Energy Transfer and Trapping in Isolated Photosystem II Reaction ...

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J. Phys. Chem. 1996, 100, 11488-11495

Energy Transfer and Trapping in Isolated Photosystem II Reaction Centers of Green Plants at Low Temperature. A Study by Spectral Hole Burning M. L. Groot,†,‡ J. P. Dekker,† R. van Grondelle,† F. T. H. den Hartog,‡ and S. Vo1 lker*,†,‡ Department of Biophysics, Faculty of Physics and Astronomy, Free UniVersity, 1081 HV Amsterdam, The Netherlands, and Center for the Study of Excited States of Molecules, Huygens and Gorlaeus Laboratories, UniVersity of Leiden, 2300 RA Leiden, The Netherlands ReceiVed: January 31, 1996; In Final Form: April 26, 1996X

Spectral hole burning has been performed on the Qy-region of the isolated reaction center of photosystem II, the D1-D2-cytochrome b559 complex (PSII RC), between 665 and 688 nm, at liquid He temperatures. The “effective” homogeneous line width Γ′hom at 682 nm, in the red wing of the Qy-band, follows a T1.3(0.1 power law between 1.2 and 4.2 K characteristic of glasses and extrapolates to Γ′0 ) (2πT1)-1 for T f 0 with T1 ) (4 ( 1) ns, the fluorescence lifetime of the pigments. At these low temperatures, the red-absorbing “trap” pigments are unable to transfer energy to other pigments. The spectral distribution of the traps has been determined from hole depth vs λexc experiments. Their linear electron-phonon coupling strength was found to be rather weak, S ) 0.73 ( 0.05. For λexc< 678 nm, “downhill” energy transfer takes place. Spectral distributions of pigments characterized by decay times of 200 and 12 ps have further been identified in this spectral region. The data have been used to reconstruct the fluorescence excitation and absorption spectra.

1. Introduction The primary process in photosynthesis occurs within a lipid membrane. It involves the absorption of light by antenna complexes and the transfer of excitation energy to a primary electron donor within a reaction center where the energy is trapped by a sequence of electron-transfer reactions.1,2 In photosystem II (PSII) reaction centers (RC) of higher plants, these electron-transfer reactions produce a very high oxidizing potential (∼1 V) which is used for water oxidation accompanied by oxygen evolution. This is a major difference between bacterial and plant photosynthesis.3 An important step forward in the research of the structure and dynamics of PSII has been the isolation of the D1-D2cytochrome b559 complex (PSII RC), of which the photoactivity is limited to the primary charge separation.4,5 This PSII RC consists of the D1 and D2 subunits, cytochrome b559 polypeptides, and the psbI gene product. It contains about six chlorophylls a (Chl a), two pheophytins a (Pheo a), one or two β-carotenes, and no plastoquinones because they are lost during isolation.6-9 After excitation, which may happen directly by light or indirectly by energy transfer through one of the accessory Chl a or Pheo a pigments, an electron is transferred from the primary donor, called P680*, to the first electron acceptor (a Pheo a molecule) in less than 30 ps.10-15 The primary radical pair P680+Pheo- decays then by charge recombination in about 40100 ns, depending on temperature.16,17 In the absence of quinones, electron transfer beyond Pheo a is blocked. A thermodynamic model for the PSII RC kinetics was recently proposed18 on the basis of primary exciton equilibration and charge separation reactions. It describes the experimental triplet and fluorescence quantum yields obtained as a function of temperature, from which a distribution of free energy differences (∆G ≈ 20-80 meV) between the singlet-excited P680* and the radical-pair state P680+Pheo- results.18 A * To whom correspondence should be addressed. † Free University. ‡ University of Leiden. X Abstract published in AdVance ACS Abstracts, June 1, 1996.

