Chromophore−Chromophore and Chromophore−Protein Interactions

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Chromophore-Chromophore and Chromophore-Protein Interactions in Monomeric Light-Harvesting Complex II of Green Plants Studied by Spectral Hole Burning and Fluorescence Line Narrowing Jo¨rg Pieper,*,† Margus Ra¨tsep,‡ Klaus-Dieter Irrgang,§,| and Arvi Freiberg‡,⊥ Max-Volmer-Laboratories for Biophysical Chemistry, Technical UniVersity Berlin, Berlin, Germany, Institute of Physics, UniVersity of Tartu, Tartu, Estonia, Department of Life Science & Technology, Laboratory of Biochemistry, UniVersity for Applied Sciences, Berlin, Germany, and Institute of Molecular and Cell Biology, UniVersity of Tartu, Tartu, Estonia ReceiVed: January 28, 2009; ReVised Manuscript ReceiVed: April 7, 2009

Persistent nonphotochemical hole burning and delta-FLN spectra obtained at 4.5 K are reported for monomeric chlorophyll (Chl) a/b light-harvesting complexes of photosystem II (LHC II) of green plants. The hole burned spectra of monomeric LHC II appear to be similar to those obtained before for trimeric LHC II (Pieper et al. J. Phys. Chem. B 1999, 103, 2412). They are composed of three main features: (i) a homogeneously broadened zero-phonon hole coincident with the burn wavelength, (ii) an intense, broad hole in the vicinity of ∼680 nm as a result of efficient excitation energy transfer to a low-energy trap state, and (iii) a satellite hole at ∼649 nm which is correlated with the low-energy 680 nm hole. Zero-phonon hole action spectroscopy reveals that the low-energy absorption band is located at 679.6 nm and possesses a width of ∼110 cm-1 which is predominantly due to inhomogeneous broadening at 4.5 K. The electron-phonon coupling of the abovementioned low-energy state(s) is weak with a Huang-Rhys factor S in the order of 0.6 and a peak phonon frequency (ωm) of ∼22 cm-1 within a broad and strongly asymmetric one-phonon profile. The resulting Stokes shift 2Sωm of ∼26.4 cm-1 readily explains the position of the fluorescence origin band at 680.8 nm. Thus, we conclude that the 679.6 nm state(s) is (are) the fluorescent state(s) of monomeric LHC II at 4.5 K. The absorption intensity of the lowest Qy state is shown to roughly correspond to that of one out of the eight Chl a molecules bound in the monomeric subunit. In addition, the satellite hole structure produced by hole burning within the 679.6 nm state is weak with only one shallow satellite hole observed in the Chl b spectral range at 648.8 nm. These results suggest that the 679.6 nm state is widely localized on a Chl a molecule, which may belong to a Chl a/b heterodimer. These characteristics are different from those expected for Chl a612, which has been associated with the fluorescent state at room temperature. Alternatively, the 679.6 nm state may be assigned to Chl a604, which is located in a cluster with several Chl b molecules resulting in a relatively weak excitonic coupling. 1. Introduction The light-harvesting complex of photosystem II (LHC II) is the major antenna complex of green plants, which serves to collect solar radiation with a broad spectral and spatial cross section as well as to efficiently transfer the acquired excitation energy to the reaction center complex, where the photochemical processes of photosynthesis are initiated by a primary charge separation (for a review, see, e.g., van Amerongen and Croce1). Besides light-harvesting, LHC II has a number of photoprotective functions including (i) nonradiative dissipation of excess energy by nonphotochemical quenching,2,3 (ii) efficient quenching of chlorophyll (Chl) triplet states by carotenoid (Car) * Author to whom most correspondence should be addressed. Address: Technical University, PC14, Strasse des 17. Juni 135, 10623 Berlin, Germany. Phone: +49-30-31427782. Fax: +49-30-31421122. E-mail: [email protected]. † Technical University Berlin. ‡ Institute of Physics, University of Tartu. § University for Applied Sciences. | Author to whom correspondence about sample preparation and biochemical analysis should be addressed. Address: University of Applied Sciences, Life Sciences and Technology, Seestr. 64, 13347 Berlin, Germany. Phone: 0049-30-4504-3910. E-mail: [email protected]. ⊥ Institute of Molecular and Cell Biology, University of Tartu.

