J. Phys. Chem. B 2001, 105, 8607-8615
8607
Optical Dephasing in the Light-Harvesting Complex II: A Two-Pulse Photon Echo Study F. Hillmann,*,† J. Voigt,† H. Redlin,‡ K.-D. Irrgang,§ and G. Renger§ Humboldt-UniVersity of Berlin, Institute of Physics, InValidenstrasse 110, D-10115 Berlin, Germany, Max-Born-Institute for Nonlinear Optics and Short Pulse Spectroscopy, Berlin, Germany, and Max-Volmer-Institute for Biophysical Chemistry and Biochemistry, Technical UniVersity of Berlin, Berlin, Germany ReceiVed: March 23, 2001; In Final Form: June 25, 2001
Time-resolved and integrated two-pulse photon echo signals were measured at 5 K in the wavelength range from 640 to 690 nm (with an increment of 5 nm) in samples of solubilized light-harvesting complex II (LHC II) from spinach. Furthermore, the dephasing time was determined at 680 nm in the temperature range 5 e T e 300 K. The following results and conclusions were gathered from these experiments: (a) the photon echo intensity as a function of the temporal distance between both excitation pulses exhibits a nonexponential decay; (b) data analysis on the basis of a superposition of several dephasing processes leads to three characteristic dephasing time domains (A, B, and C) with markedly different wavelength dependencies of dephasing time and relative amplitude, T2A e 1.7 ps from 640 to 675 nm, T2B ) 4-13 ps over the whole wavelength region of 640 to 690 nm, and T2C g 40 ps from 675 to 685 nm; (c) the dephasing times T2A and T2B are attributed to the kinetics of excitation energy transfer and to multiexciton processes above 670 nm, and (d) the longest dephasing time T2C reaches values up to 350 ps at 684 nm. The temperature dependence of the dephasing rate (T2C)-1 at 680 nm is characterized by two different domains: below 20 K the rate steeply decreases due to pure dephasing, similar to the well-known T1.3 dependence of the homogeneous line widths at low temperatures in organic glasses. At temperatures above 20 K a linear increase of (T2C)-1 with temperature and a comparatively weak slope are observed, ascribed to uphill energy transfer. The results obtained in this study are discussed in comparison with data in the literature that were gathered from hole burning, transient absorption measurements, and spectra simulations.
1. Introduction The very first event of photosynthesis is light absorption of pigments that form regular arrays by incorporation into a protein matrix1 or in case of chlorosomes in green sulfur bacteria by selfaggregation of bacteriochlorophyll c (d or e) molecules.2 Different types of pigment protein complexes either act as antenna systems for light harvesting only or built up reaction centers (RC) that also contain the photoactive pigment PRC for photochemical charge separation (for reviews see, e.g., Renger1,3 and van Grondelle et al.4). The main functions of antenna systems can be characterized by three physically different processes: (i) the absorption of solar energy by the pigments, (ii) the spatial energy transfer from the absorbing pigments to the RC, and (iii) the energy relaxation from the level of the primary excited states to the energetically lower level of the RC. Describing the energy states of the interacting pigments by exciton states, i.e., linear combinations of molecular states of the pigments, the processes ii and iii are strongly related. Each relaxation process within the exciton manifolds proceeds via a spatial energy transfer between the pigment molecules constituting the exciton states. Therefore, the processes ii and iii are usually summarized by the term excitation energy transfer (EET) which includes both processes. All adaptation of photosynthetic organisms to different illumination conditions occurs * Corresponding author. E-mail:
[email protected]. Fax: (+49) 30 2093 7659. † Humboldt-University. ‡ Max-Born-Institute. § Max-Volmer-Institute.