S0022-3654(96)00326-7 CCC: $12.00

consequence of this model is that the radical-pair recombination reaction to P680* may only take place at temperatures T > 50 K. The increase of the fluorescence quantum yield observed when lowering the temperature below 50 K was explained by assuming the presence of accessory pigments (those which do not constitute P680) energetically degenerate with P680. Because of inhomogeneous broadening, at very low temperatures, the accessory pigments lying energetically below P680 will act as “traps” for the excitation energy and decay in a time determined by their fluorescence lifetime of about 4 ns. This is in agreement with fluorescence lifetimes of ∼4-6 ns observed at T ∼ 20 K by Roelofs et al.,19 but these authors suggested that the nanosecond lifetimes reflect charge recombination fluorescence. If this would be the case, ∆G should be much smaller than 20 meV, in contradiction to ref 18. The steadystate fluorescence at 4 K observed at λ > 683 nm by Kwa et al.,20 on the other hand, was attributed to a long-wavelengthemitting Chl a molecule. By contrast, from persistent spectral hole-burning experiments at 1.6 K it was concluded that all accessory pigments, when excited, transfer their energy to P680.21 Holes burnt at ∼682 nm yielded decay times of ∼50 ps, which were interpreted as energy transfer from Pheo a to P680 implying the absence of accessory trap pigments in PSII RC. A lack of consensus in the literature concerning PSII RC has not only been reported for energy-transfer processes within the Qy-region, but also for the charge separation rate. The reason for these controversies has its origin, probably, in the large overlap of strongly inhomogeneously broadened absorption bands between 660 and 690 nm. As a consequence, (sub-) picosecond time-resolved experiments are difficult to interpret. Several groups have claimed that “slow” (∼20 ps) energy transfer from 670 to 680 nm absorbing pigments13,19,22 is followed by “fast” charge separation (∼2 ps).10,13,22 Such times were supported by transient and permanent hole burning at 1.6 K.11,21 The group at Imperial College,14,15 however, has reported a “very fast” (∼100 fs) equilibration process between pigments at 670 and 680 nm, in addition to slow (∼20 ps) charge separation at room temperature.23 Very recently, (sub-) pico© 1996 American Chemical Society

A Study by Spectral Hole Burning second transient-absorption experiments at ∼680 nm, at 77 K have yielded decay times that were interpreted as fast (1-2 ps) “intrinsic” charge separation and slow (80-100 ps) “energy transfer-limited” charge separation,24 the latter arising from a trap state energetically degenerate with P680.18 “Downhill” energy transfer times of 400-500 fs and ∼14 ps were additionally observed when exciting at ∼670 nm.24 In order to verify whether low-lying trap pigments exist with a lifetime of about 4 ns which are not involved in any kind of energy transfer or charge separation at low temperature and to solve the contradictions related to energy transfer in PSII RC, we have performed persistent spectral hole-burning (HB) experiments between 1.2 and 4.2 K over the whole Qy-region from 665 to 688 nm. This technique, because of its high spectral resolution and wavelength- and burning-fluence selectivity, is an attractive tool for studying the excited-state dynamics of complexes with strongly overlapping spectral bands. HB yields not only T1- and T2-dephasing times as a function of wavelength and temperature but also spectral distributions of pigments characterized by their dynamic properties. Since we have probed the holes by means of fluorescence excitation spectroscopy, an electronicaly excited pigment may only be detected if it fluoresces or if it transfers its excitation energy to another pigment which in turn fluoresces. Because P680* undergoes very fast charge separation in a few picoseconds, it practically does not fluoresce. Thus, only the accessory pigments are sensitive to hole burning detected in this way. While performing these HB experiments, we have indeed found holes in the red wing of the Qy-region for which the width extrapolated to temperature T f 0 corresponds to a fluorescence lifetime of ∼4 ns. Thus, “4 ns” trap pigments are present in PSII RC, at least at very low temperatures up to 4.2 K, their dynamics being controlled by “pure” dephasing processes. We have also obtained the spectral distribution of these 4 ns traps (section 3.1). Further toward the blue of the Qy-region, we have identified two types of pigments characterized by energy transfer times of 200 and 12 ps (section 3.2). From the spectral distributions of the three identified pigments and from two additional distributions indirectly obtained, one for “P680” and another one for pigments of unidentified decay time, we were able to reconstruct the fluorescence excitation and absorption spectra (section 3.3). 2. Experimental Section The reaction center of photosystem II, called D1-D2cytochrome b559 (PSII RC), was isolated from the CP47-RC complex by means of a short Triton X-100 treatment as described in refs 9 and 25. The latter was obtained from spinach using the nonionic detergent n-dodecyl β,D-maltoside.26 Prior to the low-temperature measurements, the samples were diluted in a buffer containing 20 mM BisTris (pH 6.5), 20 mM NaCl, 0.03% (w/v) n-dodecyl β,D-maltoside, and 85% (w/v) glycerol. They were stored at 77 K when not used. The PSII RC complexes contained 6.4 Chl a and 1.6 β-Car per 2 Pheo a.9 To obtain glasses of good optical quality at liquid helium temperature, the samples were slowly cooled (in about half an hour) from room temperature to 77 K by keeping the cuvette (thickness 1 mm) in an empty 4He bath cryostat of which the outer mantle was filled with liquid nitrogen. Cooling from 77 to 4.2 K was achieved in a few minutes by filling the cryostat with liquid helium. The temperature was varied between 4.2 and 1.2 K and controlled by the vapor pressure of the 4He. The temperature was measured using a calibrated carbon resistor in contact with the sample with an accuracy better than 0.01 K.

