Two-Photon Study on the Electronic Interactions between the First

ARTICLE pubs.acs.org/JPCA

Two-Photon Study on the Electronic Interactions between the First Excited Singlet States in CarotenoidTetrapyrrole Dyads Pen-Nan Liao,† Smitha Pillai,‡ Devens Gust,‡ Thomas A. Moore,‡ Ana L. Moore,‡ and Peter J. Walla*,†,§ †

Technische Universit€at Braunschweig, Institute for Physical and Theoretical Chemistry, Department for Biophysical Chemistry, Hans-Sommer-Strasse 10, 38106 Braunschweig, Germany; ‡ Department of Chemistry & Biochemistry and Center for Bioenergy and Photosynthesis, Arizona State University, Tempe, Arizona 85287-1604, United States § Max Planck Institute for Biophysical Chemistry, Department of Spectroscopy and Photochemical Kinetics, Am Fassberg 11, 37077 G€ottingen, Germany ABSTRACT: Electronic interactions between the first excited states (S1) of carotenoids (Car) of different conjugation lengths (811 double bonds) and phthalocyanines (Pc) in different CarPc dyad molecules were investigated by two-photon spectroscopy and compared with Car S1chlorophyll (Chl) interactions in photosynthetic light harvesting complexes (LHCs). The observation of Chl/Pc fluorescence after selective twophoton excitation of the Car S1 state allowed sensitive monitoring of the flow of energy between Car S1 and Pc or Chl. It is found that two-photon excitation excites to about 80% to 100% exclusively the carotenoid state Car S1 and that only a small fraction of direct tetrapyrrole two-photon excitation occurs. Amide-linked CarPc dyads in tetrahydrofuran demonstrate a molecular gear shift mechanism in that effective Car S1 f Pc energy transfer is observed in a dyad with 9 double bonds in the carotenoid, whereas in similar dyads with 11 double bonds in the carotenoid, the Pc fluorescence is strongly quenched by Pc f Car S1 energy transfer. In phenylamino-linked CarPc dyads in toluene extremely large electronic interactions between the Car S1 state and Pc were observed, particularly in the case of a dyad in which the carotenoid contained 10 double bonds. This observation together with previous findings in the same system provides strong evidence for excitonic Car S1Pc Qy interactions. Very similar results were observed with photosynthetic LHC II complexes in the past, supporting an important role of such interactions in photosynthetic down-regulation.

’ INTRODUCTION The photosynthetic apparatus is a fascinating biophysical machine that not only allows extremely efficient light-harvesting and funneling of excitation energy to the reaction center using pigment densities that would usually result in significant concentration quenching but also enables a subtle balancing of the amount of the excitation energy that actually reaches the reaction center under the wide range of solar irradiation conditions that occur in nature.1 The roles of carotenoids (Car) in the photosynthetic apparatus are quite diverse as they participate in photosynthetic light harvesting as well as in down-regulation of reaction center function and other types of photoprotection. As light harvesters they absorb in the blue spectral region via optical transition to their second excited state (Car S0 f Car S2) and transfer a large fraction of this energy either directly to chlorophyll (Chl) (Car S2 f Chl) or from the optically forbidden first excited state (Car S1 f Chl) after very fast internal conversion (Car S2 f Car S1). The function of carotenoids in downregulation of photosynthetic reaction center function is less clear, partly because most regulation mechanisms that have been proposed so far involve the optically forbidden, first excited state r 2011 American Chemical Society

of the carotenoid (Car S1), that cannot be investigated by conventional absorption or fluorescence spectroscopy. On the basis of various experimental results at least three different electronic down-regulation mechanisms have been proposed for the carotenoid-mediated quenching: (I) Chl f Car S1 energy transfer with subsequent fast (∼10 ps) internal conversion (Car S1 f Car S0) of the energy into harmless heat,2,3 (II) excitonic Car S1 T Chl Qy interactions that result in an energy trap for the pigment pool and a strong reduction in the lifetime of the participating Chl,46 and (III) Car S1Chl electron transfer to form a transient charge-separated state79 (Figure 1). In addition to the mechanistic uncertainty, it is also unclear whether the changes in electronic interactions are induced by conformational changes within the pigmentprotein complexes1012 or by interactions at their periphery.13 There is still a debate concerning Special Issue: Graham R. Fleming Festschrift Received: December 24, 2010 Revised: February 22, 2011 Published: March 22, 2011 4082

dx.doi.org/10.1021/jp1122486 | J. Phys. Chem. A 2011, 115, 4082–4091

The Journal of Physical Chemistry A

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Figure 1. Electronic CarChl interactions that have been proposed as regulation mechanisms for light-harvesting in photosynthesis.29.

which pigmentprotein complexes are actually involved and what role important regulators such as PsbS or the xanthophyll-cycle carotenoids play.1417 Two-photon spectroscopy is a useful technique for gaining more insight into some of these questions because it allows selective direct excitation of the optically forbidden Car S1 state. This can be done even in complex biological environments such as entire, living plants.5,1825 Direct excitation is possible because the optical selection rules are different for one- and two-photon excitation. Car S0 f Car S1 is two-photon allowed and a subsequent observation of chlorophyll fluorescence is a clear measure of the Car S1 f Chl energy flow under different conditions. The quenching mechanism via excitonic Car S1Chl Qy interactions has been proposed as a photosynthetic downregulation mechanism based on a Car S1 two-photon study in which the electronic Car S1Chl interactions in living plants were determined during photosynthetic up- and downregulation.5 Such changes in the Car S1Chl interactions can be easily measured by a quantitative comparison of the Chl fluorescence intensity detected upon selective excitation of the carotenoid dark states, FlTPE, and direct one-photon excitation of the Chl, FlOPE 1 -Chl φCarS Coupling µ

FlTPE FlOPE

ð1Þ

1-Chl In general, φCarS Coupling is a parameter describing quantitatively the Car S1 f Chl energy flow in different samples. It is closely related to the electronic Car S1Chl interactions in a particular sample and is directly proportional to the rate of the