molecules,4 and (iii) regulation of the distribution of excess energy between photosystems II and I.5 The structure of LHC II was first analyzed by electron crystallography with a resolution of 3.4 Å.6 LHC II was found to form a trimer of subunits, up to 13 Chl, and at least two carotenoid molecules per monomeric subunit were initially identified. However, the resolution did not permit an unambiguous identification of Chl a and Chl b molecules. In a tentative assignment, seven Chl molecules in close contact to carotenoids were predicted to be Chl a based on the criterion of efficient quenching of chlorophyll triplets by carotenoids. The remaining more distant five molecules were believed to be Chl b. This assignment placed the closest center-to-center distances of ∼9-14 Å into pairs of Chl a-Chl b molecules; i.e. the strongest excitonic interactions were expected for Chl a-Chl b heterodimers, suggesting a relatively weak delocalization of excitonic states of LHC II.7 The identities of several Chl molecules (Chls a1, a2, a4, a5, b5, and b6 according to the nomenclature of ref 6) were confirmed by spectroscopic studies of LHC II mutants lacking single pigments,8-10 while different identities were observed in the case of Chl b39 as well as Chls a7, b1, and b3.8 A mixed occupancy was reported for several binding sites.8

10.1021/jp900836p CCC: $40.75  2009 American Chemical Society Published on Web 07/16/2009

Monomeric Light-Harvesting Complex II of Green Plants TABLE 1: Nomenclature of Chl Molecules in LHC II According to refs 6, 11, and 12 Chl type

Ku¨hlbrandt et al., 19946

Liu et al., 200411

Standfuss et al., 200512

a a a a a a a a b b b b b b

a1 a2 a3 a4 a5 a6 b2 b3

610 612 613 602 603 604 611 614 601 607 608 609 606 605

Chl 1 Chl 2 Chl 3 Chl 4 Chl 5 Chl 6 Chl 7 Chl 8 Chl 9 Chl 10 Chl 11 Chl 12 Chl 13 Chl 14

a7 b1 b5 b6

More recently, X-ray diffraction studies revealed the structures of trimeric LHC II from spinach11 and pea12 at nearly atomic resolution. According to these studies, each monomeric subunit binds 14 Chl molecules arranged in two layers close to the stromal and lumenal surfaces of the thylakoid membrane, respectively, and four carotenoid molecules. The identities of 8 Chl a and 6 Chl b molecules as well as the orientation of their transition dipole moments can now be unambigously assigned, while mixed occupancy was excluded. New nomenclatures for the Chl molecules were introduced in refs 11 and 12 (see Table 1 for a compilation) to account for new assignments of Chl identities and so far unresolved pigments. In the following, the notation of ref 11 will be used throughout this paper. The shortest center-to-center distances of ∼8-10 Å are reported for both Chl a-a and Chl b-b homodimers as well as for Chl a-b heterodimers. In addition, close center-to-center distances of up to ∼12 Å are also observed between Chl b molecules of different subunits. The Chls are preferentially bound via liganding and hydrogen bonding to the protein backbone formed by three membrane spanning and two amphipathic R-helices. However, there is also coordination of Chls via water molecules and in one exceptional case to the lipid phosphatidylglycerol (PG) located in the core of the LHC II trimer. It adds to the complexity of the system that the LHC II trimer can be constituted by various permutations of three different proteins referred to as Lhcb1-3, which are encoded in the nucleus by different genes and exhibit slightly different spectrocopic properties.13 All possible combinations of the three proteins except for the Lhcb3 homotrimer have been shown to be stable in reconstitution experiments.13 The functional role of the different Lhcb1-3 proteins is so far unknown, however the extent of their expression appears to depend on illumination conditions.14 Although it is widely believed that the trimeric form of LHC II is also dominant in ViVo, especially regulatory processes are most likely associated with changes in the LHCII structure12,15 and in its macroorganization.16-18 In this regard, monomerization of LHC II trimers can be induced under excess light conditions by a thermo-optic mechanism to prevent photodamage.16,17 There is also recent evidence that in ViVo a significant number of LHC II complexes is organized in ordered arrays of interacting trimers, whose spectroscopic properties resemble those of lamellar aggregates of LHC II.19 As to the light-harvesting function of isolated LHC II, a number of time-resolved spectroscopic studies revealed that Chl