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Figure 1. Qy-region of the absorption spectrum A(λ) (see section 2) of the isolated reaction center of photosystem II (PSII RC) at 1.2 K. The curve traced through the data has been used for the reconstruction of Figure 9b.

Broad-band absorption and fluorescence excitation spectra at 1.2 K were taken in Leiden with a tunable CW dye laser (Coherent 599-21, DCM dye, bandwidth ∼30 GHz ) 1 cm-1 without intracavity assembly, amplitude stabilized to 0 may include a contribution from spectral diffusion because the holes were probed with a delay after burning of a few minutes.27,28 Since the hole widths of a few hundred megahertz to a few gigahertz were much larger than the laser bandwidth (Γlaser ≈ 2 MHz), we took Γ′hom ) 1/2Γhole,Pt/Af0. The hole profiles were well fitted with Lorentzian curves.

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Figure 2. (a) Half the hole width 1/2Γhole as a function of burningfluence density Pt/A at 682.0 nm and 1.2 K. The data extrapolate to the effective homogeneous line width Γ′hom for Pt/A f 0. Inset: Hole burnt at Pt/A ) 0.54 mJ/cm2 . (b) Temperature dependence of Γ′hom between 1.2 and 4.2 K at 682.0 nm. Γ′hom follows a T1.3(0.1 power law which extrapolates to Γ′0 ) (2πT1)-1 ) 45 ( 10 MHz for T f 0. T1 corresponds to the fluorescence lifetime τfl ) (4 ( 1) ns of chlorophylllike molecules.

3. Results and Discussion 3.1. Trap Pigments Absorbing Furthest to the Red (λ g 680 nm). In order to determine the dynamic behavior of the accessory pigments absorbing furthest to the red within the Qyregion of the PSII RC complex, we have performed HB experiments with megahertz resolution as a function of temperature between 1.2 and 4.2 K. The value of the effective homogeneous line width Γ′hom was obtained, as described at the end of section 2, by extrapolating half the hole width 1/2Γhole for burning-fluence densities Pt/A f 0. Such a plot is shown in Figure 2a for holes burnt at 682 nm, at 1.2 K. Between 678 and 688 nm we have found narrow holes, of the order of a few hundred megahertz, with a wavelength-independent width at a given burning-fluence density. A typical hole is shown in the inset of Figure 2a. It was obtained at a low value of Pt/A ) 0.54 mJ/cm2 for which 1/2Γhole 280 MHz is close to Γ′hom. The temperature dependence of Γ′hom for pigments absorbing at 682 nm is given in Figure 2b. The data follow

Γ′hom ) Γ′0 + aT1.3(0.1

(1)

between 1.2 K and 4.2 K. A T1.3 power law, as obtained here, is characteristic for dephasing in doped organic glasses29 and indicates that for PSII RC the pigment-protein interaction is similar to that in amorphous systems. The extrapolated value of Γ′hom for T f 0 is given by Γ′0 ) (2πT1)-1, in which T1 )

Figure 3. (a) Relative hole depth D (%) as a function of excitation wavelength λ (dashed line) together with the profile of the fluorescence excitation spectrum (solid line). The holes were burnt with Pt/A ) 0.54 mJ/cm2 at 1.2 K. (b) Spectral distribution of the 4 ns trap pigments (dashed line) reconstructed in the fluorescence excitation spectrum. The distribution was obtained after multiplying the two curves in Figure 3a by each other and normalizing the height to the fluorescence excitation spectrum (solid line).

(4 ( 1) ns proves equal to the fluorescence lifetime τfl of Chl a and Pheo a molecules. Because T1 ) τfl, these HB results demonstrate that there are pigments absorbing furthest to the red within the Qy-region which indeed are traps. These 4 ns living pigments undergo pure dephasing and decay, but do not transfer energy to other pigments like P680, at least at low temperatures up to 4 K. Such traps were predicted for T < 50 K from the kinetic model of ref 18. To determine the spectral distribution of these 4 ns trap pigments we have measured not only the hole width but also the relative depth of the holes D (%) as a function of excitation wavelength between 677 and 687 nm at a constant, low burningfluence density (Pt/A ) 0.54 mJ/cm2, see Figure 2a) at 1.2 K. In this wavelength region the holes change their depth but not their width, indicating that this method selects those pigments that are involved in a specific dynamic process, in this case characterized by a 4 ns decay time. The values of D (%) vs λ are reproduced in Figure 3a together with the profile of the fluorescence excitation spectrum between 675 and 690 nm. The dashed curve traced through the hole depth data is a guide to the eye. By multiplying this curve by the profile of the fluorescence excitation spectrum, one obtains an approximate Gaussian profile30 with a maximum at (681.8 ( 0.3) nm and a fwhm ) (143 ( 5) cm-1, which represents the spectral distribution of the 4 ns fluorescing pigments. This distribution is shown in Figure 3b where its height has been normalized to the fluorescence excitation spectrum.30 By