at the level of EET. These processes permit both (i) highly efficient funneling to PRC at low light intensities and (ii) dissipation of superfluous excitation by transfer to nonphotochemical traps. Accordingly, a great variety of different types of antenna systems has been developed during evolution whereas on the other hand only few types of RC exist as “standard blocks” of the photosynthetic apparatus. The mechanism of EET depends on the nature of the pigments, their mutual orientation in the ensemble, and the coupling to the protein environment. Therefore, a thorough, theoretical description of these processes requires detailed knowledge on spatial pigment arrangement, strength of electronic pigment-pigment interaction and the mode and strength of electron-phonon coupling to the matrix. Accordingly, detailed structural information is an indispensable prerequisite to understand EET mechanism(s) in photosynthetic and biomimetic antenna systems. Highly resolved X-ray structures are known for light harvesting complex LH2 from anoxygenic purple bacteria.5,6 On the basis of these data, an effective Frenkel exciton Hamiltonian was formulated and, in combination with a collective electronic oscillator (CEO) algorithm, the electronic coupling between all pigments of LH2 was calculated. As a result, the Coulomb (Fo¨rster type) and exchange (Dexter) contributions were obtained. The latter was found to be significant only in the B850 ring (10-15% between next neighbors).7 Compared with LH2 less detailed structural information is available for the plant antenna system. In green plants the antenna system is built up by different integral pigment proteins with light harvesting complex II (LHC IIb, in the following
10.1021/jp011107r CCC: $20.00 © 2001 American Chemical Society Published on Web 08/18/2001
8608 J. Phys. Chem. B, Vol. 105, No. 36, 2001 referred as LHC II) as the most abundant constituent that binds about 50% of the total chlorophyll (Chl) content of the thylakoid membrane. In solubilized form, LHC II normally attains a trimeric state which is assumed to be also the predominant form in the native thylakoid membrane.8 This LHC II together with minor pigment protein complexes (each containing no more than 6% of the total Chl) constitutes the peripheral and proximal antenna of photosystem II in green plants.9-12 The composition of a monomeric LHC II subunit with seven Chl a, five Chl b, and at least three carotenoids (Car) has been determined to nearly atomic resolution (3.4 Å) by electron diffraction of twodimensional crystals.13 The structure of ref 13 shows only two Car; the site(s) of other Car are not resolved. An inspection of the results readily shows that the mode of pigment array markedly differs from that of LH2. Unfortunately, the LHC II structure is not refined enough to permit an unambiguous pigment assignment of Chl a and Chl b. An attempt to identify Chl a and Chl b on the basis of functional properties led to a model13 that was later questioned in some details.14,15 Nevertheless, the identities of five Chl molecules (Chl a1, a2, a3, b5, and b6) could be confirmed by spectroscopic data gathered from LHC II mutants whereas Chl b3 was found to be a Chl a.16 Different approaches can be used to analyze structurefunction relationships in pigment protein complexes. The most widely performed studies are time-resolved spectroscopic measurements, especially transient absorption (TA) experiments. As a result of these investigations consensus exists that ultrafast EET takes place from Chl b to Chl a with kinetics of ∼150 and 600 fs and 1-10 ps, while EET between Chl a molecules occurs on a time scale of a few ps within LHC II.15,17-23 Excited singlet states of Car were shown to be transferred extremely fast to Chl a and Chl b within the range of 50-100 fs.24 Likewise, the transfer of Chl triplets to carotenoids as an essential protective mechanism to photodynamic destruction by suppressing singlet oxygen formation25 was recently shown to take place in less than 1 ns and thus being much faster than data reported before.26 The mechanism of the EET processes is not fully understood because the distances between certain Chl molecules within LCH II are rather short so that effects owing to excitonic interaction cannot be ignored. A recent quantum mechanical study revealed that especially the interacting Chl a/Chl b pairs give rise to a significant redistribution of the molecular oscillator strength while the energy levels are only slightly influenced and presumably determined by Chl-protein interactions.27 On the basis of spectra simulation (linear absorption and circular dichroism), these calculations also offer additional information on pigment organization in LHC II.28 The existence of delocalized excited cluster states provides a consistent description of the fluorescence quantum yield of solubilized LHC II as a function of the photon density of excitation pulses.29 Most of the laser spectroscopic techniques used for the analysis of time dependent populations of excitonic (or molecular) states are incoherent methods. Complementary information on excitation dynamics can be gathered from results obtained by application of coherent spectroscopic approaches. Thus, theoretical work on four-wave mixing was enforced30-32 and the accumulated photon echo33,34 was established. Recently, three pulse photon echo peak shift methods have been used to analyze the excited-state dynamics in the antenna complex LH2 from anoxygenic purple bacteria35,36 and in LHC II of green algae.37 Two-pulse photon echo (2PE) measurements were performed to study EET and charge separation steps in photosynthetic RC preparations (D1/D2/Cyt b559) from spin-
Hillmann et al. ach.38 In 2PE measurements, a polarization is generated in an inhomogeneously broadened pigment ensemble that interacts twice with the coherent radiation of a retarded laser pulse. Thus, a population is created either in the excited or the ground state and, immediately, a second polarization. After a time delay corresponding to the period between both laser pulses, the phase development of the microscopic dipole moments within the ensemble gives rise to a macroscopic polarization and generation of echo photons. The intensity of the photon echo as a function of the laser pulse retardation characterizes the total dephasing time T2 of the polarization.39 T2 depends on both the excitedstate lifetime T1 and the pure dephasing time T2*, according to the relation:40
1 1 1 ) *+ T2 T 2T1
(1)
2