J. Phys. Chem. B, Vol. 113, No. 31, 2009 10871 b f Chl a excitation energy transfer (EET) is ultrafast with kinetic components of ∼150 fs, 600 fs, and 10 ps at room temperature20-25 and exhibits only a weak dependence on temperature.24-26 The kinetics of Chl b f Chl a EET are phenomenologically quite similar in trimeric and monomeric LHC II, suggesting that the dominating EET steps occur within the monomeric subunit of LHC II.23 The spectral characteristics of EET were shown to be slightly different for the Lhcb1-3 proteins.25 The availability of high-resolution structural models with an unambigous assignment of pigment identities has been followed by a number of theoretical simulations.27-29 Low-temperature (77 K) time-resolved and frequency domain spectroscopic data were simulated using a modified Redfield approach.28 In this study, the femtosecond kinetics are attributed to fast EET within excitonically coupled clusters of Chl b and Chl a molecules, respectively, while the slower picosecond components are ascribed to relatively long-lived monomeric electronic states which are mainly found in the intermediate wavelength region of 660-670 nm. Consideration of intersubunit interactions led to improved fits of the frequency domain spectroscopic data. The terminal (fluorescent) excitonic state is placed into the cluster of strongly coupled Chl a molecules a610-a611-a612 in this model. At first glance, this appears to be in line with site-directed mutagenesis studies, which found the lowest excitonic state of LHC II to be located at Chl a612 at room temperature.8,10 However, at low temperatures of T < 120 K, this assignment is in contradiction with a previous hole burning study30 that found the lowest excitonic state of LHC II at ∼680 nm to be widely localized on one Chl molecule and weakly coupled to other Chl molecules. A weak satellite hole due to excitonic coupling was observed at ∼649 nm, which is almost coincident with the major Chl b absorption band of trimeric LHC II. Therefore, it is instructive to revisit the problem of assignment and nature of the lowest excitonic state and to compare results obtained for monomeric and trimeric LHC II. In the frequency domain, spectral hole burning (SHB) and complementary siteselective techniques are well-suited for analyses of excited state positions as well as for gauging the extent of homogeneous or inhomogeneous broadening and of electron-phonon coupling at low temperatures (for reviews, see Reddy et al.,31 Jankowiak et al.,32 and Reinot et al.33). Previous detailed hole burning studies of trimeric LHC II30,34 have established that the lowest excitonic energy level lies at ∼680 nm at 4.2 K, which is located about 4 nm to the red of the main absorption band at ∼676 nm. The 680 nm state was shown to be characterized by moderate electron-phonon coupling with a Huang-Rhys factor S of ∼0.9 as well as by a strongly asymmetric one-phonon profile with a mean phonon frequency ωm of ∼18 cm-1 and a width of ∼105 cm-1.35,36 Fluence-dependent hole burning experiments30 revealed three low-energy states located at 677.1 ( 0.2, 678.4 ( 0.2, and 679.8 ( 0.2 nm, each possessing an inhomogeneous width of 80 ( 10 cm-1. The absorption intensity of each of these low-energy states is equivalent to that of about one Chl a molecule per LHC II trimer so that each state should be widely localized on one Chl a molecule of a subunit. A high degree of localization is also in line with the weak satellite hole structure produced by hole burning within the ∼680 nm state and by the small pressure shift rates observed for holes in this spectral region.30 The three closely spaced low-energy states can most probably be identified with the lowest states of the Lhcb1-3 proteins constituting the LHC II trimer, which have later been shown to exhibit different spectroscopic properties.13,25