A Study by Spectral Hole Burning

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comparing the position and the width of this 4 ns trap distribution with the broad (∼120 cm-1 width) permanent satellite hole at 681.6 nm reported by Tang et al.,21 we conclude that our distribution and their satellite hole most probably are related to the same pigments. The authors of ref 21 attributed the pigments absorbing at 681.6 nm to Pheo a, because a hole burnt at 663 nm caused simultaneously two satellite holes, the very broad one at 681.6 nm and a second one at 545.7 nm, the wavelength of the Qx-band of Pheo a. As mentioned in the introduction, and in contradiction to our results, holes burnt at ∼682 nm were reported to yield decay times of ∼50 ps,21 much shorter than the 4 ns determined by us. The discrepancy may be due to too high burning-fluence densities and temperatures used: the holes of ref 21, which were measured at a single temperature of 1.6 K, correspond to a value of Γ′hom ) 3.2 GHz. This is a factor of 10 larger than our value of Γ′hom at the same temperature and wavelength. Although no burning-fluence density is mentioned in ref 21, the same group reported in ref 11 a hole of a similar width burnt with a Pt/A value of the order of 2000 times larger than that in our experiments. As may be seen from our Figures 2a,b, it is necessary to extrapolate the hole widths for Pt/A f 0 and T f 0 to get a reliable value of T1. We have also determined the linear electron-phonon coupling strength S for the 4 ns trap pigments. The value of S was obtained from HB experiments carried out under saturating conditions, i.e. at very high burning-fluence densities by which, besides the zero-phonon holes, side holes become visible (see Figure 4a). To burn such deep holes we had to use the CW laser without the intracavity assembly (bandwidth of ∼1 cm-1, see section 2). The hole widths (∼4 cm-1) are then not only limited by twice the laser bandwidth, but additionally, they are power broadened. The area of the zero-phonon hole Azph was divided by the sum of Azph and the area of the pseudo-phonon side hole Apsh, and the ratio was extrapolated to zero burningfluence density (see Figure 4b). This yields the so-called Debye-Waller factor R which, under these conditions, is related to S by

R ) (Azph/(Azph + Apsh))Pt/Af0 ) 2e-2S/(1 + e-2S)

Figure 4. (a) Deep holes burnt under saturating conditions, at two burning-fluence densities Pt/A, at 682.0 nm and 1.2 K. (b) Burningfluence density dependence of the ratio Azph/(Azph + Apsh), where Azph is the area of the zero-phonon hole and Apsh is the area of the pseudophonon side hole, at 682 nm and 1.2 K. The extrapolated value for Pt/A f 0 yields the Debye-Waller factor R which is related to S, the electron-phonon coupling strength. S ) 0.73 ( 0.05 (see text).

(2)

Examples of deep holes and side holes burnt at 682 nm are shown in Figure 4a for two different Pt/A values. We attribute the side hole appearing at ∆ν ∼ (16 ( 1) cm-1 from the zerophonon hole to a low-frequency mode of the protein.31 From the ratio of the hole areas as a function of Pt/A (see Figure 4b), we have obtained S ) 0.73 ( 0.05, a value which agrees with that reported for the Pheo a Qy-state.21 It represents a rather weak electron-phonon coupling strength when compared to S ∼ 2, the value reported for P680 of PSII in ref 11 and for P870/ P960 of purple bacteria.32 Finally, we remark that by burning at 682 nm we also found a satellite hole at ∼545 nm, the wavelength of the Pheo a Qxstate. Thus the 4 ns trap pigments consist, at least, of Pheo a. On the other hand, from fluorescence spectra at 4 K it was concluded that the most red emission arises from Chl a.20 Probably both pigments are present at λ > 680 nm. 3.2. Pigments Absorbing at Higher Energies within the Qy-Region (λ < 680 nm). By burning at wavelengths shorter than 678 nm, at Pt/A ∼ 0.5 × 10-3 J/cm2, the narrow holes representing the 4 ns trap pigments (see Figure 2a) become very shallow (D < 2%) and disappear (see Figure 3a). However, it is still possible to detect holes at these shorter wavelengths if 103 times higher burning-fluence densities are used. This is illustrated in Figure 5, where 1/2Γhole has been plotted as a function of Pt/A for holes burnt at 676 nm, at 1.2 and 4.2 K.