The present paper describes results that were obtained from integrated and time-resolved 2PE measurements in solubilized LHC II. The total dephasing time values T2 at 5K gathered from photon echo signals monitored in the whole region of Qy transitions (640-690 nm) provide information on dephasing processes due to excited-state relaxation from Chl b to the lowest excited singlet of Chl a. The data are discussed within the framework of the energy levels calculated on the basis of the structural model of Ku¨hlbrandt et al.13 2. Experimental Section Preparation of Samples. The LHC II samples were isolated by solubilization of salt washed PS II membrane fragments from spinach in the presence of β-dodecyl maltoside (β-DM) and separation by sucrose density gradient centrifugation as described in detail by Irrgang et al.41 The Chl a/Chl b (w/w) ratio was determined to be 1.29 ( 0.05 using the method of Porra et al.42 The samples were diluted with a glass forming buffer solution containing 70% (w/v) glycerol and 0.025% (w/v) β-DM to minimize undesirable aggregation of the LHC II trimers.43 The scanning of silver stained SDS/urea/PAGE41 revealed a high purity (≈87%) of the LHC II samples. In addition, a small fraction of the minor Chl a/Chl b proteins is present, i.e., CP14/ 15, CP22, CP24, and CP26 as outlined in ref 41. PS II core complex proteins were not detectable. The optical density at 676 nm at room temperature was about 1.0 in a cuvette with a path length of 1 mm. Experimental Apparatus. Optical pulses were generated at 800 nm by a regenerative amplifier (BMI-Alpha 1000 S) based on Ti:sapphire, pumped by a frequency doubled Nd:YLF laser at a repetition rate of 1 kHz and seeded by stretched pulses of a femtosecond oscillator (Coherent-Mira 900 B). These pulses were compressed and split into two beams, the first of which was wavelength shifted by second harmonic generation followed by an optical parametrical generator/amplifier (OPG/OPA). The second beam was used as a probe signal with variable delay for time-resolved echo measurements by an upconversion arrangement. The OPA output pulses were characterized by the following parameters: a wavelength ranging from 640 to 700 nm, a spectral full width at half-maximum (fwhm) of 5-6 nm, a duration of 150-250 fs, and a pulse energy of up to 7 µJ. To achieve the self-diffracting two-pulse geometry the OPA output pulses were split into a reference signal and two excitation beams, one of them with a variable delay. The excitation beams were crossed (angle 5.7°) and focused on a spot of 250 µm in diameter (fwhm) at the region of spatial overlap thus giving rise to a maximum power of 40 GW/cm2. To adjust the intensity
Optical Dephasing in LHC II
Figure 1. Time-resolved signals monitored at three different states of the mechanical chopper switching the excitation pulses. The figure illustrates three cases of excitation: only pulse 1 (open triangles), only pulse 2 (filled squares), and both pulses (open circles) with a fixed delay time of 2 ps.
a variable reflective neutral density filter was used. The instrument response function was independently determined by transient grating measurements on a thin cadmium sulfide crystal. The data could be fitted very well with a third-order correlation function of Gaussians according to G3(τ) ) ∫I1(t)I2(t + τ)2 dt. The size of the excited area and the quality of spatial overlap were checked by scanning the plane of the sample with a photodiode behind a 10 µm pinhole. The excitation beams with wave vectors k1 and k2, respectively, were polarized parallel to each other. In our experiments we measured the photon echo intensity emitted in the direction 2k2-k1 as a function of the delay time of pulse 2 relative to pulse 1 for a time window of up to 100 ps. Low-temperature measurements were performed using a helium flow cryostat (Oxford Instruments). To minimize any interference by photo damage of the samples, each trace was measured at another position of the sample, even though a repetition of the measurement at the same position yielded identical results. The pulse intensity was fixed at a value that gives rise to an echo signal with a dynamic range of about 1 order of magnitude for delay times beyond the temporal overlap of the excitation pulses. With respect to this condition, the typical intensity used was 1 × 1016 photons cm-2 pulse-1; the maximum was 3 × 1016 photons cm-2 pulse-1. At this high excitation energy it is obviously necessary to include multiexciton processes for the interpretation of the results. One experimental key point in obtaining reliable information from time-integrated photon echo measurements is the elimination of signals due to scattered light. Scattering causes not only a constant background thus diminishing the resolution but may also give rise to artificial decaying signals if the scattered light is affected by transient absorption analogously to a pump-probe experiment. Such an effect that is not suppressed by lock-in techniques was observed either if the detector was slightly misplaced with respect to the direction 2k2-k1 or if the spatial selectivity adjusted by pinholes was insufficient. Time-resolved measurements were performed to check for the photon echo origin of the integrated signal and to exclude disturbing artifacts. The time course of the signal was recorded at several fixed delay times by an upconversion setup, where the sum frequency of the diffracted signal and a probe pulse at 800 nm with variable delay and a duration of ≈350 fs was generated in a phase matched BBO crystal. A synchronized mechanical chopper switching the excitation pulses in combination with single pulse detection permitted a distinction between photon echo and scattered light. Pulses were chopped with frequencies of 200 and 250 Hz, respectively. Every single value of the measured trace could be assigned precisely to one of the four possible chopper states (beam 1 closed, beam 2 closed, and both beams closed or opened). Figure 1 shows the time-resolved signal at a fixed delay time τ of 2 ps for the three chopper states with at
J. Phys. Chem. B, Vol. 105, No. 36, 2001 8609
Figure 2. Semilogarithmic plot of the photon echo intensity at 680 nm as a function of delay time between excitation pulses monitored by two different operation modes: (i) subtraction of the scattered background (full squares, I)3.0 × 1016 photons cm-2 pulse-1) and (ii) background free (open circles, I)1.5 × 1016 photons cm-2 pulse-1) using the upconversion arrangement detecting the signal only at t)2τ, the temporal position of the photon echo.