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Similar site-selective measurements on LHC II monomers, which probe the significance of intersubunit interactions and protein heterogeneity along with their effect on the low-energy excitonic structure of the monomeric LHC II subunit, are so far lacking. In the present paper, we address these problems by detailed 4.5 K hole burning and line-narrowed fluorescence experiments on monomeric LHC II. Upon monomerization by lipolysis, intersubunit excitonic interactions and EET among subunits of the LHC II trimer can be assumed to vanish, while effects due to protein heterogeneity, i.e., the presence of spectroscopically different Lhcb1-3 proteins, remain. The results presented can serve as benchmarks for future theoretical simulations of EET in LHC II. 2. Materials and Methods Sample Preparation. LHC II was purified from spinach photosystem II membrane fragments by sucrose density gradient centrifugation in the presence of n-β-dodecylmaltoside as described previously.37 This trimeric LHC II (see below) was shown to be phosphorylated on threonine (Thr) residues by using anti P-Thr (Zymed) in Western blotting experiments (data not shown). The Qy-absorption spectrum at room temperature exhibited two bands with maxima at 652 ( 1 and 675 ( 1 nm. The protein composition of LHC II was analyzed by SDS/ urea/PAGE at 4 °C in combination with silver staining as reported earlier.38,39 Samples equivalent to 5 µg of Chl were loaded onto the gels. Furthermore, Lhcb 1, 2, 3, 5, and 6 antibodies directed against synthetic peptides derived from the primary structures of the mature forms of the various LHC II apoproteins (Agrisera, Sweden), monoclonal anti Lhcb4, polyclonal anti PsbS, and polyclonal anti PsbA were used to analyze the protein composition of the preparations in detail by immunoblotting. No PsbA and Lhcb4 were identified; PsbS contributed less than 1% and Lhcb 5/6 less than 5% to the total amount of protein. The relative contributions of Lhcb 1, 2, and 3 were 40, 40, and 20 ( 10%, respectively, as semiquantitatively analyzed by densitometric scanning of the immunodecorated bands on polyvinylidene fluoride membranes. In addition, the protein composition of all samples has been analyzed by MALDI-TOF-mass spectrometry as described recently in Leupold et al.40 with some modifications. The protein content of LHC II samples was quantitatively determined following the method of Brown et al.41 The size of the complex was analyzed by gel filtration column chromatography (Superose HR 10/30, Amersham Biotech) using a FPLC system, density gradient centrifugation, or analytical ultracentrifugation. The molecular weight determined was 175 ( 20 kDa for the holocomplex including Chl a/b, carotenoids, and the n-β-dodecylmaltoside shell surrounding the pigment-protein complex. This mass is only consistent with the trimeric form of LHC II. Chl concentrations were determined as recommended by Porra et al.42 The carotenoid content as well as the Chl/ carotenoid ratio was calculated from measurements of the sample absorptions at 470, 646, 663, and 750 nm in 80% v/v acetone according to Porra et al.42 and Wellburn and Lichtenthaler.43 The Chl a/b ratio (w/w) was determined to be 1.4 ( 0.5 (n ) 22). At the employed detergent concentration of 0.025% w/v n-β-dodecylmaltoside, the trimeric form was highly stable as described recently in Pieper et al.36 Monomerization of trimeric LHC II was induced as described by Nussberger et al.,44 however with some modifications: samples were incubated in 50 mM Hepes-NaOH, pH 7.5, 10 mM MgCl2, 5 mM CaCl2, 0.04% w/v β-DM with

Pieper et al. phospholipase A2 from Apis mellifera (1360 U/ mg protein, Sigma) for 16 h at room temperature in the dark at a Chl/phospholipase A2 ratio (w/w) of 4.95/1. Incubation was performed under slight rotary shaking. Sample integrity and phospholipase A2 removal were tested by chromophore and protein analyses. The Chl a/b ratio (w/w) of 1.40 ( 0.5 determined after monomerization is quite similar to that of trimeric LHC II (see above), so that there is no indication for loss or decoupling of Chl. Monomeric LHC II was diluted in a glass forming buffer solution containing 50 mM Hepes-NaOH (pH 7.5), 10 mM MgCl2, 5 mM CaCl2, 0.04% w/v n-β-DM, and 70% w/w glycerol. This solvent composition ensures that the samples are virtually free of aggregation effects as discussed in ref 45. The Chl concentration of samples used for hole burning and fluorescence measurements was 4 µg/mL. Experimental Setup. Hole burning measurements with burn wavelengths between 640 and ∼680 nm were carried out using a Spectra Physics model 375 dye laser (line width of 676 nm. As discussed in the latter reference, this is qualitatively consistent with a shorter excited state lifetime and, thus, with an efficient depopulation of Qy states at shorter wavelengths than 676 nm by EET. Figure 2 shows almost saturated hole spectra for three representative λB values in this spectral region along with the absorption spectrum of monomeric

Figure 2. 4.2 K absorption (black line) and typical hole burned spectra of monomeric LHC II obtained with different burn wavelengths. Hole spectra were obtained with a burn fluence of about 50 J/cm2 and a read resolution of 0.4 nm. The burn wavelengths (labeled by arrows) were 648.8, 660, and 670 nm, respectively. The position of the antihole of hole A is marked by an asterisk. Hole spectra are separated by a ∆A of 0.05 for ease of inspection. A thick, broken arrow marks the position of a pseudophonon sideband hole which is located ∼20 cm-1 to the red of the ZPH at 670 nm. Letters A and B label the broad satellite holes at 679.6 and 648.8 nm. Small arrows label vibronic holes in the region of hole A.