Figure 5. Half the hole width 1/2Γhole as a function of burning-fluence density Pt/A, at 676 nm, for 1.2 and 4.2 K. The extrapolated value for Pt/A f 0 yields Γ′hom at each temperature.

Notice that, while the values of Pt/A ∼ 0.5-1 J/cm2 are indeed 3 orders of magnitude higher than in Figure 2a (holes at 682 nm), the holes are about four times broader. As in Figure 2a, Γ′hom at a given temperature is obtained from the extrapolation of 1/2Γhole for Pt/A f 0. In order to select pigments by their dynamic properties, i.e. by their hole widths and depths at a given Pt/A value, we have measured 1/2Γhole as a function of Pt/A at various wavelengths. Such a plot is shown in Figure 6a. Notice that the widths of holes burnt at 676 and 678 nm follow the same curve (open

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Figure 7. Temperature dependence of Γ′hom of pigments absorbing at 676 nm compared to that of the 4 ns trap pigments at 682 nm (Figure 2b). At 676 nm, Γ′0 ) (2πT1)-1 > (2πτfl)-1 with T1 ) (200 ( 10) ps (see text).

Figure 6. (a) Burning-fluence density dependence of 1/2Γhole under saturating conditions (Pt/A values up to 5 J/cm2), for three excitation wavelengths at 1.2 K. (Upper curve) 676 nm (open circles) and 678 nm (closed circles). (Lower curve) 685 nm (diamonds). Notice that pigments absorbing at 676 and 678 nm, having the same hole widths, belong to the same spectral distribution (see text). (b) 1/2Γhole vs burningfluence density (for Pt/A < 0.15 J/cm2), at 678 nm (closed circles) and 685 nm (diamonds), at 1.2 K. Notice that pigments absorbing at 678 and 685 nm, having the same hole widths, belong to the same spectral distribution. Thus, pigments absorbing at 678 nm may belong to either of the two distributions depending on their decay time (see text).

and closed circles) and saturate to a width of ∼4 GHz, which is much larger than that of holes burnt at 685 nm with 1/2Γhole ∼ 2 GHz for Pt/A f ∞ (diamonds). Thus, the pigments absorbing at 676 and 678 nm belong to the same spectral distribution characterized by a faster dynamics than the pigments absorbing at 685 nm which decay in 4 ns. If, on the other hand, we follow the values of 1/2Γhole in the region of Pt/A < 0.15 J/cm2 (see Figure 6b), we notice that the widths of holes burnt at 678 nm have crossed over from the 676 nm curve to the 685 nm curve. We have to conclude that pigments absorbing at 678 nm also belong to the 4 ns trap distribution (see section 3.1). This is understandable if we look at the spectrum in Figure 3b, where at 678 nm not only pigments belonging to the 4 ns distribution absorb, but also others that can only be burnt at much larger Pt/A values and undergo a faster dynamics. In Figure 7 the dephasing between 1.2 and 4.2 K of pigments absorbing at 676 nm has been plotted and compared to that of the 4 ns trap pigments absorbing at 682 nm (from Figure 2b). We observe that for both types of pigments Γ′hom follows a T1.3 dependence, i.e. a glasslike behavior, but the extrapolated value Γ′0 for T f 0 is different at the two wavelengths. At 676 nm, Γ′0 ) (2πT1)-1 > (2πτfl)-1 with T1 ) (200 ( 10) ps. Apparently, the decay of pigments absorbing at 676 nm is 20

times faster than that of pigments in the red wing. The direct fluorescence to the ground state will, therefore, be very weak. Because holes burnt between 672 and 678 nm at a constant Pt/A value yielded the same hole widths, we call the spectral distribution of these pigments the “200 ps” distribution. Its maximum is at ∼675 nm and has a width of ∼140 cm-1 (see section 3.3 and Table 1). We attribute the 200 ps decay to downhill energy transfer to the lower-lying P680 and 4ns pigments. When burning at 672 nm, two types of holes are observed, as illustrated in Figure 8a. The first type belongs to pigments of the 200 ps distribution. It has a width 1/2Γhole ∼ 2 GHz at Pt/A ≈ 1 J/cm2 at 1.2 K. When burning at about five times higher burning-fluence density this type of hole saturates (see also Figure 6a) and its base line shifts downward suggesting that a much broader hole has been formed. This second type of hole is very broad (lowest one in Figure 8a). It has a width 1/ Γ 2 hole ≈ 13 GHz, about 15 times larger than Γ′hom of the 200 ps distribution. Between ∼5 and 10 J/cm2 the holes do not broaden significantly. Since pure dephasing up to 1.2 K contributes only little to the hole width (at most 0.2 GHz), T1 ≈ (2π 13 GHz)-1 ) (12 ( 2) ps. The latter type of holes is observed between ∼665 and ∼673 nm, from which we have estimated that the “12 ps” distribution is centered at ∼669 nm and has a width of about 200 cm-1 (see section 3.3 and Table 1). Similar holewidths corresponding to a decay time of 12 ps at 1.6 K were reported in the literature when burning at ∼665 nm.21 Also picosecond-time-resolved experiments at higher temperatures (from 15 K to room temperature) yielded decay times of 10-20 ps in this wavelength region.13,19,24,33 We attribute the fast decay at 1.2 K to downhill energy transfer from the 12 ps pigment distribution to the 200 ps, 4 ns, and P680 distributions. In Figure 8b we have summarized our hole-burning results by plotting the values of Γ′hom as a function of excitation wavelength, for given values of Pt/A, together with the fluorescence excitation spectrum. The regions of constant holewidths represent the spectral distributions of pigments which have been labeled with the values of their corresponding decay times: 4 ns, 200 ps, and 12 ps. 3.3. Reconstruction of Spectra from the Spectral Distributions of Pigments Characterized by Their Decay Times. To reconstruct the fluorescence excitation spectrum at 1.2 K from the spectral distributions of pigments obtained from our hole-burning experiments, we have to make assumptions about