least one opened beam under conditions of a good spatial alignment of all components. Measuring 1000 single pulses per delay position each data point in Figure 1 represents the mean value of 200 or 300 recorded values, conditional on the chopper pattern. The results reveal that the photon echo intensity is of the same order of magnitude as the scattered light or even smaller despite extensive effort to minimize scattering. However, it is clearly seen that signals occurring at times of t)0 and t)τ are only due to scattering because at these times already one of the beams is sufficient to generate the same signal as both beams. Thus, the photon echo signal can be gathered from two independent methods: (i) by subtracting the scattered signals of the excitation pulses from the total integrated signal using the mean values at the corresponding chopper states and (ii) by using the upconversion arrangement where the background free signal is only detected at the temporal position of the photon echo at t ) 2τ. The very good agreement between the results of both methods is a proof that the integrated signal is not affected by artifacts and only consists of the photon echo. As shown in Figure 2, both methods yield very similar results even if the excitation intensity is varied by a factor of 2. As an alternative procedure to i, a Fourier analysis of the measured trace can be performed to obtain the Fourier component at the difference frequency 50 Hz. Its absolute value is proportional to the photon echo signal, in analogy to lock-in techniques. Both approaches are equivalent but permit different noise filtering procedures. 3. Data Analysis Measurements were performed on several time scales with a delay time of up to 100 ps. Data recorded with both described methods (see Experimental Section) at different time scales were included in the analysis. The description of all data obtained on similar conditions by the same parameter set is a proof for the reproducibility of the experiment. In general, all measured photon echo traces are characterized by an initial peak at a delay time of zero with a width determined by the instrument response function followed by a nonexponential decay (Figure 2). The details of the signal shape depend on wavelength and temperature. In analogy to the interpretation of multiexponentially decaying transient absorption data15,22,23 within the framework of various energy transfer and relaxation processes, the measured 2PE data were analyzed in terms of a superposition of several dephasing processes. We assumed that the 2PE intensity of each process as a function of the excitation pulse retardation τ follows the theoretical expression originating from inhomogeneously broadened two-level systems given by39
8610 J. Phys. Chem. B, Vol. 105, No. 36, 2001
Hillmann et al.
( ){ ( )}
δω 4τ τ , 1+Φ T2 xπ 2 x exp(-t 2) dt (2) Φ(x) t xπ 0
Iecho(τ g 0) ) A exp -
∫
where δω denotes the width of inhomogeneous broadening and A is the proportionality constant, in the following referred to the amplitude. For two-level systems, Iecho (τ200 ps (661 nm, 77K) MC-TA22 120 fs, 7.5 ps, 135 ps, 3.4 ns (663 nm, 77K) MC-TA15 320 fs, 4 ps (277K) TG35 350 fs, 3 ps, 6 ps (277K) 3PE-PS35 280 fs, 11.5 ps, 160 ps, 3.4 ns (669 nm, 77K) MC-TA15 750 fs, 11 ps, 215 ps (672 nm, 77K) MC-TA15 240 fs, 18 ps, >0.5 ns (672 nm, 77K) MC-TA22 T2(4.2 K))12 ps HB45 T2(4.2 K) )100 ps HB45 450 fs, 7.3 ps, 120 ps, 3.4 ns (678 nm, 77K) MC-TA15 2.9 ps, 17 ps (77K) SC-TA22 1.9 ps, 20 ps, >1 ns (682 nm, 77K) MC-TA22 450 fs, 60 ps, 3.4 ns (682 nm, 77K) MC-TA15 T2(4.7 K, 681-682 nm) )150 ps HB45
a For comparison, the column at the right side compiles some excitation energy transfer and total dephasing times (italic bold) at different excitation wavelengths and temperatures (in parentheses). b SC-TA: single-color transient absorption. TC-TA: two-color transient absorption. MCTA: multicolor transient absorption. TG: transient grating. 3PE-PS: three-pulse photon echo peak shift. HB: hole burning.
Figure 5. Relative amplitudes of the three observed dephasing domains at 5 K as a function of excitation wavelength.
However, it affects only few molecules and therefore the number of probable pigment arrangements can be drastically reduced by simulations of experimental results. With respect to excited-state dynamics three wavelength regions of Qy transitions in LHC II can be roughly distinguished. The range from 640 to about 660 nm is primarily characterized by excitation of Chl b and ultrafast energy transfer to neighbored Chl a. In the excitonic model picture28 this process can be described as a relaxation within excitonic states of Chl a/Chl b heterodimers.49 From about 660 to 675 nm the processes are dominated by excitation of Chl a, energy equilibration between Chl a molecules, and transfer toward the lowest excited Chl a state(s). The lowest excited Chl a states in each of the three subunits of trimeric LHC II are localized in the range from 677 to 680 nm. These states are almost unaffected by excitonic coupling.45 At room temperature excitons are thermally equilibrated among all spectral Chl forms within the isolated LHC II attaining the highest exciton density at around 680 nm,38 which nicely corresponds with the energy level of the primary electron donor within the RC (P680). In the case of isolated LHC II the EET into the RC cannot take place, and the sample in its
Figure 6. Total dephasing times of the three observed dephasing domains at 5 K as a function of excitation wavelength.