Car S1 f Chl energy flow that is—according to Fermi’s Golden Rule—proportional to the square of the electronic interactions (more details about this coupling parameter are described in our previous study5). Excitonic interactions were proposed as a quenching mechanism not only because of the observation of red shifts in the absorption and fluorescence of the participating pigments but in particular because a significant increase in energy transfer was observed in both directions, Chl f Car S1 as well Car S1 f Chl, during down-regulation.25 This finding, which seems to be very puzzling at the first glance, can easily be explained by excitonic interactions between the Car S1 and the Chl since then an excitation of one pigment must always lead directly to the excitation of the other pigment (for details, see ref 25). The complexity of the Car S1 T Chl interactions in natural systems makes it desirable to have well-defined model systems that demonstrate regulation mechanisms (IIII) and that provide additional evidence for the existence of excitonic interaction between the optically forbidden state of carotenoids, Car S1, and tetrapyrroles. Fortunately, phthalocyanine (Pc)carotenoid dyads with carotenoids of different conjugation lengths dissolved in different solvents have been shown to be ideal test systems for the various regulation mechanisms shown in Figure 1. In a study by Berera et al. using amide-linked dyads it was shown that even the addition of only one double bond can turn the carotenoid from a light harvester (Car S1 f Pc) into a very strong quencher (Pc f Car S1) in good agreement with mechanism (I), which has often been referred as the molecular gear shift mechanism.26 More recently, it was also discovered by the same group that spectroscopic results in phenylamino-linked dyad systems in toluene can so far only be explained by quenching of the excited state of Pc via excitonic Car S1Pc Qy interactions (mechanism II).27 The dyad system that has been proposed to demonstrate excitonic Car S1 T Pc Qy interactions shows immediate appearance of the transient absorption of the carotenoid S1 state upon chlorophyll excitation. This type of experiment only monitors the flow of energy in the direction Pc f Car S1. However, if indeed excitonic interactions occur between the two pigments, energy flow should be occurring, as mention above, in both directions (Pc f Car S1 as well as Car S1 f Pc) as both molecules share excitation energy.25 Therefore, to provide additional evidence for or against excitonic quenching in these dyad systems, we present here a 1Pc quantitative comparison of φCarS Coupling for a series of dyads. The CarS1Pc φCoupling value directly reflects the degree of energy flow in the CarS1Chl 1Pc direction Car S1fPc. (φCarS Coupling and φCoupling , see eq 1, represent basically the same kind of parameters but are named differently because in one case Pc is interacting with Car S1 whereas in the other case the Chl a is interacting with Car S1.) In addition, we have quantitatively compared these results to those for the major photosynthetic light-harvesting complex LHC II to search for further indications for the presence of one or more of the mechanisms (IIII) in this complex. Finally, we have also compared all samples to Chl a and Pc to quantify the relative contribution of selective Car S1 two-photon excitation and Chl/Pc two-photon excitation in such experiments. 1Pc Our results demonstrate that the determination of φCarS Coupling reflects sensitively the Car S1 f Pc energy flow. Depending on the sample it is found that two-photon excitation excites to about 80% to 100% exclusively the carotenoid state Car S1 and that only a small fraction of direct tetrapyrrole two-photon excitation occurs. In phenylamino-linked dyads in toluene very 4083

dx.doi.org/10.1021/jp1122486 |J. Phys. Chem. A 2011, 115, 4082–4091

The Journal of Physical Chemistry A strong Car S1 f Pc energy flow can be observed in addition to the previously reported energy flow in the direction Pc f Car S1.27 This observation of a quenching-correlated bidirectional energy flow Pc Qy T Car S1 provides strong evidence that these systems are very good models for studying excitonic Car S1 T Chl Qy interactions. The observed interactions resemble closely the quenching-correlated interactions observed for the major light-harvesting complex II (LHC II). Finally, we have also observed strong Car S1Pc Qy electronic interactions in those systems that have previously been shown to demonstrate electron transfer from Car S1 to Pc, indicating that excitonic Car S1 T Chl/Pc Qy interactions might be precursors for the formation of radical cations.

’ MATERIALS AND METHODS Sample Preparation. The synthesis of all molecules used in this study has been described previously (for structural formulas and corresponding excited state energies of the pigments used in this study see Figures 2 and 3).26,27 The amide Pc model compound (Pc1), amide-linked 9 double bond dyad (9DB), and amide-linked 11 double bond dyad (11 DB) were dissolved in tetrahydrofuran (THF). (See spectra in Figure 4.) In addition, similar dyads with slightly different phthalocyanines and aminolinkers were also used in this study. The corresponding phenylamino Pc model compound (Pc2), phenylamino-linked 8 double bond dyad (8DB), and phenylamino-linked 10 double bond dyad (10DB) were separately dissolved in two solvents with different polarities: THF and toluene. Chlorophyll a and chlorophyll b (Sigma) were dissolved in acetone. Native LHC II was isolated from spinach as previously described.28 A buffer solution of 50 mM Tris pH 7.5 0.3% NG (n-nonyl-β-Dglucopyranoside) was used to dilute the LHC II samples. All samples were adjusted to an absorbance of 0.33/mm at the peak of the Chl Qy or Pc Qy band except the amide-linked dyads, which were adjusted to 0.5/mm. Such absorbances turned out to be ideal to observe strong two-photon signals. Measurements using different concentrations confirmed that self-absorption ef-

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fects were negligible using these absorbances. Quenched LHC II was prepared by detergent removal with SM-2 absorbent (Bio-Rad). A range of quenched states was obtained by using different incubation times. Sample preparation was done under dim light, and samples were stored at 24 °C until measurement. The absorption and fluorescence spectra were recorded with a Lambda 25 UV/vis spectrometer from PerkinElmer and a Cary Eclipse fluorescence spectrophotometer from Varian, respectively, and cuvettes with an optical path length of 3 mm were used. Two-Photon Spectroscopy. A confocal setup that allowed the measurement of fluorescence observed after both twophoton excitation (TPE) and one-photon excitation (OPE) has been described previously in detail.5 Briefly, the 1188 nm idler beam from an optical parametric amplifier (OPA 9450, Coherent) was chosen for two-photon excitation. This twophoton wavelength corresponds approximately to the maximum of the two-photon absorption spectrum of the Car S1 for most carotenoids (see Figure 2). A 1100 nm long pass filter (FEL1100, Thorlabs) and a hot mirror (L46-386, Edmund Optics) were used to thoroughly reject the visible light of the signal beam to

Figure 3. Structural formulas of the compounds used in this study.

Figure 2. Energies of one- and two-photon excitation used in the present work along with the state energies of the carotenoids as well as Pc and Chl a. Please note that the state energies for carotenoids in 9DB, 11DB, 8DB, and 10DB are not exactly known. It is only known for sure that the Car S1 state of 9DB and 11DB must be higher and lower, respectively, than the Qy state of Pc1. As a representative example the two-photon spectrum of β-carotene (orange line) is also shown along with a one-photon spectrum of Chl a (green line) that resembles closely the two-photon spectrum of Chl a.29. 4084



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Figure 4. Absorption and fluorescence spectra of the Pc1 model compound (black line) and the amide-linked 9DB (red line) and 11DB (blue line) dyads in THF. Dashed lines are the corresponding fluorescence spectra.

prevent the occurrence of chlorophyll fluorescence due to direct one-photon excitation. The fluorescence sensitized by TPE (FTPE) was detected by an ultrafast photodiode (designed by Professor D. Schwarzer) which was connected to a lock-in amplifier synchronized with a mechanical chopper positioned in the idler beam path. The fluorescence signal was collected and recorded by a computer connected to the lock-in amplifier for further data evaluation. The one-photon excitation was carried out with a conventional PAM fluorometer (FMS1, Hansatech). The modulated beam with a wavelength of 594 nm was used for chlorophyll or Pc OPE. This wavelength is the standard excitation wavelength for Chl fluorescence measurements by PAM fluorometers. (For more details see refs 5 and 22) Also the fluorescence observed after one-photon excitation (FOPE) was detected by the PAM fluorometer. A control experiment was done to determine the contribution of fluorescence after twophoton excitation from pure 8 or 10 double bonds carotenoid model compounds. It clearly showed that no FTPE is observed from these compounds (data not shown).