A Study by Spectral Hole Burning

Figure 8. (a) Holes burnt at various burning-fluence densities at 672 nm and 1.2 K. (From top to bottom) Pt/A ∼ 1 J/cm2, 5 J/cm2, and 10 J/cm2. The hole at Pt/A ∼ 10 J/cm2 has a width 1/2Γhole ) 13 ( 2 GHz which corresponds to a transfer time T1 ) (2πΓ′0)-1 ) (12 ( 2) ps. (b) Values of Γ′hom at different spectral positions within the fluorescence excitation spectrum. Burning-fluence densities are given together with the name of their spectral distribution.

the shape of these distributions, their positions, heights, and widths. With respect to the shape, we have assumed that at liquid He temperature it is Gaussian because the spectral distributions are inhomogeneously broadened (holes can be burnt into them). The position and width of the 4 ns trap pigments distribution have been accurately determined as described in section 3.1 (see Figures 3a,b) with a maximum at (681.8 ( 0.3) nm and a fwhm ) (143 ( 5) cm-1. For the positions and widths of the 200 ps and 12 ps pigment distributions, we have used an iterative procedure. In a first approximation, we have taken for the value of fwhm the wavelength region over which the holes have equal width when burnt at constant Pt/A. By the choice of the burning-fluence density Pt/A, we select pigments that decay with a specific time constant, cf. the discussion of Figures 6a,b above. For the maximum of each distribution we have taken the center of the region over which the holes have an equal width. Although by adjustment of their relative heights the 4 ns, 200 ps, and 12 ps distributions fill in the fluorescence excitation spectrum reasonably well over a large wavelength region, they leave a gap around 679 nm. This gap can only be filled in if we assume at least one distribution in the 679 nm region with a total width of ∼100 cm-1. We have not attempted to interpret this gap because we have not identified further decay times by hole burning in this region. From time-resolved pump-probe experiments at room temperature in the same wavelength region, it was concluded that very fast equilibration of the order of 100 fs takes place between pigments absorbing in the red and in the blue of the Qy-region.14,15 If such a fast equilibration would also occur at liquid He temperatures, we would expect extremely

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Figure 9. (a) Reconstruction of the fluorescence excitation spectrum at 1.2 K by a superposition of inhomogeneously broadened bands. The latter were taken as Gaussians; they represent the spectral distributions of pigments characterized by a specific decay time. Those labeled with 4 ns, 200 ps, and 12 ps were obtained from the widths and depths of holes at different wavelengths. The distribution represented by dots was added in order to fill in the fluorescence excitation spectrum without gaps (see text). (b) Reconstruction of the absorption spectrum Ia(λ) at 1.2 K (see section 2) by superposition of the four inhomogeneously broadened (Gaussian) distributions of Figure 9a, but with different heights, and an indirectly obtained distribution which we attribute to P680 (see text, section 3.3). The area of the fluorescence excitation spectrum has been scaled to 0.25 of that of the absorption spectrum.18 The latter Ia(λ) is proportional to the absorbance A(λ) ) OD only at low OD. Since OD ∼ 0.14, the proportionality is valid (see sections 2 and 3.3).