LHC II. The hole burned spectra were obtained with a burn fluence of about 50 J/cm2 and λB values of 649 (blue line), 660 (red line), and 670 nm (green line). The arrows in Figure 2 indicate the burn wavelengths. These spectra are composed of three main features: (i) a narrow zero-phonon hole coincident with the burn wavelength, (ii) an intense hole in the vicinity of 680 nm (hole A) which appears as a result of efficient EET to a low-energy trap state (see below), and (iii) a weak satellite hole (hole B) at about 649 nm which builds on the low-energy 680 nm hole. The most pronounced feature of all hole spectra shown in Figure 2 is hole A located at 14715 ( 5 cm-1 (679.6 nm). Analogous to trimeric LHC II,30 it is found almost 4 nm lower in energy than the main absorption band at 676 nm. A Gaussian fit (full black lines in Figure 3) was based on the peak position and low-energy wing of hole A because the antihole of nonresonantly burned holes is typically blue-shifted and thus their red wing is less affected by interference with the antihole. This fit yielded a full width at half-maximum (fwhm) of 110 ( 10 cm-1. The non-Gaussian rise of the high-energy wing of hole A is due to the superposition with its antihole labeled by an asterisk. The position and width of hole A appear to be almost

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Figure 3. Low-energy region of hole burned spectra obtained using a burn wavelength of 670 nm and different fluences between 1 and 50 J/cm2. The green spectrum is the same as in Figure 1. The peak position and low-energy wing of hole A are fit to a Gaussian profile (black lines) centered at 14715 cm-1 (679.6 nm) and having a width of 110 cm-1. The position of the antihole of hole A is indicated by an asterisk. At the highest burn fluence employed, the ZPH at 670 nm is accompanied by a pseudophonon sideband hole (marked by a thick, broken arrow) with a peak frequency of ∼20 cm-1.

independent of burn wavelength (see Figure 2). Slight differences in the shape of hole A observed for different burn wavelengths are most likely due to an overlap with vibronic features. These narrow features represent ZPH within the lowest energy state which absorb at λB via intramolecular Chl a vibrations (see small arrows and labels in Figure 2). Furthermore, as shown by the Gaussian fits in Figure 3, hole A is almost independent of burn fluence (see Figure 3). Especially, the shift between low fluence and saturated hole A observed here is much smaller than the corresponding 1.5 nm blue-shift reported for trimeric LHC II.30 In summary, these results indicate that hole burning preferentially becomes effective after efficient EET to a low-energy trap state(s) which is most probably uncorrelated with higherenergy Qy states located at λB. Thus, the absorption band of the lowest energy state(s) of monomeric LHC II lies at about 679.6 nm and carries a width of about 110 cm-1. This width is predominantly due to inhomogeneous broadening, as will be discussed below. In ref 30, hole A was found to be fluencedependent in the case of trimeric LHC II. Therefore, comparative measurements were carried out for trimeric LHC II (not shown) which reproduced the blue-shift of hole A with increasing burn fluence using the same fluence values applied to obtain the monomer data presented in Figure 3. Information about higher-energy Qy states can be gathered from the narrow ZPH coincident with the burn wavelengths (see Figure 2). The holewidths were examined in detail for shallow ZPH with a fractional depth of less than 10% in order to avoid saturation broadening.51 However, the widths of the ZPH are in the order of the experimental resolution of 0.4 nm and thus have to be described by a Voigt profile, which is the convolution of a Gaussian resolution function and a Lorentzian hole profile. Then, the widths (fwhm) of the resulting Lorentzian contribution remain broader than expected from pure dephasing/spectral diffusion at 4.5 K (see, e.g., ref 52), so that they can be attributed to the lifetime (T1) of the Qy states excited directly at λB. Using T1 ) (πcΓhole)-1, where c is the speed of light and Γhole (cm-1) the Lorentzian holewidth, one obtains lifetimes of 1.1 ( 1.0, 2.8 ( 1.0, and 3.5 ( 1.0 ps for λB ) 649, 660, and 670 nm, respectively. These lifetimes T1 most probably represent the depopulation of the excited states probed at λB via Chl b f Chl a and Chl a f Chl a excitation energy transfer, respectively. Assuming a weak temperature dependence, the 1.1 ps lifetime

Pieper et al.