’ RESULTS AND DISCUSSION Amide-Linked Dyads in Tetrahydrofuran. Further Evidence for the Molecular Gear Shift Mechanism in These Dyads. In Figure 6a the relative Pc fluorescence intensities

observed after direct Pc one-photon excitation of model Pc1 and the amide-linked 9 double bond (9DB), and 11 double bond (11DB) dyads in THF are also shown. The concentrations of all samples were adjusted to give essentially equal absorbance in the region of the Pc Qy peak (see Figure 4). The corresponding numerical values are shown in Table 1. As can be seen from Figure 6a and Table 1 the fluorescence quantum yield of the 11DB dyad is more than 17 times smaller than that of the model Pc1 whereas the 9DB dyad fluorescence yield is about 90% that of Pc1. This is expected because these compounds have been shown to demonstrate the molecular gear shift mechanism (I); the 11DB dyad fluorescence is significantly quenched due to effective Pc f Car S1 energy transfer. Figure 6b shows the Pc fluorescence intensities observed after Car S1 two-photon excitation, FlTPE. The 9DB dyad shows significantly more fluorescence intensity than the other two

Figure 5. Absorption and fluorescence spectra of (a) LHC II (magenta) and Chl a (olive), (b) the Pc2 model compound (black) and the phenylamino-linked 8DB (red) and 10DB (blue) dyads dissolved in toluene, and (c) same as (b) dissolved in THF. Solid lines are absorption spectra, whereas dashed lines are the corresponding fluorescence spectra. The maxima of the fluorescence intensities of Pc2 in toluene and THF were normalized to 1. The fluorescence intensities of all other samples (including LHC II and Chl a) were correspondingly scaled in order to be directly comparable with the fluorescence intensities of Pc2 in the corresponding solvents. The fluorescence spectrum of Chl a was recorded with excitation at 662 nm whereas the other samples were excited at 670 nm.

samples (model Pc1 and the 11DB dyad). This observation provides clear evidence that significant energy transfer in the 9DB dyad occurs in the direction Car S1 f Pc. Again, this result is in perfect agreement with the molecular gear shift mechanism (I). In the 9DB dyad effective two-photon excitation of Car S1 with subsequent energy transfer to Pc, Car S1 f Pc, occurs, giving rise to the high values for FlTPE. In contrast, FlTPE for the 11DB dyad is much smaller than FlTPE for 9DB because here almost no Car S1 f Pc energy transfer occurs. Instead, Pc f Car S1 energy transfer occurs. Finally, the two-photon sensitized Pc fluorescence observed with the pure Pc1 is also significantly smaller than FlTPE of 9DB, because an excitation Car S0 f Car S1 is by far more two-photon allowed than a direct two-photon excitation of tetrapyrroles. These results provide clear evidence 4085



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Figure 6. (a) Pc fluorescence after OPE and (b) after TPE of the Pc1 1-Pc model compound and amide-linked 9DB and 11DB dyads. φCarS Coupling OPE of 11DB is difficult to determine accurately because the F is too small. The Qy peaks were all normalized (see Figure 4).

Table 1. The Numerical Values of Pc Fluorescence after OPE and TPE of Pc1 and Amide-Linked 9DB and 11DB Dyads FlOPE/a.u.

FlTPE/a.u.

Pc1

1.000 ( 0.002

1.00 ( 0.08

9DB

0.908 ( 0.002

2.27 ( 0.11

11DB

0.041 ( 0.001

0.55 ( 0.06

that the observation of Pc fluorescence sensitized by selective two-photon excitation of Car S1 is a sensitive monitor of the extent of Car S1 f Pc energy flow and that direct Pc two-photon excitation occurs only to a minor extent. The data shown in Figure 6b provide clear evidence that two-photon excitation excites about 8090% exclusively the carotenoid S1 state, Car S1, and that only a small fraction of direct tetrapyrrole two-photon excitation occurs. (A detailed calculation for the specificity of exclusive Car S1 two-photon excitation relative to Pc or Chl twophoton excitation can be found in the Mathematical Appendix.) Phenylamino-Linked Dyads in Toluene and Comparison with Chl a and LHC II. Evidence for Quenching of the Excited State of Pc/Chl a by Excitonic CarotenoidTetrapyrrole Interactions. In Figure 7 the relative Pc fluorescence intensities observed after direct Pc one-photon excitation of the model Pc2 and phenylamino-linked 8 double bond (8DB) and 10 double bond (10DB) dyads in toluene are shown. In the same plot the fluorescence intensities observed with pure Chl a in acetone and LHC II in different quenched states in aqueous solution are also shown. Again, all samples had concentrations corresponding to essentially equal absorbance in the region of the Qy peak, allowing a direct comparison of their relative fluorescence intensities (see also Materials and Methods, Figure 5a,b, and the Mathematical Appendix). From Figure 7 it is obvious that even the 8DB carotenoid has a much more significant quenching effect on the Pc fluorescence than did the carotenoid of the amide-linked 9DB dyad described in the previous section and that the 10DB carotenoid has a very strong quenching effect on the Pc fluorescence. Chl a in acetone has a similar fluorescence intensity to that of a solution of the model Pc2 at the same absorbance. The Chl

Figure 7. Chlorophyll/Pc fluorescence intensity observed after OPE at 594 nm of Pc2, phenylamino-linked 8DB dyad (black), phenylaminolinked 10DB dyad (black), Chl a (red), and LHC II in various quenched states (blue). The three right-most LHC II samples have been treated by aggregation quenching so that their fluorescence was 3, 6, and 13 times reduced in comparison to unaggregated LHC II, respectively. In order to directly compare the Chl a fluorescence intensity from LHC II with the fluorescence intensity of pure Chl a the fluorescence intensities of LHC II were corrected by a factor that accounts for direct Chl b excitation and subsequent energy transfer to Chl a (for details see Mathematical Appendix).