TABLE 1: Position of the Maxima and Widths of the Spectral Distributions of Pigments Characterized by Their Decay Time, at 1.2 Ka distribution

λmax (nm)

fwhm (cm-1)

4 ns trap (from HB) P680 679 (unidentified decay) 200 ps (from HB) 12 ps (from HB)

681.8 ( 0.3 680.8 ( 0.5 678.8 ( 0.3 675.1 ( 0.4 669.1 ( 0.5

143 ( 5 150 ( 5 101 ( 3 142 ( 5 196 ( 6

a The fluorescence excitation and absorption spectra in the Q -region y of PSII RC were reconstructed by superposition of the spectral distributions discussed in section 3.3 (see Figure 9a,b).

broad holes of ≈50-100 cm-1 width. Such holes would be very shallow and, therefore, probably not detectable within the noise. The four distributions of pigments are given in Figure 9a together with the fluorescence excitation spectrum. The three dashed curves represent the spectral distributions estimated from hole burning, whereas the curve with thin points is that of

11494 J. Phys. Chem., Vol. 100, No. 27, 1996 hypothetical pigments absorbing at ∼679 nm with one or more unidentified decay time(s). The superposition of all four distributions is given by the curve with thick points. It reproduces the fluorescence excitation spectrum well, with the exception of the far blue wing which should probably be accounted for by additional distributions or vibronic bands. Next, we made an attempt to reconstruct the absorption spectrum Ia(λ), which at low concentration (OD ) 0.14) is proportional to the absorbance A(λ) (see section 2), starting from the distributions obtained in the fluorescence excitation spectrum. The reconstruction was carried out in three steps. First, the fluorescence excitation spectrum was scaled in such a way that its area corresponds to 25% of that of the absorption spectrum Ia(λ) (see Figure 9b). The reason for this is based on the results of ref 18 where it was calculated that after nonselective excitation at ∼610 nm at 4 K, about 75% of the excitation is used for charge separation. Thus, the remaining 25% will decay through the accessory pigments to the lowestlying 4 ns trap and should be detectable in fluorescence excitation. As a consequence, the height of the 4 ns trap distribution is fixed to that of the fluorescence excitation spectrum. Second, we have assumed that the red wing of the absorption spectrum at 1.2 K consists of 4 ns trap pigments and P680 pigments only, at least for λ > 683 nm. This assumption is based on various types of experiments: (1) our HB experiments at 1.2 K which have shown that the red wing of the fluorescence excitation spectrum only consists of 4 ns trap-pigments, (2) triplet-minus-singlet absorption difference spectra34 which show a broad bleaching at 680.6 nm (width 6.5 nm) and were attributed to depletion of the P680 ground state, (3) magnetophotoselection experiments at 6 K35 which have proven the presence of P680 pigments in the red edge of the absorption spectrum, and (4) transient hole-burning experiments at 4.2 K in the 681 nm region in which it was reported that the absorption maximum of P680 lies at 680.3 nm,11 or 681.7 nm,21 with an inhomogeneously broadened width of about 140 cm-1.11,21 Since the 4 ns distribution is fixed in position, width, and height, the P680 distribution, under the assumptions made here, follows automatically because it has to fill in the red wing of the absorption spectrum. The maximum of P680 then appears at (680.8 ( 0.5) nm with a width of (150 ( 5) cm-1. Its height was matched to that of the absorption spectrum (see Figure 9b). In this procedure, the spectral location of P680 is not measured by us but follows from a comparison of the band shapes of the red wings of the fluorescence and absorption spectra. It is gratifying that the spectral distribution of P680 thus derived is so similar to that reported in the literature by other techniques.11,21,34,36 The very small shoulder at 667 nm in the wing of the distribution, reported in ref 34, does not appear in the reconstructed spectrum of P680. Our results do not give any indication on the nature of P680, i.e. whether it is a multimer of several weakly coupled pigments37 or a dimer,35 nor on the charge separation time. Third, to reconstruct the rest of the absorption spectrum we have used the positions and widths of the 200 ps, 12 ps, and the 679 nm-distribution of pigments from the fluorescence excitation spectrum and only varied their relative heights until they filled in the absorption spectrum. This means that, for simplification, we have assumed that no other distributions are present in this part of the absorption spectrum at 1.2 K, for which we do not have any proof. If at 1.2 K, like at room temperature,14,15,37 very fast equilibration would occur over a large part of the spectrum, we would expect very broad and shallow holes to be difficult to detect. The results of the

Groot et al.