Figure 4. Hole depths of holes A and B measured at their peak positions as a function of burn fluences. The depth of hole B is approximately proportional to that of hole A with a constant factor of 10.

determined at 649 nm may be similar to the intermediate 600 fs component reported for Chl b f Chl a EET in LHC II at room temperature (see, e.g., ref 26). The equivalent of the faster ∼150 fs EET component, which slows down to ∼310 fs at 12 K,24 is not directly observed by hole burning, because it is most probably hidden by the broad hole B discussed in the next paragraph. The spectra shown in Figure 2 (see also Figure 6 below) reveal that hole burning at wavelengths shorter than 676.0 nm generally produces a broad and shallow high-energy satellite hole at 648.8 ( 0.5 nm (hole B) which is ∼0.7 nm to the blue of the main Chl b absorption band. Its position is not only independent of burn wavelength, but hole B is also observed when burning at lower energies, i.e., when the burn wavelength is located to the red of hole B itself. Generally, two interpretations for the appearance of hole B can be given (see ref 30): (a) hole B is an excitonic satellite hole of hole A at ∼680 nm, or (b) at high burn fluence, the strutural changes produced by hole burning at 680 nm are not confined to the vicinity of the corresponding Chl a molecule but may also affect the protein environment of neighboring Chl b molecules regardless of their actual excitonic coupling strength. However, with increasing burn fluence, the depth of hole B remains widely proportional to that of hole A within the fluence range under study (Figure 4). Therefore, it is reasonable to assume that hole B is an excitonic satellite hole of hole A at ∼679.6 nm. This means that the width of hole B of 130 ( 10 cm-1 is contributed to by inhomogeneous broadening and has to be described by a Voigt profile, which is the convolution of a Gaussian inhomogeneous component and a Lorentzian homogeneous contribution. Assuming that Γinh is similar to the width of hole A of ∼110 cm-1, the homogeneous contribution to hole B corresponds to a lifetime T1 of ∼240 fs at 4.5 K. This T1 component of ∼240 fs appears to be equivalent to the fast Chl b f Chl a EET component determined in transient absorption measurements. Hole B was not observed for a burn wavelength of ∼680 nm, which is located in the vicinity of the lowest energy Qy state (not shown). The latter finding indicates that the coupling of this state with higher energy states is weak. At λB ) 670 nm, another broad hole feature accompanies the ZPH at its lowenergy side (indicated by a dashed arrow in Figure 2). Since this hole is observed for this burn wavelength only and lies ∼20 cm-1 to the red of the ZPH, it is most probably a pseudoPSB; i.e., it represents the phonon wing of the ZPH at 670 nm. Constant fluence hole burning (ZPH action) spectroscopy was employed in the region of hole A in order to investigate the spectral position and inhomogeneous width of the lowest state’s

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Figure 5. 4.2 K absorption (red line) and ZPH action spectrum (red diamonds) of monomeric LHC II. The action spectrum obtained with a constant burn fluence of 3 mJ/cm2 and read resolution of 0.1 nm is shown in the left upper corner (see left and upper λ-scale) and fit by a Gaussian shape (black line) centered at 14715 ( 10 cm-1 (679.6 nm) with a width of 110 ( 10 cm-1. The position of the ZPH action spectrum within the absorption spectrum is indicated by black diamonds.

Figure 6. Comparison of nearly saturated hole burning spectra of monomeric (upper, blue line) and trimeric LHC II (lower red line) obtained with λB ) 660 nm, a burn fluence of 50 J/cm2, and a read resolution of ∼0.4 nm. The burn wavelength is marked by an arrow. Letters A, B, and C label the broad satellite holes at 679.6, 648.8, and 672.5 nm. The spectra are separated by offsets for ease of inspection; the respective baselines with ∆A ) 0 are shown in the vicinity of hole A by horizontal black lines. The lowest curve (purple) is the difference of the above hole burned spectra of monomeric and trimeric LHC II, which corresponds to satellite hole C. A fit of the difference spectrum is given as the black, smooth curve (see text for details of the subtraction and fit procedures).