nonaggregated form exhibits a distinct fluorescence with a typical lifetime of about 4 ns.43,47 The separation into three wavelength domains and their characteristic features provide a qualitative basis for the interpretation of the experimental data. Figure 5 shows that the wavelength range where the normalized amplitudes of domains A and B (C is below the detection limit) are almost independent of wavelength coincides with the range where mainly Chl b is excited (645-655 nm). The decay of the photon echo is dominated by the fast (1.1-1.7 ps) dephasing domain A with a relative extent of about 80%. The second domain B of 4.18.2 ps comprises the remaining part of 20%. At very low temperatures (Tf0) the total dephasing in organic amorphous systems is dominated by the lifetime of the excited states T1.40
8612 J. Phys. Chem. B, Vol. 105, No. 36, 2001 At temperatures between 0.3 and at least 20 K the influence of pure dephasing increases and leads usually to a T1.3 power law of the homogeneous line width.48a However, this rule fails if the lifetimes of the primary excited states are restricted by fast energy transfer processes with time constants noticeably smaller that T2*. If one assumes that contributions owing to pure dephasing can be neglected, i.e., if the relation T1,T2* is satisfied, the total dephasing times T2 are equal to the double of T1 values (see eq 1) even at higher temperatures. The comparison of T2 values from 2PE measurements and T1 times from incoherent transient absorption (TA) experiments can be used as a proof, if this assumption is true for the observed dephasing processes faster than 10 ps at 5 K. Accordingly, excited-state lifetimes of 550-850 fs and 2.0-4.1 ps should be directly obtained from the determined total dephasing times. When comparing these values with EET times gathered from TA measurements it has to be taken into account that 2PE data only reflect decay times of the primary excited states, in contrast to TA data that include also secondary transfer steps. Since the first incoherent transfer step leads to phase destruction, 2PE spectroscopy is not sensitive to spectral diffusion. This property offers the possibility to distinguish primary and secondary processes but simultaneously restricts the comparability of 2PE data with results obtained by multicolor TA experiments. On the other hand, single-color TA and transient grating experiments are more suitable for comparison because of similar spectral conditions concerning excitation and probing. In samples excited and probed at 650 nm three components were resolved with time constants of 100 and 500 fs and 3.1 ps and relative amplitudes of 37, 33, and 29%, respectively.22 These data reveal that about 70% of the 650 nm excitation disappear in less than 1 ps. Furthermore, the simulation of three-pulse photon echo peak shift data from the LHC II of the green algae Chlamydomonas reinhardtii35 suggested Chl b intraband energy transfer with times of 300 and 800 fs as well as Chl b to Chl a transfer with time constants of 150 and 600 fs and 4 ps. A comparison with the above-mentioned reported values reveals that the data gathered from the present 2PE measurements correspond in both energy transfer times and normalized extent with the results obtained by using complementary analytical tools. With respect to the kinetics it has to be kept in mind that dephasing times below 1 ps must be interpreted with caution owing to the coherence artifacts (see Figure 3). Regardless of this particular problem the close similarity with other data highly supports the assumption that optical dephasing at wavelengths around 650 nm is almost entirely caused by population decay of the excited Chl b states due to EET. If one takes into account that LHC II contains five Chl b molecules per monomer9 a relative amplitude of about 80% of the fast dephasing domain A is in perfect agreement with the widely accepted idea that no more than one Chl b molecule in each subunit losses its excitation energy via EET with times longer than 2 ps. A more complex pattern emerges for λ>660 nm where absorption by Chl a becomes significant. In the region of 660670 nm component A is characterized by a normalized extent of about 85% and clearly shorter dephasing times of 400-600 fs that is close to the limit of our experimental time resolution. The T1 times obtained by neglecting pure dephasing are 200300 fs. These values are in line with those gathered from TA measurements. Times of 20 ps monitored in TA measurements are most likely due to secondary EET steps and hence not reflected in the 2PE data (vide supra). Decay associated spectra in multicolor TA studies22,23 exhibit only small amplitudes in the range from 660-665 nm when samples were excited at 650 nm. Gradinaru et al.15 assigned the absorption at 662 nm to a single Chl a7 of LHC II. In the 670-675 nm domain significant changes arise. The sub-picosescond dephasing component A of 500-600 fs drastically drops down in its normalized amplitude from 85% at 670 nm to 36% at 675 nm (see Figure 5) accompanied by a corresponding increase of dephasing component B (6-7 ps). Moreover, a markedly longer component C with a dephasing time of g40 ps gradually emerges as an additional feature. Domains A and B corresponding to excited-state lifetimes of 250-300 fs and 3.0-3.5 ps are in good agreement with recent transient grating measurements37 at 670 nm resulting in population decay times of 320 fs (60%) and 4 ps (30%). Furthermore three-pulse photon echo