550 nm gave essentially the same result as 530 nm (data not shown).

**3.3 A PCP triplet state dynamic** 

mJ/cm2.

In our spectral analysis, we distinguish four regions of interest: The carbonyl region (1800- 1630 cm-1); the C=C stretch region, characteristic of the polyene backbone of carotenoids and chlorophylls giving rise to bands between 1610 and 1525 cm-1; the CH-deformations and possible lactone-ring modes in the region of 1450-1380 cm-1 and the fingerprint region below 1380 cm-1 with e.g. CH-out-of-plane, C-C and C-O stretches and their combinations.

As we can see in Fig. 6B, the two DADS show many spectral similarities. The weak bands at 1623,1633 cm-1 (bleach) and strong bands at around 1555/1530 cm-1 (bleach/ESA) are Per C=C stretching modes. Typically, carotenoid C=C stretching modes are around 1520 cm-1 – e.g. spheroidene, ß-carotene (Noguchi, Hayashi et al. 1990; Hashimoto, Koyama et al. 1991) – and down-shifted by 20 cm-1 to around 1500 cm-1 in the T1 state (Hashimoto, Koyama et al. 1991). In Per we observe slightly higher frequencies, indicating a decrease in bond-order, as observed normally for peripheral C=C stretches (Nagae, Kuki et al. 2000). Moreover, the broad band extending from ~1480 cm-1 to lower frequencies (bleach) due to CH-deformation

Time-Resolved FTIR Difference Spectroscopy Reveals the Structure and Dynamics

a/BChl-a-

modes to the lactone vibration of Per.

**Carbonyl modes of Per** 

peaking around 1740 cm-1.

The spectral assignments leads to the conclusions that:

of Carotenoid and Chlorophyll Triplets in Photosynthetic Light-Harvesting Complexes 243

perfectly overlapped with the T-S FTIR spectrum of Chl-*a* in THF (Breton, Nabedryk et al. 1999). Chl-*a* triplets are known to exhibit a down-shift in the ESA, while Chl-*a* cations are up-shifted (Breton, Nabedryk et al. 1999; Breton 2001; Noguchi 2002). A hypothetical charge transfer state leading to Per (+)/Chl-*a(*-),would be expected to downshift the 9-keto vibration of Chl-*a* anion by about 55 to 92 cm-1 as observed for BPheo/BPheo- in THF (Mantele, Wollenweber et al. 1988), Pheo/Pheo- in PSII (Okubo and Noguchi) and BChl-

shifts by 30 cm-1 and strongly suggests that only Chl-*a* triplets exist on typical triplet carotenoid lifetimes and we can exclude the presence of Chl-*a* cations and anions. From the 9-keto-mode-amplitudes at 1699, 1686 cm-1, we conclude that both Chl-*a* molecules of the quasi-symmetric PCP monomer are involved in the triplet state dynamics at RT, however, to a different extent depending on the excitation wavelength. The 9-keto mode is the strongest mode in T-S Chl-*a* FTIR difference spectra, about three times more intense than the 10a-ester mode (Breton, Nabedryk et al. 1999; Breton 2001), so we assign the other strong carbonyl

The remaining three ground state bleach modes – again best resolved after 530 nm excitation– at 1745 cm-1 (DADS1), 1741 (DADS2) and 1720 cm-1 (DADS2) represent the lactone carbonyl modes of three distinguishable Per conformers; however the 1741 cm-1 (DADS2) conformer is absent under 670 nm excitation. (Fig. 7) The Per ester group, which could be a possible candidate for these frequencies is located at one of the cyclo-hexane endgroups, and isolated from the conjugated backbone. For this reason, we do not expect contributions of the ester group after electronic excitation. The carbonyl-stretch of a fivemembered lactone has a typical frequency of 1765±5 cm-1 (Kristallovich, Shamyanov et al. 1987), which can downshift by 20 cm-1 in conjugation with a π-system, as in Per, and even further in a polar environment or with hydrogen bonding of the carbonyl to the protein pocket. Moreover, the 25 cm-1 down-shift of the ESA corresponding to the 1745/1720 cm-1 bands would be large compared to 5 cm-1 for ester groups (as reported e.g. for chlorophyll) (Breton 2001). The observed ground state bleach modes are in agreement with Per resonant (530 nm) Raman data for PCP (unpublished data, Papagiannakis, E. and Robert B.) which display only two broad bands at 1745 and 1720 cm-1 in the carbonyl region. In addition, the resonant Raman spectrum of Per in methanol shows only one broad carbonyl frequency




conformers, respectively, experiencing different protein environment.

Chl-*a*, and both triplet signals decay with typical carotenoid tripet lifetimes.

(Hartwich, Geskes et al. 1995). In our data the 9-keto vibration of Chl-*a* down

modes is characteristic of carotenoids (Bernhard and Grosjean 1995). The reported modes of Chl-*a* in PCP (Kleima, Wendling et al. 2000) at 1610, 1553 and 1526 cm-1 might also contribute to these bands to a minor extent. Additional recognizable modes are at ~1450 cm-1 the methylene C-H deformation and the methyl asymmetric bending modes, as well as the symmetric methyl bending mode of the Per backbone peaks at 1408/1380 cm-1 (bleach/ESA) (Bernhard and Grosjean 1995).