absorption band. The action spectrum is shown in the upper left-hand corner of Figure 5. The fractional depths of the ZPH were smaller than 10% so that the action spectrum should not be significantly affected by saturation effects. Furthermore, there is no marked contribution from the ZPH of higher energy states because of their much shorter lifetimes due to efficient downward EET. The action spectrum can be fit by a Gaussian with a peak position at 14715 ( 10 cm-1 (679.6 nm) and a fwhm of 110 ( 10 cm-1. These values are in close agreement with the fit of hole A shown in Figure 2, which was based on the peak position and the shape of the low-energy wing of hole A. Thus, we conclude that the ZPH action spectrum represents the same low-energy Qy state(s) as hole A. The action spectrum finally establishes that hole A is mainly inhomogeneously broadened. The widths of the ZPH in the action spectrum shown in Figure 5 are limited by the read resolution of ∼2 cm-1. This means that the ZPH widths are resolution-broadened while their relative intensities are preserved; i.e., the shape of the action spectrum is not distorted by this type of measurement. Higher resolution experiments have not been performed, because it had been shown in detail for trimeric LHC II that the holewidths for λB in the near vicinity of 680 nm follow a T1.3 temperature dependence with an effective dephasing time of ∼100 ps at 4.5 K.30 This dependence is the well-known signature for spectral dynamics stemming from coupling to glass-like double-well potentials of the protein matrix (see, e.g., ref 52). Figure 6 shows nearly saturated hole burning spectra of both trimeric and monomeric LHC II, obtained with a burn fluence of about 50 J/cm2 and a burn wavelength of 660 nm. The composition of these spectra is qualitatively quite similar with the narrow ZPH at 660 nm and the satellite holes A and B at ∼680 and ∼649 nm, respectively. Monomerization of LHC II mainly affects the shape and spectral position of hole A, which is slightly red-shifted by ∼1.2 nm and markedly broadened from ∼80 to ∼110 cm-1; see above. Both effects can be explained by a change in the local environment of the chromophore responsible for the Qy state at ∼680 nm. Another difference is the presence of an additional satellite hole C at ∼671 nm for

trimeric LHC II. This indicates that the excitonic interaction responsible for the appearance of hole C is lost upon monomerization and provides a possibility to obtain the shape and spectral position of the corresponding absorption band (see the Discussion). In the case of trimeric LHC II, hole burning at wavelengths longer than ∼676 nm produces phonon sideband (PSB) holes located ∼18 cm-1 to the red of the narrow ZPH.30 However, it can be shown that spectral hole burning at the red-edge of the absorption profile may lead to a breakdown of the expected mirror symmetry between the lineshapes of real- and pseudoPSB and, thus, to the observation of a seemingly narrower onephonon profile.35,50 This effect is different from variations of the relative PSB intensities with, e.g., burn fluence53 or with the position of λB within an inhomogeneously broadened absorption band.54 Therefore, we follow the approach of refs 46-48 and employ delta-FLN spectroscopy for a comparative investigation of electron-phonon coupling on trimeric and monomeric LHC II. FLN spectra selectively excited within the fluorescence origin band of monomeric LHC II are shown in frame A of Figure 7 (black curve) for a representative excitation wavelength of 684 nm. As expected, the spectrum is composed of a sharp ZPL located at the excitation wavelength and the broad phonon wing peaking about 22 cm-1 to the red of the ZPL. In these spectra, the ZPL is still contaminated with scattered light originating from the excitation laser. Additional FLN spectra were recorded after hole burning at the excitation wavelength with different burn fluences ranging from 1.8 to 32 mJ/cm2 (blue curve in Figure 7A). Delta-FLN spectra are readily obtained after subtracting the postburn from the preburn FLN spectra. The delta-FLN spectrum shown as the red curve in Figure 7A was produced by a relatively high burn fluence of 32 mJ/cm2 to improve the signal-to-noise ratio in the region of the PSB. A similar spectrum that was obtained in the short burn time limit with a much lower burn fluence of 1.8 mJ/cm2 is shown in the inset of Figure 7A, which yields a ZPL unperturbed by saturation effects. Under these conditions, the intensity ratio between ZPL and PSB reflects the Huang-Rhys factor S; see below. Delta-FLN measurements under comparable experimental conditions were carried out for trimeric LHC II (see data in