peak shift data37 at 670 nm were simulated using lifetimes of 350 fs and 3 and 6 ps. From TA experiments excited at 672 nm transfer times of 420 fs and 18 ps were obtained22 while excitation at 669 nm led to values of 280 fs and 11.5 and 160 ps, respectively15 (omitting observed times >500 ps coming from secondary transfer processes). A comparison of the different results reveals that at these wavelengths markedly different time constants are obtained when coherent (2PE) and incoherent (TA) methods are applied. This phenomenon can be explained by the assumption that energy equilibration between energetically and spatially close neighboring Chl molecules around 670 nm destroys the phase relation within 3-3.5 ps (T1 time for EET steps leading to equilibration) while significant spectral diffusion occurs later at times above 10 ps. It is interesting to note that quantum mechanical simulations of linear absorption and circular dichroism28 lead to the conclusion that near 670 nm remarkable excitonic coupling effects are expected to give rise to significant excited-state delocalization and an increased transition dipole strength. The absorption around 670 nm was originally assigned to Chl a3 and Chl a5.28 However, the large distance between these molecules is hardly reconcilable with the observed dephasing component of 6-7 ps. Recently Rogl et at.16 reported that Chl b3 might be actually a Chl a. In this case Chl a3 would be in sufficiently close neighborhood to another Chl a. Alternatively, observed differences between the time constants gathered from 2PE and TA experiments, respectively, when samples are excited at around 670 nm could originate from multiexcitonic effects. It is well-known that at higher excitation energies multiexcitonic interactions take place,49 predominately in the Chl a absorption bands, leading to considerable effects
Optical Dephasing in LHC II
Figure 7. Absorption spectrum of the solubilized LHC II sample at 5 K (solid line) and two selected excitation pulse spectra centered at 670 and 684 nm (dashed lines). The filled squares show the spectral behavior of the reciprocal value of the dephasing time T2B at the corresponding excitation wavelengths.
as reflected by excited-state absorption or fluorescence quenching.29 From TA experiments time domains of 2-3 ps, 12-36 ps, and several hundred picoseconds were reported that are assigned to singlet-singlet or singlet-triplet annihilation, respectively.20,22,,23 The spectral course of dephasing time in the intermediate, picosecond decay component (domain B) above 660 nm can be interpreted as an indication of the influence of multi-excitonic effects. A compilation of spectra in Figure 7 shows that the dephasing rate (T2B)-1, i.e., the reciprocal value of the dephasing time of domain B, roughly follows the absorption spectrum in the range from 660 to 684 nm (Figure 7). Similar phenomena are not observed for the dephasing domains A and C (data not shown). Interestingly, an analogous clear feature is missing in the wavelength region of Chl b absorption. The maximum of (T2B)-1 coincides with the absorption maximum near 676 nm, whereas the value of the normalized amplitude of the dephasing domain B increases only above 670 nm and reaches its highest level at the position of the lowest exciton state around 680 nm (see Figure 5). This behavior is reasonable because this wavelength is characterized by the highest exciton density.46 Furthermore, excited states with the lowest energy have the longest lifetime and therefore an enhanced probability of an exciton-exciton interaction. Optical dephasing around 680 nm is strongly influenced by the lowest exciton state of the LHC II.45 At 680 nm two dephasing time domains are observed: 10 ps (78%) and 135 ps (22%). The longest dephasing time C dramatically increases toward longer wavelengths and reaches a maximum value of about 350 ps at 684 nm with a relative amplitude of about 50% (see Figures 5 and 6C). At wavelengths longer than 685 nm we have barely resonant excitation and the photon echo signal drastically decreases in intensity. Therefore we refrain from presenting these data. The sharp spectral shape of the longest dephasing component in Figure 6C and results from hole burning experiments45 are indicative of a high spectral sensitivity of T2 times in this wavelength region. Accordingly, it is important to take into consideration the spectral shape of the excitation. Actinic laser flashes of femtosecond pulse duration are necessarily spectrally rather broad (see dashed-lined profiles in Figure 7). On the contrary, HB experiments are performed with lasers characterized by very small fwhm values. The comparatively sharp wavelength dependence of T2 readily explains differences between T2 times gathered from 2PE and by HB experiments, respectively. For example, total dephasing times of 44 and 135 ps were obtained from 2PE measurements at excitation wavelength of 679 and 680 nm, respectively, whereas T2 values of 40 ps (678 nm) and 100 ps (679 nm) were determined by HB.45 At the first glance, HB might be superior to 2PE techniques with the disadvantage of spectrally broad femtosecond pulses.
J. Phys. Chem. B, Vol. 105, No. 36, 2001 8613
Figure 8. Semilogarithmic plot of the photon echo intensity as a function of delay time at 680 nm and three selected temperatures (5, 10, and 20 K). These data were obtained by the upconversion operation mode, i.e., detecting the signal intensity only at t)2τ, the temporal position of the photon echo.