1-Chl/Pc Figure 8. φCarS of Pc2, phenylamino-linked 8DB dyad, phenylaCoupling mino-linked 10DB dyad (black), Chl a (red), and LHC II in various quenched states (blue). The three right-most LHC II samples have been treated by aggregation quenching so that their fluorescence was 3, 6, and 13 times reduced in comparison to unaggregated LHC II, respectively.

fluorescence intensity of unquenched LHC II is already smaller than that of pure Chl a. Finally, aggregation-induced quenching leads to a significant reduction in the fluorescence intensities in LHC II, as has been reported previously.24,30 In Figure 8 the electronic Car S1f Pc and Car S1 f Chl CarS1-Chl 1-Pc energy flow, φCarS Coupling and φCoupling , determined from the Pc or Chl fluorescence intensities observed after direct Pc/Chl onephoton excitation, FOPE, and after selective Car two-photon excitation, FTPE, are shown for the same samples as in Figure 7. CarS1-Chl 1-Pc Both φCarS Coupling and φCoupling were calculated using eq 1. In the case of the Pc model compound and Chl a the small values 1-Chl/Pc reflect the contribution of direct Pc or Chl of φCarS Coupling 4086



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1 -Chl/Pc Table 2. The Numerical Values of FOPE and OCarS of Coupling Pc2, Phenylamino-Linked 8DB Dyad, Phenylamino-Linked 10DB Dyad, Chl a, and LHC II in Various Quenched States

FOPE/a.u.

1-Chl/Pc φCarS /a.u. Coupling

Pc2

1.000 ( 0.002

1.00 ( 0.03

8DB dyad

0.415 ( 0.002

14.05 ( 0.15

10DB dyad

0.058 ( 0.000

25.70 ( 0.78

Chl a

1.044 ( 0.004

2.64 ( 0.06

LHCIIa LHCII, ∼3 fluo. reductiona

0.630 ( 0.006 0.230 ( 0.010

5.64 ( 0.14 9.65 ( 0.79

LHCII, ∼6 fluo. Reductiona

0.101 ( 0.007

15.69 ( 1.58

LHCII, ∼13 fluo. reductiona

0.047 ( 0.008

25.65 ( 5.15

a

Corrected for contribution from Chl b excitation. The relative values 1-Chl/Pc for φCarS have been normalized so that the value for Pc2 is 1. Coupling

two-photon excitation to the observed signal. The most striking 1-Pc feature of the data is the very large φCarS Coupling in the phenylaminolinked dyads, especially, in the 10DB dyad. The value found for 1-Chl/Pc in this compound is among the largest that have so φCarS Coupling far been observed in carotenoidtetrapyrrole systems.5 Our findings are in very good agreement with the recent study27 in which it was found that there is an instantaneous (faster than 100 fs) population of Car S1 in these phenylaminolinked dyads in toluene when Pc is excited. Also in that study it was found that the instantaneous population of Car S1 is the highest in the 10DB dyad. This remarkable kinetic behavior and other findings led the authors to the conclusion that in these systems excitonic Car S1Pc Qy interactions are present. Theoretically, excitonic interactions between the optically forbidden Car S1 state and Chl/Pc pigment excited states are originating from the same electronic coupling that enables effective energy transfer Car S1 f Chl/Pc Qy, which has been experimentally observed in many studies.2426,3135 It only depends on the strength of the coupling and the energy difference between the states whether the energy is localized on a single pigment and hops to another pigment or whether the energy is excitonically shared by the pigments. The pumpprobe studies by Kennis and co-workers reflect energy flow in the direction Pc f Car S1.27 In turn, the electronic Car S1Pc Qy 1-Pc parameter, φCarS Coupling, investigated in the present study reflects energy transfer in the opposite direction, Car S1 f Pc. If only unidirectional energy transfer between the two pigments occurred, this would correspond to a simple energy transfer from one pigment with higher energy excited state to another with lower energy excited state. That is, either significant Pc f Car S1 or Car S1 f Pc ET would be observed, as it is in the molecular gear shift mechanism (Figure 1, mechanism I). But if energy transfer occurs in both directions simultaneously, as in the present case of phenylamino-linked dyads in toluene, this is a very strong indication for excitonic interactions in these systems as both molecules actually share excitation energy (Figure 1, mechanism II). 1-Pc The large difference in the φCarS Coupling values observed for the pure Pc and the two dyads again demonstrates the high sensi1-Pc tivity of φCarS Coupling for determining electronic PcCar S1 interactions and provides evidence for the high specificity of Car S1 excitation by two-photon excitation using the correct conditions. A larger contribution of direct two-photon excitation of Pc 1-Pc would result in much higher φCarS Coupling values also for the pure 1-Pc Pc model compound. On comparison of φCarS Coupling of Pc2

1-Pc Figure 9. Comparison φCarS Coupling of Pc2 and phenylamino-linked 8DB and 10DB dyads in toluene (red circles) and in THF (black squares).

and the 8DB dyad in toluene, it can be concluded that the Pc fluorescence observed after two-photon excitation is at least 1 (1.00/14.05) = 93% due to selective Car S1 excitation and subsequent energy transfer, Car S1 f Pc (for details see Mathematical Appendix). 1-Pc In Figure 8 the φCarS Coupling values determined for the phenylamino-linked dyads in toluene are also compared with the 1-Chl φCarS Coupling values observed with pure Chl a in acetone, as well as with the major light harvesting complex II of higher plants (LHC II) in various states of aggregation. Since these complexes show increasing quenching with increasing aggregation and show also other characteristic features of the down-regulation of photosynthesis in planta, they are regarded by several authors to be good model systems for important aspects of natural photosynthetic regulation.2,36 Indeed it was shown that in aggregated LHC II as well as in living plants the parameter 1-Chl φCarS Coupling , indicative for Car S1 f Chl energy transfer, increases with increasing quenching of the Chl fluorescence. As mentioned previously, this—among other experimental observations—was an important indication for the presence excitonic interactions as a potential mechanism of photosynthetic down-regulation. 1-Chl The comparison of the quenching correlation φCarS Coupling in LHC II CarS 1 -Pc with the quenching correlation φCoupling in the phenylaminolinked dyads shows that both phenomena have remarkable similarities. In summary, the phenylamino-linked dyads in toluene are very good model compounds for the investigation of quenching of the fluorescence of tetrapyrroles by excitonic interactions and provide clear evidence that such interactions can actually occur between tetrapyrroles and the optically forbidden first excited state of carotenoids, Car S1. Phenylamino-Linked Dyads in Tetrahydrofuran. Are Excitonic CarotenoidTetrapyrrole States Precursors to CarotenoidTetrapyrrole Charge Transfer States? Another mechanism that has been reported to play a role in the regulation of light harvesting in photosynthesis is electron transfer from carotenoids to chlorophyll molecules. In the recent study by Kennis and co-workers27 it was reported that clear spectroscopic signatures of radical species indicating photoinduced electron transfer were present when the phenylamino-linked dyads were investigated in the relatively polar solvent tetrahydrofuran. For these dyads, also indication of instantaneous Car S1 population after direct Pc excitation was found, suggesting excitonic Car 4087



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1 -Pc Table 3. The Numerical Values of OCarS Coupling of Pc2 and Phenylamino-Linked 8DB and 10DB Dyads. a 1-Pc φCarS Coupling/a.u.