#### **Carbonyl region – molecular probes**

The carbonyl region contains contributions from Per and Chl-*a* since both have carbonyl groups in conjugation with their electronic system. These carbonyl modes are very sensitive to electronic changes and can be used as molecular probes for the individual chromophores. The carbonyl modes that can be expected in this region are the lactone mode of Per, and the 10a-ester and 9-keto modes of Chl-*a* (Fig. 7).

After global analysis, the DADS in the carbonyl region show five major frequencies in the ground state bleach (best resolved with 530 nm excitation): 1745 cm-1 (DADS1), 1741, 1720 cm-1 (DADS2), 1699 and 1686 cm-1 (both DADS) as seen Figure 7.

Fig. 7. Comparison of the carbonyl modes of the PCP DADS at 670, 530 and 480 nm excitations.

#### **Carbonyl modes of Chl-a**

The bands at ~1699, 1686 cm-1 (bleach), shown in Fig. 6 and 7, match the 1697 and 1681 cm-1 ones reported from fluorescence line narrowing (FLN) spectra at 4 K (Kleima, Wendling et al. 2000). The ESA of the two bands has shifted down to 1670 and 1657 cm-1, respectively. Such a down-shift is typical for the 9-keto vibration of Chl-*a* in T-S FTIR spectra and can be perfectly overlapped with the T-S FTIR spectrum of Chl-*a* in THF (Breton, Nabedryk et al. 1999). Chl-*a* triplets are known to exhibit a down-shift in the ESA, while Chl-*a* cations are up-shifted (Breton, Nabedryk et al. 1999; Breton 2001; Noguchi 2002). A hypothetical charge transfer state leading to Per (+)/Chl-*a(*-),would be expected to downshift the 9-keto vibration of Chl-*a* anion by about 55 to 92 cm-1 as observed for BPheo/BPheo- in THF (Mantele, Wollenweber et al. 1988), Pheo/Pheo- in PSII (Okubo and Noguchi) and BChla/BChl-a- (Hartwich, Geskes et al. 1995). In our data the 9-keto vibration of Chl-*a* down shifts by 30 cm-1 and strongly suggests that only Chl-*a* triplets exist on typical triplet carotenoid lifetimes and we can exclude the presence of Chl-*a* cations and anions. From the 9-keto-mode-amplitudes at 1699, 1686 cm-1, we conclude that both Chl-*a* molecules of the quasi-symmetric PCP monomer are involved in the triplet state dynamics at RT, however, to a different extent depending on the excitation wavelength. The 9-keto mode is the strongest mode in T-S Chl-*a* FTIR difference spectra, about three times more intense than the 10a-ester mode (Breton, Nabedryk et al. 1999; Breton 2001), so we assign the other strong carbonyl modes to the lactone vibration of Per.

### **Carbonyl modes of Per**

242 Infrared Spectroscopy – Life and Biomedical Sciences

modes is characteristic of carotenoids (Bernhard and Grosjean 1995). The reported modes of Chl-*a* in PCP (Kleima, Wendling et al. 2000) at 1610, 1553 and 1526 cm-1 might also contribute to these bands to a minor extent. Additional recognizable modes are at ~1450 cm-1 the methylene C-H deformation and the methyl asymmetric bending modes, as well as the symmetric methyl bending mode of the Per backbone peaks at 1408/1380 cm-1 (bleach/ESA)

The carbonyl region contains contributions from Per and Chl-*a* since both have carbonyl groups in conjugation with their electronic system. These carbonyl modes are very sensitive to electronic changes and can be used as molecular probes for the individual chromophores. The carbonyl modes that can be expected in this region are the lactone mode of Per, and the

After global analysis, the DADS in the carbonyl region show five major frequencies in the ground state bleach (best resolved with 530 nm excitation): 1745 cm-1 (DADS1), 1741, 1720

1800 1775 1750 1725 1700 1675 1650

1800 1775 1750 1725 1700 1675 1650

**1720**

**<sup>1670</sup> <sup>13</sup><sup>s</sup>**

**<sup>1720</sup> <sup>1699</sup>**

**1686**

**1657**

**670 nm**

**530 nm**

**480 nm**

**1745**

Fig. 7. Comparison of the carbonyl modes of the PCP DADS at 670, 530 and 480 nm

**1741**

wavenumber (cm -1 )

The bands at ~1699, 1686 cm-1 (bleach), shown in Fig. 6 and 7, match the 1697 and 1681 cm-1 ones reported from fluorescence line narrowing (FLN) spectra at 4 K (Kleima, Wendling et al. 2000). The ESA of the two bands has shifted down to 1670 and 1657 cm-1, respectively. Such a down-shift is typical for the 9-keto vibration of Chl-*a* in T-S FTIR spectra and can be

(Bernhard and Grosjean 1995).