TABLE 2: Total Dephasing Times and Relative Amplitudes (in Parentheses) at an Excitation Wavelength of 680 nm and Different Temperatures temperature (K)
T2B (ps)
T2C (ps)
5 10 20 40 60 90 298
10 (78%) 4.9 (72%) 4.1 (56%) 2.6 (38%) 2.0 (26%)
135 (22%) 54 (28%) 44 (44%) 38 (62%) 32 (74%) 27 12
This is true when the dephasing times are not too long because in case of high T2 values extended effort is necessary to avoid limitations by the width of the burn laser or the spectral resolution. With increasing wavelength, the dephasing time determined by high-resolution HB reaches a value of 150 ps at 681-682 nm and 4.7 K, whereas 2PE reveals dephasing times up to 350 ps at 684 nm. Accordingly, both techniques provide complementary approaches that are of optimal precision in different time domains. To obtain further information about the nature of the dephasing domains 2PE measurements were performed at 680 nm from 5 K up to room temperature. Figure 8 shows three selected 2PE traces at constant excitation intensities and different temperatures recorded in upconversion mode. At 5 K the 2PE signal of this excitation wavelength is characterized by two dephasing times of 10 and 135 ps. Both dephasing time constants decrease with increasing temperature as listed in Table 2. Furthermore, an inspection of Figure 8 readily shows that the amplitude of the photon echo at fixed delay times decreases at higher temperatures. Due to this decline of the photon echo intensity the experimental error of the dephasing times increases from 15% at 5 K up to 40% at room temperature. Reliable conclusions on the absolute signal strength could not be achieved because the measured absolute photon echo intensity strongly depended on the specific experimental conditions, e.g., the noise level. Therefore, the temperature dependence of the relative amplitude of dephasing domain B is more informative. The results illustrated in the inset of Figure 9 show that the relative amplitude of the dephasing domain B exhibits a pronounced decrease with increasing temperature. This dependence can be very well fitted by an exponential curve. The observed trend is basically understandable by considering that the electronphonon coupling of the lowest excited state at 680 nm is rather weak with a Huang-Rhys factor of about 0.8.45 Energetically higher Chl a states excited by the spectrally broad pulses can be involved in multiexciton processes and are obviously more sensitive to electron-phonon interactions than the lowest state. This finding is a further indication that the dephasing domain B in this spectral range is determined by multiexcitonic effects. Above 20 K both dephasing rates (T2B)-1 and (T2C)-1 are linearly
8614 J. Phys. Chem. B, Vol. 105, No. 36, 2001
Figure 9. Dephasing rate (T2B)-1 as a function of temperature at 680 nm. The inset shows the relative amplitude of the dephasing domain B as a function of temperature, i.e., the amplitude of the dephasing domain B normalized separately at each temperature to the sum of the amplitudes of the dephasing domains B and C.
Figure 10. Dephasing rate (T2C)-1 as a function of temperature at 680 nm. The inset represents the same dependence at an enlarged temperature range.
dependent on temperature according to the high-temperature limit of the Bose-Einstein distribution function (Figures 9 and 10). It is important to note that despite a nonlinear rise of the effective absorption at 680 nm with increasing temperature50 and despite exceeding the glass temperature, the dephasing rate (T2C)-1 exhibits an almost linear dependence on temperature with a comparatively weak slope (inset of Figure 10). A linear temperature dependence is characteristic for uphill energy transfer caused by an absorption of phonons. The marked change of the temperature dependence at around 20 K (see Figure 10) is in good agreement with HB results.44 According to the latter data the electron-phonon coupling at 680 nm is characterized by a mean phonon frequency of 15 cm-1, which corresponds to a critical temperature of 22 K. Below this value the curve becomes noticeable steeper. This effect is much less pronounced for the faster dephasing domain B shown in Figure 9. In contrast to the results at shorter excitation wavelengths the dephasing at 680 nm around 5 K is strongly affected by pure dephasing, because fast EET processes originating from the lowest energy states are not possible at low temperatures. Our 2PE data are not sufficient to decide whether the dependence in this lowtemperature region satisfies a T1.3 relation as is typical for pure dephasing and was found for homogeneous line widths at low temperatures in LHC II45 and in organic glasses.48b At least, the trend of the 2PE data below 20 K is not in contradiction with a T1.3 dependence. 5. Concluding Remarks The present study describes results on time integrated and time-resolved two-pulse photon echo (2PE) measurements in solubilized LHC II from spinach. Special efforts were made to minimize interfering effects owing to scattered light. Data are presented for the Qy bands of Chl a and Chl b (wavelength region 640-690 nm) at 5 K and for the temperature dependence of 2PE signals at 680 nm. A comparison of the results with published data that were gathered from measurements with complementary techniques