tetrahydrofuran

toluene

1.00 ( 0.03

1.00 ( 0.03

8DB dyad

12.93 ( 2.17

14.05 ( 0.15

10DB dyad

10.17 ( 2.08

25.70 ( 0.78

Pc2

1-Pc The relative values for φCarS Coupling have been normalized so that the value for Pc2 is 1.

a

S1Pc Qy interactions also in THF. We thus also measured 1-Pc φCarS Coupling for the phenylamino-linked dyads in THF and compared the results with the results obtained for the same compounds in toluene. As can be deduced from Figure 9, the 8DB and 10DB dyads show significant Pc fluorescence after selective Car S1 excitation, although the phenylamino-linked 10DB dyad has significantly less Car S1Pc coupling in THF than in toluene. However, the fact that in the study of Kennis and co-workers27 energy transfer in the opposite direction (from Pc to Car) was observed for both compounds in the same solvent, coupled with 1-Pc the observation of quite large φCarS Coupling values in the present study indicates a significant contribution of excitonic interactions in these compounds also in THF. As mentioned above, in the study by Kennis and co-workers27 also clear evidence for CarPc charge separation was found for these dyads in THF. Since both excitonic interactions and electron transfer have also been reported in plants and light-harvesting complexes, it seems that such charge separation is often associated with the presence of excitonic interactions between these two pigments. This finding supports the hypothesis that mixed or in other words excitonic CarChl states might be direct precursors to CarChl charge separation, as has been suggested by previous studies.6,9

’ CONCLUSION The present study confirms the observation that depending on the carotenoid conjugation length, linkage structure, and solvent environment all electronic CarChl interactions that have been proposed as regulation mechanisms for light harvesting in photosynthesis (Figure 1) can be observed and investigated with carotenoidphthalocyanine dyads. The amide-linked dyads in THF display the so-called molecular gear shift mechanism (Figure 1, mechanism I).3 Effective Car S1 f Pc energy transfer was observed in the 9DB dyad whereas the Pc fluorescence in the 11DB dyad was strongly quenched. So the 9DB dyad resembles the situation of a dark-adapted plant in which the excitation energy collected by the carotenoids is effectively transferred to the chlorophylls and from there to the photosynthetic reaction center. The 11DB dyad, in contrast, resembles the situation of a light-adapted plant in which the excess excitation energy collected by the chlorophylls is effectively transferred to the carotenoids and dissipated from Car S1 within a few picoseconds as heat by internal conversion Car S1 f Car S0. In phenylamino-linked dyads in toluene extremely large electronic Car S1Pc interactions are observed, particularly in the case of the 10DB dyad. The electronic Car S1Pc coupling 1-Pc value, φCarS Coupling , found for this compound is among the largest that has so far been observed in carotenoidtetrapyrrole systems.5 This finding, together with the previous findings of Kennis and co-workers27 that in the same system effective,

instantaneous Pc f Car S1 energy transfer exists, provides good evidence for the presence of quenching excitonic Car S1 T Pc Q y interactions. If energy transfer occurs in both directions simultaneously, as in the present case of phenylamino-linked dyads in toluene, this can only be explained by excitonic interactions as both molecules actually share excitation energy in such a situation (Figure 1, mechanism II). The findings with these compounds thus provide clear evidence that such interactions can actually occur between tetrapyrrole excited states and the optically forbidden first excited state of carotenoids, Car S1. In addition, these compounds are well suited as model systems for investigating such interactions and their role in photosynthetic light harvesting. In particular the 10DB dyad resembles the situation of a light-adapted plant in which the excess excitation energy is effectively trapped from the entire pigment pool by the lower Car S1Chl Qy excitonic state and then dissipated in a short time. This short-time dissipation occurs because the lifetime of the excitonic state is greatly reduced in comparison to pure chlorophylls due to the contribution of the short living Car S1 state (Figure 1, mechanism II).4,5 In the phenylamino-linked dyads in THF indications for excitonic Car S1Pc Qy interactions were also found. The fact that in this system as well as in the photosynthetic apparatus Car S1Chl/Pc charge separations were found simultaneously with excitonic Car S1Chl/Pc Qy interactions suggests that excitonic CarChl/Pc interactions might be direct precursors to CarChl/Pc charge separation, as has been suggested in previous studies.6,9 This model system resembles the situation of a light-adapted plant in which the excess excitation energy is first effectively trapped from the entire pigment pool by the lower Car S1Chl Qy excitonic state and then dissipated by a charge separation between the carotenoids and chlorophylls as proposed by Fleming and co-workers (Figure 1, mechanism III).9 So in summary, our study provides evidence that the investigated dyad systems in different environments establish the feasibility of each of these proposed regulatory mechanisms and can serve as model systems to investigate these mechanisms in detail in pure, well-defined 1:1 tetrapyrrole to carotenoid ratio compounds. Another important aspect of our results is that they provide clear evidence that the observed Chl/Pc fluorescence after selective two-photon excitation of the Car S1 state is a sensitive monitor the flow of energy between Car S1 and Pc or Chl in different samples and that direct Pc or Chl two-photon excitation contributes minimally to the observed signals. It is found that two-photon excitation populates about 8090% exclusively the carotenoid S1 state under the chosen conditions and that only a small fraction of direct Pc or Chl two-photon excitation occurs.

’ MATHEMATICAL APPENDIX Because in this study much attention was paid to the observation of quantitative fluorescence intensities from the various samples under strictly comparable conditions, quantitative information on the specificity of Car S1 two-photon excitation can be obtained. Some algebraic operations that have been used in the calculations are discussed in detail below. Correcting for the Contribution of Chl b Excitation in FOPE of LHC II Samples. As already mentioned, the concentrations of

most samples were adjusted to have an absorbance of 0.33/mm at the peak of the Chl a or Pc Qy band. This was done to allow direct comparison of the relative fluorescence intensities FOPE 4088



Figure 10. Fit of absorption spectra of pure Chl a (red line) and Chl b (blue line) to the absorption spectrum of LHC II (black line). Dark cyan line is the sum of the spectra of Chl a and Chl b. The relative absorbances observed for Chl a and Chl b at 594 nm in the fit were used for the correction of the portion of the LHC II FOPE values that are due to the contribution of Chl b excitation and energy transfer to Chl a.