**Carbonyl region – molecular probes** 

10a-ester and 9-keto modes of Chl-*a* (Fig. 7).


0.0



dOD (a.u.)

excitations.

**Carbonyl modes of Chl-a** 

cm-1 (DADS2), 1699 and 1686 cm-1 (both DADS) as seen Figure 7.

**42 s**

The remaining three ground state bleach modes – again best resolved after 530 nm excitation– at 1745 cm-1 (DADS1), 1741 (DADS2) and 1720 cm-1 (DADS2) represent the lactone carbonyl modes of three distinguishable Per conformers; however the 1741 cm-1 (DADS2) conformer is absent under 670 nm excitation. (Fig. 7) The Per ester group, which could be a possible candidate for these frequencies is located at one of the cyclo-hexane endgroups, and isolated from the conjugated backbone. For this reason, we do not expect contributions of the ester group after electronic excitation. The carbonyl-stretch of a fivemembered lactone has a typical frequency of 1765±5 cm-1 (Kristallovich, Shamyanov et al. 1987), which can downshift by 20 cm-1 in conjugation with a π-system, as in Per, and even further in a polar environment or with hydrogen bonding of the carbonyl to the protein pocket. Moreover, the 25 cm-1 down-shift of the ESA corresponding to the 1745/1720 cm-1 bands would be large compared to 5 cm-1 for ester groups (as reported e.g. for chlorophyll) (Breton 2001). The observed ground state bleach modes are in agreement with Per resonant (530 nm) Raman data for PCP (unpublished data, Papagiannakis, E. and Robert B.) which display only two broad bands at 1745 and 1720 cm-1 in the carbonyl region. In addition, the resonant Raman spectrum of Per in methanol shows only one broad carbonyl frequency peaking around 1740 cm-1.

The spectral assignments leads to the conclusions that:


Time-Resolved FTIR Difference Spectroscopy Reveals the Structure and Dynamics

**1683**

**1700**

H-PCP. The gray line represents the 13 µs DADS in A-PCP.

the triplet sublevels (Carbonera, Giacometti et al. 1999).

**1720**

**1670**

**1666**

of Carotenoid and Chlorophyll Triplets in Photosynthetic Light-Harvesting Complexes 245

**1538 1382**

1800 1700 1600 1500 1400 1300 1200

Wavenumber, *cm-1*

The second component in A-PCP has been observed below 200 K by Carbonera *et al*. (Carbonera, Giacometti et al. 1999) and at 77K by Kleima *et al*., while only a 10 µs component was present at room temperature in the latter work (Kleima, Wendling et al. 2000). We reproduced Kleima's result at room temperature under aerobic and anaerobic conditions, by measuring the triplet decay in the visible using a diluted A-PCP solution. We found only the 10 µs component under aerobic and anaerobic conditions, which excludes an effect related to oxygen (data not shown). Thus, it seems that the appearance of the ~40 µs component is likely related to a protein conformational change induced by cooling and/or high concentrations used for FTIR, rather than a temperature effect on the equilibration among

We previously proposed that the Per conformers involved in the photoprotective mechanism were likely Per 612/622 and Per 614/624. In a recent TR-EPR study (Di Valentin, Ceola et al. 2008), participation of the Per 612/622 pair in the 3Chl-*a* quenching was considered unlikely on the basis of the similarity of the 3Per triplet spectra in Main Form A-PCP (MFPCP) and High Salt A-PCP (HSPCP), since the latter does not bind the Per 612/622 pair. Comparison between experimental and calculated EPR spectra led the authors to conclude that the triplet was mainly (~80%) localized on the Per 614/624 pair, which has the shortest center-to-center distance to the Chl-*a*. This conclusion is consistent with the result of MD calculations showing that in the triplet state, the highest spin density is localized in the center of the Per backbone (Di Valentin, Ceola et al. 2008). Thus, it seems likely that in the step-scan FTIR experiments, the main Per conformer at 1745(-)/~1720(+) cm-1 corresponds to Per 614, Per 624, or both. Hence, we conclude that in A-PCP and H-PCP, Per 614 and/or 624 likely constitute the principal 3Chl-*a* quenchers, and that their specific interaction with

Chl-*a* promotes the mixing of the 3Chl-*a* and 3Per states during the lifetime of 3Per.

Fig. 8. Decay-Associated Difference Spectra (DADS) that result from a global analysis of step-scan FTIR data of H-PCP and A-PCP. The black line represents the 10 µs component in

**1558**

**1407**

 A-PCP (13 s) H-PCP (10 s)




**1770**

**1725**

**1745**

Delta Absorption, *a.u.*

0.0

0.5

1.0

dynamics shows a strong wavelength dependence suggesting increased population of this Per conformer upon direct Per excitation. These spectral changes are accompanied by an overall increase of signal amplitude observed from 670 to 480 and to 530 nm. This wavelength dependence indicates that Per triplet formation proceeds via different pathways.