Figure 10. Energy-level scheme of PSII RC corresponding to the data obtained in this work at 1.2 K. The arrows denote the possible decay channels from the spectral distributions of pigments identified by hole burning and from the indirectly obtained P680 (see section 3.3). No distributions of pigments have been included which are not directly or indirectly determined here.

reconstruction under the given assumptions are shown in Figure 9b. The absorption spectrum is well reproduced by only five pigments distributions, of which four were taken from the fluorescence excitation spectrum, while the P680 pigment distribution arose from a need to fill in a gap left over in the red part of the absorption spectrum as compared to the fluorescence excitation spectrum. The P680 pigments are not detected by hole burning because they do not fluoresce owing to the fast charge separation within a few picoseconds. Nevertheless it is surprising that, under the conditions assumed, the five distributions reproduce a large part of the absorption spectrum rather well without the need for additional distributions. As in the fluorescence excitation spectrum, the extreme blue wing of the Qy-region of the absorption spectrum is not accounted for by the five distributions mentioned. In this spectral region there probably are vibronic bands or other pigments which could not be identified by hole burning. In Table 1 the positions and widths are given for (1) the three spectral distributions deduced from our HB experiments at 1.2 K, (2) the distribution which we have attributed to P680, and (3) another distribution of hypothetical pigments with an unidentified decay time with a maximum at ∼679 nm and a width of ∼100 cm-1. We have summarized the results obtained in this work in the energy-level diagram of Figure 10. Each spectral distribution of pigments within the Qy-region of PSII RC is characterized by a specific decay time. These distributions were obtained by persistent hole burning probed by fluorescence excitation at 1.2 K. The possible decay pathways are also given in the figure. We think that the “12 ps”-pigments decay via energy transfer to the 200 ps, the 4 ns, and the P680 pigments, whereas the 200 ps pigments decay via energy transfer to the 4 ns and the P680 pigments. The 4 ns pigments decay only to the ground state via emission of fluorescence and nonradiative decay. From the experiments we cannot conclude anything about the internal dynamics of P680. Within the framework of the multimer model,37 P680 reflects the core pigments of the PSII RC including the pheophytins and may show dynamics on a subpicosecond time scale before charge separation occurs.

A Study by Spectral Hole Burning Because of the low temperatures of our experiments, we have not considered any energy transfer processes back from P680 to the various pigments absorbing at higher energy which become important at room temperature. 4. Conclusions We have proved by means of megahertz-resolution holeburning spectroscopy that trap pigments, absorbing in the red wing of the isolated PSII RC complex and with a fluorescence lifetime of 4 ns, exist at liquid He temperatures. From the hole depth as a function of λexc we have obtained the spectral distribution of these trap pigments. This set of molecules has an approximate Gaussian distribution centered at 681.8 nm with a width of ∼140 cm-1. From HB experiments performed under near-saturating conditions, and by taking the ratio of Azph to the sum of Azph plus Apsh, extrapolated for zero burning-fluence density Pt/A f 0, we have determined the linear electronphonon coupling strength, S ) 0.73 ( 0.5. These trap pigments consist, at least, of pheophytin a molecules because a hole burnt at 682 nm yields a satellite hole at ∼545 nm, the spectral position of their Qx-band. According to ref 20, also Chl a should be present at 682 nm, which we have not identified here. By measuring holewidths and hole depths as a function of excitation wavelength for λexc < 678 nm, we have verified that downhill energy transfer takes place. Between 672 and 678 nm it competes with pure dephasing and occurs in 200 ps, whereas between 665 and 672 nm it is much faster and occurs in 12 ps. Because hole burning is selective as a function of wavelength and burning-fluence density, we were able to determine the spectral distributions of pigments characterized by their decay times. After determining the positions and widths of these distributions, the fluorescence excitation and absorption spectra at 1.2 K were reconstructed. Although not identified here by hole burning, we inferred that the P680 spectral distribution should be centered at 680.8 nm and have a width of ∼150 cm-1. It is satisfying to see that the results for P680 are consistent with results from the literature obtained by other techniques.11,21,34,36 In addition, we obtained a distribution centered at 678.8 nm with a width of ∼100 cm-1 which corresponds to hypothetical pigments of one or more unidentified decay times. The results presented demonstrate that hole burning, because of its high spectral resolution and wavelength- and burningfluence selectivity, represents a powerful tool for disentangling the low-temperature excited-state dynamics of photosynthetic complexes with strongly overlapping spectral bands. Acknowledgment. We thank C. Eijckelhoff and H. van Roon for providing us with the PSII RC samples and M. P. Bakker for assistance in some of the hole-burning experiments. Further, we acknowledge J. H. van der Waals for valuable remarks regarding the manuscript. The investigations were supported by the Netherlands Foundation for Physical Research (FOM) and Chemical Research (SON) with financial aid from the Netherlands Organization for Scientific Research (NWO). References and Notes (1) van Grondelle, R.; Dekker, J. P.; Gillbro, T.; Sundstrom, V. Biochim. Biophys. Acta 1994, 1187, 1. (2) Fleming, G. R.; van Grondelle, R. Phys. Today 1994, 47, 48.

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