and FTPE after excitation of samples having the same amount of Chl a or of Pc/Car in the various samples. However, the onephoton sensitized fluorescence intensity, FOPE, observed with unquenched LHC II is then higher than that of pure Chl a because of the additional presence of Chl b (Chl b is also excited by the one-photon wavelength of 594 nm and then transfers almost all of its excitation energy to Chl a). Therefore the FOPE of LHC II was corrected to account for this additional Chl b contribution. Assuming that energy transfer from Chl b to Chl a is 100% efficient, the measured Chl a fluorescence is a factor (AChlb(594 nm) þ AChla(594 nm))/ AChla(594 nm) = 2.24 too high. The relative absorbances AChlb(594 nm) and AChla(594 nm) were derived from fitting the sum of the absorption spectra of pure Chl a and Chl b, varying their spectral shift and amplitude, to the absorption spectrum of LHC II (Figure 10). For the correction of FOPE, the OPE fluorescence intensities measured with LHC II were divided by this factor of 2.24. In addition, the fluorescence intensities were also corrected for the overestimation due to the red shift of LHC II fluorescence spectrum compared with Chl a. (Figure 5a) The LHC II spectrum was blue-shifted 13 nm to coincide with Chl a. It is found that FOPE was overestimated 41% after we compared the area above 700 nm (a 700 nm long pass filter was used for the fluorescence detection) in the original spectrum to the shifted one. The resulting relative fluorescence intensities of Chl a and unquenched LHC II as well as the Pc2 model compound (Figure 7) are similar, even though the Chl fluorescence in LHC II is already significantly quenched in comparison to pure Chl a. CarS1-Chl 1-Pc For a relative comparison of φCarS Coupling and φCoupling of all comOPE values were also used for the LHC II pounds the corrected F samples. It is important to note, however, that FOPE as well CarS1-Chl 1-Pc as φCarS Coupling and φCoupling can only be exactly compared for samples that are containing either only Pc or only Chl a because Pc and Chl a have different transition dipole moments and thus fluorescence intensities. Determination of the Specificity of the Selective Car S1 Two-Photon Excitation of the Amide-Linked Dyads in THF from the Observed Fluorescence Intensities. One attractive feature of the Pc-Car dyads is that they represent in contrast to

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photosynthetic pigment protein complexes very well-defined 1:1 tetrapyrrole to carotenoid ratio systems that allow a quantitative determination of the extent of the various interactions between these two pigments. So they are also an ideal test system to quantify the extent of selective Car two-photon excitation versus direct tetrapyrrole two-photon excitation for specific wavelengths and distinct dye systems. In the following, we will derive from the experimentally observed data the actual fraction of selective Car S1 and direct Pc two-photon excitation in 9DB taking into account the limited 1-Pc energy transfer from Car S1 to Pc, φCarS Coupling , and the smaller relative fluorescence quantum efficiency in the amide-linked 9DB dyad, φrel Fl (9DB), in comparison to the pure Pc model compound. According to the relative fluorescence intensity observed from the 9DB dyad and the Pc model compound after one-photon excitation (see Figure 6a and Table 1) the relative fluorescence quantum yield of Pc is somewhat reduced in the 9DB dyad OPE ð9DBÞ=FlOPE ðPc1Þ ¼ 91% φrel Fl ð9DBÞ ¼ Fl

ð2Þ

Therefore, the Pc fluorescence that would be expected to be observed from direct Pc two-photon excitation in the 9DB dyad in the absence of any Car S1 two-photon excitation or Car S1 f Pc energy transfer would be reduced by the same factor in comparison to the direct two-photon excited fluorescence observed with the pure Pc model, FlTPE (Pc1) TPE FlTPE ðPc1Þφrel nocarS1 f PcET ð9DBÞ ¼ Fl Fl ð9DBÞ ¼ 0:91 ð3Þ

The actual experimentally observed value FlTPE(9DB) (Figure 6b) is of course significantly larger because in addition there is effective two-photon excitation of Car S1 state and subsequent energy transfer to Pc (with the energy transfer 37 1 -Pc Therefore the actually observed total efficiency φCarS ET-Efficiency. fluorescence of the 9DB dyad, FlTPE(9DB), is the fluorescence intensity that is expected in the absence of any Car S1 f Pc energy transfer, FlTPE TPEfPcET(9DB), plus the actual contribution by additional Car S1 excitation and energy transfer to the Pc TPE CarS1 Pc FlTPE ð9DBÞ ¼ FlTPE nocarS1 f PcET ð9DBÞð1 þ nCarS1 φET-Efficiency Þ

¼ 2:27 ð4Þ nTPE CarS1,

Here, is the actual number of selectively two-photon excited carotenoid molecules per directly two-photon excited Pc or in other words the factor by which it is more likely that carotenoid molecules are excited than tetrapyrrole molecules 1-Pc for certain two-photon excitation conditions. φCarS ET-Efficiency is the already mentioned quantum efficiency for the energy transfer to a Pc molecule after excitation of a Car S1 state, Car S1 f Pc. CarS1-Pc Equation 4 basically tells that if (1þ nTPE CarS1φET-Efficiency ) more Pc molecules are excited by direct Pc excitation and Car S1 f Pc energy transfer than only by direct Pc two-photon excitation the observed Pc fluorescence will be exactly by that factor larger than FlTPE noCarS1fPcET(9DB). As a consequence the factor nTPE CarS1 can be calculated from the measured values by using eqs 3 and 4 CarCarS1 -Pc TPE nTPE ð9DBÞ=ðFlTPE ðPc1Þφrel CarS1 ¼ ðFl Fl ð9DBÞÞ 1Þ=φET-Efficiency

ð5Þ The actual fractions or percentages of Car and Pc molecules, xCarS1 and xpc, that are excited by two-photon excitation under 4089



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the chosen conditions are then TPE xCarS1 ¼ nTPE CarS1 =ðnCarS1 þ 1Þ

1 -Pc φCarS Coupling ðDyadÞ ¼

and

xPc ¼ 1=ðnTPE CarS1 þ 1Þ

Dyad

ð6Þ

¼

The quantum efficiency for energy transfer can be calculated from the rate of energy transfer, kET, and the lifetime of Car S1, τCarS1, in the presence of energy transfer 1 -Pc φCarS ET-Efficiency ¼ kET τCarS1

Pc FlTPE ðPc2Þ nTPE nTPE Pc φFl Pc 1 -Pc ¼ φCarS ðPc2Þ ¼ ¼ Coupling Pc nOPE nOPE FlOPE ðPc2Þ Pc Pc φFl