Hillmann et al. (time-resolved transient absorption, hole burning, three pulse photon echo peak shift) and interpretation within the framework of a model on excited-state level structure of LHC II based on quantum mechanical calculation leads to the following general conclusions: (a) the majority of excited singlet states generated by absorption of Chl b rapidly decays via EET in the subpicosecond time domain with no more than one Chl b molecule in the complex exhibiting lifetimes g2 ps, (b) in the range of 660-670 nm the exciton dynamics is dominated by equilibration among the ensemble of Chl a molecules and multiexciton interaction, and (c) at 5 K and excitation wavelengths above 677 nm the excited states are eventually trapped at a single Chl a molecule in each subunit of the trimeric LHC II. These “long wavelength” Chl a molecules are weakly excitonically coupled with the other pigments of the ensemble and attain rather long dephasing times exceeding 100 ps at 5 K. The electronic level structure of exciton dynamics of LHC II is inferred to be optimized for its physiological function in funneling electronically excited states to P680 of PS II. Acknowledgment. Authors are grateful to the Deutsche Forschungsgemeinschaft SFB 312 for financial support. K.-D. I. and G. R. gratefully acknowledge support from SFB 429. Moreover, F.H. thanks Dr. J. Pieper and Dr. T. Renger for helpful discussions and Dipl.-Ing. K. Palis for technical assistance. References and Notes (1) Renger, G. In Topics in Photosynthesis: The Photosystems: Structure, Function and Molecular Biology; Barber, J., Ed.; Elsevier: Amsterdam, 1992; p 45. (2) Holzwarth, A. R.; Griebnow, K.; Schaffner, K. J. Photochem. Photobiol. A: Chem. 1992, 65, 61. (3) Renger, G. In Concepts in Photobiology: Photosynthesis and Photomorphogenesis; Singhal, G. S., Renger, G., Govindjee, Irrgang, K.D., Sopory, S. K., Eds.; Kluwer Academic Publishers: Dordrecht, 1999; p 52. (4) van Grondelle, R.; Dekker, J. P.; Gillbro, T.; Sundstro¨m, V. Biochim. Biophys. Acta 1994, 1187, 1. (5) McDermott, G.; Prince, S. M.; Freer, A. A.; HawthornthwaiteLawless, A. M.; Papiz, M. Z.; Cogdell, R. J.; Isaacs, N. Nature 1995, 374, 517. (6) Koepke, J.; Hu, X.; Muenke, C.; Schulten, K.; Michel, H. Structure 1996, 4, 581. (7) Tritiak, S.; Middleton, C.; Chernyak, V.; Mukamel, S. J. Phys. Chem B 2000, 104, 4519. (8) Boekema, E. J.; van Roon, H.; Dekker: J. P. FEBS Lett. 1998, 424, 95. (9) Ku¨hlbrandt, W. Curr. Opin. Struct. Biol. 1994, 4, 519. (10) Jansson, S. Biochim. Biophys. Acta 1994, 1184, 1. (11) Paulsen, H. Photochem. Photobiol. 1995, 62, 367. (12) Green, B. R.; Durnford, D. G. N. Annu. ReV. Plant Physiol. Plant Mol. Biol. 1996, 47, 685. (13) Ku¨hlbrandt, W.; Wang, D. N.; Fujiyoshi, Y. Nature 1994, 367, 614. (14) Trinkunas, G.; Conelly, J. P.; Mu¨ller, M. G.; Valkunas, L.; Holzwarth, A. R. J. Phys. Chem B 1997, 101, 7313. (15) Gradinaru, C. C.; O ¨ zdemir, S.; Gu¨len, D.; van Stokkum, I. H. M.; van Grondelle, R.; van Amerongen, H. Biophys. J. 1998, 75, 3064. (16) Rogl, H.; Ku¨hlbrandt, W. Biochemistry 1999, 38, 16214. (17) Kwa, S. L. S.; van Amerongen, H.; Lin, S.; Dekker, J. P.; van Grondelle, R.; Struve, W. S. Biochim. Biophys Acta. 1992, 1102, 202. (18) Du, M.; Xie, X.; Mets, L.; Fleming, G. R. J. Phys. Chem. 1994, 98, 4736. (19) Savikhin, S.; van Amerongen, H.; Kwa, S. L. S.; van Grondelle, R.; Struve, W. S. Biophys. J. 1994, 66, 1597. (20) Bittner, T.; Irrgang, K.-D.; Renger, G.; Wasilewski, M. R. J. Phys. Chem. 1994, 98, 11821. (21) Bittner, T.; Wiederrecht, G. P.; Irrgang, K.-D.; Renger, G.; Wasilewski, M. R. Chem. Phys. 1995, 194, 311. (22) Visser, H. M.; Kleima, F. J.; van Stokkum, I. H. M.; van Grondelle, R.; van Amerongen, H. Chem. Phys. 1996, 210, 297. (23) Connelly, J. P.; Mu¨ller, M. G.; Hucke, M.; Gatzen, G.; Mullineaux, C. W.; Ruban, A. V.; Horton, P.; Holzwarth, A. R. J. Phys. Chem. B 1997, 101, 1902.
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