1 nOPE Pc

¼

ð7Þ

The maximum extent of the Car S1 f Pc energy transfer in the 9DB dyad can be estimated from the energy transfer in the opposite direction (Pc f Car S1) which has been observed in these compounds not to be faster than kET = (17 ps)1 even in the very quenched 11 DB dyad (see rate k1 in Table 1 of Berera et al.26). Since there is in principle no difference in the electronic Car S1 T Pc couplings that govern the Pc f Car S1 energy transfer in the 11DB dyad and the corresponding couplings that govern the Car S1 f Pc energy transfer in the 9DB dyad it can be well assumed that also the latter energy transfer has a rate that is on the order and not significant higher than kET = (17 ps)1.38 In the study by Berera et al. it was also concluded that the Car S1 lifetime of the 10 and 11 DB carotenoids in the dyads in THF is as short as τCarS1 = 44.8 ps even in the absence of Car S1 f Pc energy transfer (see rate k2 in Table 1 of Berera et al.26). If we assume that the 9DB carotenoid has a similar lifetime, this would result in a Car S1 f Pc energy transfer 1 1-Pc efficiency of φCarS ET-Efficiency = kETτCarS1 = (17 ps) 3 5 ps ∼ 30%. Even if we assume a significantly larger lifetime for the 9DB carotenoid of τCarS1 = 10 ps this would certainly not result in a Car S1 f Pc energy transfer efficiency of more than 1 1-Pc φCarS ET-Efficiency = kETτCarS1 = (17 ps) 3 10 ps ∼ 60%. CarS1-Pc So finally, using for φET-Efficiency this quite large range of values from 0.3 to 0.6 and using eq 5 and eq 6 to calculate the specificity of selective two-photon Car S1 excitation values of at least xCarS1 = 72% to xCarS1 = 83% are observed. A specificity of 7080% is well suited to compare relative electronic interactions in different tetrapyrrolecarotenoid system and pigmentprotein complexes by calculating 1 -Chl/Pc µ FlTPE/FlOPE. A smaller fraction of direct Chl/ φCarS Coupling Pc two-photon excitation will not affect a relative comparison 1-Chl/Pc values from different samples since this of φCarS Coupling 1-Chl/Pc that is the same as long will only add an offset to φCarS Coupling as the same tetrapyrrole compound and concentration are present. 1 -Chl/Pc Calculating the Specificities from OCarS . In a Coupling similar manner as in the previous section the specificity of the selective Car S1 two-photon excitation can also be deduced CarS1-Chl 1-Pc from the comparison of φCarS Coupling or φCoupling observed with CarS1-Pc CarS1-Chl pure Pc or Chl with φCoupling or φCoupling of the dyads or LHC II. CarS1-Pc 1-Pc φCarS Coupling(Pc2) and φCoupling(Dyad) observed with the pure Pc2 compound or a dyad are (see also eq 1)

ð8Þ

FlTPE ðDyadÞ FlOPE ðDyadÞ

nTPE Pc φFl

Dyad

CarS1 -Pc þ nTPE CarS1 φET-Efficiency 3 φFl Dyad

nOPE Pc φFl

TPE CarS1 Pc nTPE Pc þ nCarS1 φET-Efficiency

nOPE Pc CarS1 -Pc 1 þ nTPE CarS1 φET-Efficiency

nOPE Pc

ð9Þ

TPE TPE Here, nOPE Pc , nPc and nCarS1, are the numbers of Pc or Car molecules excited under the chosen conditions by one-photon Dyad are the fluoresand two-photon excitation and φPc Fl and φFl cence quantum yields of Pc and the dyad, respectively (For a 1-Pc detailed derivation of φCarS Coupling see also the Supporting Information to ref 5) Here we assume that the probability of exciting Pc by one- or two-photon excitation is the same for the pure Pc as well as in the dyad. In both cases the fluorescence quantum Dyad yields, φPc Fl and φFl , cancel out, which is a fundamental principle of the measurements method. nTPE Pc can be replaced by 1 since TPE was defined in the section above to be the number of nCarS 1 selectively two-photon excited carotenoid molecules per single two-photon excited Pc. Also, one-photon excitation of Car S1 was excluded since there is virtually no Car absorption at 594 nm. CarS1-Pc 1-Pc In the ratio R of φCarS Coupling(Pc2) and φCoupling(Dyad) also OPE nPc cancels out

R ¼

1 -Pc φCarS Coupling ðDyadÞ 1 -Pc φCarS Coupling ðPc2Þ

CarS1 -Pc ¼ nTPE CarS1 φET-Efficiency þ 1

ð10Þ

So this ratio provides a direct link to the number of selectively two-photon excited carotenoid molecules per two-photon excited Pc. According to eq 6 the actual fractions or percentages of Car molecules, xCarS1, that are excited by two-photon excitation under the chosen conditions is then

TPE x CarS1 ¼ nTPE CarS1 =ðnCarS1

R1 1 -Pc φCarS ET-Efficiency þ 1Þ ¼ R1 þ1 1 -Pc φCarS ET-Efficiency

ð11Þ

It is obvious that the calculated xCarS1 becomes larger if the 1-Pc parameter φCarS ET-Efficiency decreases in eq 11. Therefore, the specificity of the selective Car S1 two-photon excitation is not smaller than a situation in which we assume an energy transfer efficiency 1-Pc of 100% (φCarS ET-Efficiency = 1) x CarS1 g

R1 R1 1 ¼ ¼ 1 R1þ1 R R

ð12Þ

1-Pc Since the value of φCarS ET-Efficiency (Pc2) is normalized to 1 in Tables 2 and 3, the specificity can be directly calculated from

x CarS1 g 1

1 1 -Pc φCarS Coupling ðDyadÞ

ð13Þ

1-Pc Therefore the comparison φCarS Coupling for the dyads in toluene as CarS1-Pc well as THF with the φCoupling values for pure Pc2 demonstrate that direct two-photon excitation of Pc is almost negligible in 1-Pc these cases. For example, by comparing φCarS Coupling of Pc2 and the

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The Journal of Physical Chemistry A 8DB dyad in THF (Table 3) a specificity for exclusive Car S1 excitation of at least xCarS1 g 1 (1/12.93) = 92% can be derived. 1-Chl Also from a comparison of φCarS Coupling observed for Chl a and unquenched LHC II it can be deduced that only a minor fraction of direct Chl two-photon excitation occurs in LHC II. Using eq 11 and assuming a Car S1 to Chl energy transfer efficiency of 3060% in unquenched LHC II18 a specificity of selective twophoton Car S1 excitation of about xCarS1 ∼ 7080% in unquenched LHC II can be estimated. These values are in good agreement with previous estimates.18 As can also be deduced from the data in Table 2, in the most quenched LHC II sample the contribution of Car S1Chl interactions to the observed signals is even higher than 93%.

’ ACKNOWLEDGMENT We thank W. K€uhlbrandt and L. Wilk for providing the LHC II samples. This work was supported by the Fonds der Chemischen Industrie and the Deutsche Forschungsgemeinschaft (DFG). This work was supported by a grant from the U.S. Department of Energy (DE-FG02-03ER15393). D.G. and A.M. were supported as part of the Center for Bio-Inspired Solar Fuel Production, an Energy Frontier Research Center funded by the U.S. Department of Energy, Office of Science, Office of Basic Energy Sciences under Award Number DE-SC0001016. ’ REFERENCES (1) Demmig-Adams, B.; Adams, W. W. Science 2002, 298, 2149. (2) Ruban, A. V.; Berera, R.; Ilioaia, C.; van Stokkum, I. H. M.; Kennis, J. T. M.; Pascal, A. A.; van Amerongen, H.; Horton, P.; van Grondelle, R. Nature 2007, 450, 575. (3) Frank, H. A.; Cua, A.; Chynwat, V.; Young, A.; Gosztola, D.; Wasielewski, M. R. Photosynth. Res. 1994, 41, 389. (4) van Amerongen, H.; van Grondelle, R. J. Phys. Chem. B 2000, 105, 604. (5) Bode, S.; Quentmeier, C. C.; Liao, P.-N.; Hafi, N.; Barros, T.; Wilk, L.; Bittner, F.; Walla, P. J. Proc. Natl. Acad. Sci. U.S.A. 2009, 106, 12311. (6) Ma, Y. Z.; Holt, N. E.; Li, X. P.; Niyogi, K. K.; Fleming, G. R. Proc. Natl. Acad. Sci. U.S.A. 2003, 100, 4377. (7) Avenson, T. J.; Ahn, T. K.; Zigmantas, D.; Niyogi, K. K.; Li, Z.; Ballottari, M.; Bassi, R.; Fleming, G. R. J. Biol. Chem. 2008, 283, 3550. (8) Ahn, T. K.; Avenson, T. J.; Ballottari, M.; Cheng, Y.-C.; Niyogi, K. K.; Bassi, R.; Fleming, G. R. Science 2008, 320, 794. (9) Holt, N. E.; Zigmantas, D.; Valkunas, L.; Li, X.-P.; Niyogi, K. K.; Fleming, G. R. Science 2005, 307, 433. (10) Moya, I.; Silvestri, M.; Vallon, O.; Cinque, G.; Bassi, R. Biochemistry 2001, 40, 12552. (11) Pascal, A. A.; Liu, Z.; Broess, K.; Oort, B. v.; Amerongen, H. v.; Wang, C.; Horton, P.; Robert, B.; Chang, W.; Ruban, A. Nature 2005, 436, 134. (12) Ilioaia, C.; Johnson, M. P.; Horton, P.; Ruban, A. V. J. Biol. Chem. 2008, 283, 29505. (13) Barros, T.; Royant, A.; Standfuss, J.; Dreuw, A.; K€uhlbrandt, W. EMBO J. 2009, 28, 298. (14) Li, X. P.; Bj€orkman, O.; Shih, C.; Grossman, A. R.; Rosenquist, M.; Jansson, S.; Niyogi, K. K. Nature 2000, 403, 391–395. (15) Frank, H. A.; Bautista, J. A.; Josue, J. S.; Young, A. J. Biochemistry 2000, 39, 2831. (16) Standfuss, J.; van, A. C. T.; Scheltinga; Lamborghini, M.; K€uhlbrandt, W. EMBO J. 2005, 24, 919. (17) M€uller, P.; Li, X.-P.; Niyogi, K. K. Plant Physiol. 2001, 125, 1558.

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(18) Walla, P. J.; Yom, J.; Krueger, B. P.; Fleming, G. R. J. Phys. Chem. B 2000, 104, 4799. (19) Walla, P. J.; Linden, P. A.; Hsu, C. P.; Scholes, G. D.; Fleming, G. R. Proc. Natl. Acad. Sci. U.S.A. 2000, 97, 10808. (20) Walla, P. J.; Linden, P. A.; Ohta, K.; Fleming, G. R. J. Phys. Chem. A 2002, 106, 1909. (21) Wehling, A.; Walla, P. J. J. Phys Chem. B 2005, 109, 24510. (22) Wehling, A.; Walla, P. J. Photosynth. Res. 2006, 90, 101. (23) Bode, S.; Quentmeier, C. C.; Liao, P.-N.; Barros, T.; Walla, P. J. Chem. Phys. Lett. 2008, 450, 379. (24) Liao, P.-N.; Bode, S.; Wilk, L.; Hafi, N.; Walla, P. J. Chem. Phys. 2010, 373, 50. (25) Liao, P.-N.; Holleboom, C.-P.; Wilk, L.; K€uhlbrandt, W.; Walla, P. J. J. Phys. Chem. B 2010, 114, 15650. (26) Berera, R.; Herrero, C.; van Stokkum, I. H. M.; Vengris, M.; Kodis, G.; Palacios, R., E.; van Amerogen, H.; van Grondelle, R.; Gust, D.; Moore, T. A.; Moore, A. L.; Kennis, J. T. M. Proc. Natl. Acad. Sci. U.S. A. 2006, 103, 5343. (27) Kloz, M.; Pillai, S.; Kodis, G.; Gust, D.; Moore, T. A.; Moore, A. L.,; van Grondelle, R.; Kennis, J. T. M. Submitted for publication. (28) K€uhlbrandt, W.; Thaler, T.; Wehrli, E. J. Cell. Biol. 1983, 96, 1414. (29) Shreve, A. P.; Trautman, J. K.; Owens, T. G.; Albrecht, A. C. Chem. Phys. Lett. 1990, 170, 51. (30) van Oort, B.; van Hoek, A.; Ruban, A. V.; van Amerongen, H. FEBS lett. 2007, 581, 3528. (31) Bautista, J. A.; Hiller, R. G.; Sharples, F. P.; Gosztola, D.; Wasielewski, M.; Frank, H. A. J. Phys. Chem. A 1999, 103, 2267. (32) Andersson, P. O.; Cogdell, R. J.; Gillbro, T. Chem. Phys. 1996, 210, 195. (33) Angerhofer, A.; Cogdell, R. J.; Hipkins, M. F. Biochim. Biophys. Acta 1986, 848, 333. (34) Frank, H. A.; Cogdell, R. J. The Photochemistry and Function of Carotenoids in Photosynthesis. In Carotenoids in Photosynthesis; Young, A., Britton, G., Eds.; Chapman and Hall: London, 1993; p 252. (35) Frank, H. A.; Cogdell, R. J. Photochem. Photobiol. 1996, 63, 257. (36) Johnson, M. P.; Ruban, A. V. J. Biol. Chem. 2010, 284, 23592. (37) Please note that here the absolute transfer efficiency has to be CarS1-Pc 1-Pc used, i.e., φCarS ET-Efficiency = 1 for 100% Car S1 f Pc ET and φET-Efficiency = 0 1 -Chl/Pc for no Car S1 f Pc ET. The couplings or efficiencies, φCarS , given Coupling in Figures 8 and 9 as well as in Tables 2 and 3 are only relative values for the Car S1 f Pc energy flow that are experimentally determined and that 1 -Pc are given in arbitrary units. However, both efficiencies, φCarS Coupling 1-Chl/Pc and φCarS , are in principle proportional to each other. Coupling (38) It is reasonable that in these dyad systems the electronic coupling and thus energy transfer rate between the Car S1 state and the Pc is significantly smaller than those between the Car S1 states and (B)Chls in photosynthetic pigment protein complexes where energy transfer rates up to kET ∼ (1 ps)1 have been observed. This is because in the photosynthetic pigment protein complexes the pigments are actually in van der Waals contact whereas the model compounds are still separated by a small linker.

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Two-Photon Study on the Electronic Interactions between the First

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