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Figure 7. Experimental and simulated 4.5 K delta-FLN spectra of monomeric LHC II. Frame A: Delta-FLN spectrum excited/burned at 684.0 nm (lower red curve) with the ZPL cutoff at 2.8% of its full intensity. The black (a) and blue (b) curves show the corresponding pre- and postburn FLN spectra, respectively. FLN spectra were recorded with a fluence of 0.4 mJ/cm2, while the fluence applied for hole burning was 32 mJ/cm2. The inset shows a similar spectrum recorded with a much lower burn fluence of 1.8 mJ/cm2 within the short burn time limit, yielding a nonsaturated ZPL. Frame B: Simulation (black line) of the 4.5 K delta-FLN spectrum of frame A (green noisy curve) calculated using a Huang-Rhys factor S of 0.56, a peak phonon frequency of 22 cm-1, a half width at half-maximum (hwhm) of the Gaussian and the Lorentzian wings of 11 and 70 cm-1, respectively, and a site distribution function (SDF) with a width of 110 cm-1 peaking at 14715 cm-1. The inset shows the Huang-Rhys factors S obtained for selected burn/excitation wavelengths.

frame A of Figure 8). As discussed in detail in ref 48, the subtraction of the postburn from the preburn FLN spectra widely eliminates the scattering contribution and thus provides a reliable measure of the ZPL intensity. Both data sets can be fit according to eq 2 (see frame B of Figures 7 and 8) with widely similar asymmetric one-phonon pofiles composed of Gaussian and Lorentzian wings at the high- and low-energy sides of the peak frequency ωm (see captions of Figures 7 and 8 for parameters). This finding indicates that the structural changes upon monomerization do not significantly affect the density of vibrational states of LHC II. In contrast, the electron-phonon coupling strength is markedly reduced upon monomerization with Huang-Rhys factors S of 0.80 ( 0.08 for trimeric LHC II and 0.60 ( 0.06 for monomeric LHC II, respectively. Note that the S value of 0.8 is in close agreement with that of previous studies on trimeric LHC II.35,50 Discussion As outlined in detail in the Introduction section, LHC II has been reported to bind 8 Chl a and 6 Chl b molecules per monomeric subunit of the trimer complex.11,12 The current assignment of Chl identities (see schematic representation in Figure 9) suggests complex excitonic interactions within clusters of only Chl a or Chl b molecules but also mixed Chl a-b

Pieper et al.

Figure 8. Experimental and simulated 4.5 K delta-FLN spectra of trimeric LHC II. Frame A: Delta-FLN spectrum excited/burned at 682.0 nm (lower red curve) with the ZPL cutoff at 3.7% of its full intensity. The black (a) and blue (b) curves show the corresponding pre- and postburn FLN spectra, respectively. FLN spectra were recorded with a fluence of 0.4 mJ/cm2, while the fluence applied for hole burning was 32 mJ/cm2. Frame B: Simulation (black line) of the 4.5 K delta-FLN spectrum of frame A (green noisy curve) calculated using a Huang-Rhys factor S of 0.80, a peak phonon frequency of 22 cm-1, a HWHM of the Gaussian and the Lorentzian wings of 11 and 75 cm-1, respectively, and a site distribution function (SDF) with a width of 80 cm-1 peaking at 14705 cm-1.

clusters with dipolar couplings of up to 200 cm-1 in point dipole approximation.27-29 In general, it can be assumed that 14 Qy states contribute to the absorption spectrum of trimeric LHC II; in the case of sizable intersubunit pigment-pigment interaction and/or structural heterogeneity among the different subunits, the number may be as high as 42 Qy states. A full set of Qy states for the monomeric case has been obtained using a modified Redfield approach,28 which describes the main features of 77 K transient absorption and nonline-narrowed spectroscopic data. In the following, we compare the hole burning results obtained here for monomeric LHC II with those reported before for LHC II trimers and discuss the data in the light of the actual higher-resolution X-ray structures of LHC II.11,12 Assignment of Low-Energy Excitonic States. Spectral hole burning resonant to λB is especially sensitive to long-living excited Qy states because its quantum yield is directly proportional to the excited state lifetime (see, e.g., Kenney et al.55). Therefore, constant fluence hole burning is well-suited to investigate the position as well as the homogeneous and inhomogeneous broadening of the long-lived low-energy excited Qy states which are populated following EET. Thus, the preceding hole burning study on trimeric LHC II led to the identification of a distribution of low-energy states peaking at ∼678.4 nm30 by ZPH action spectroscopy, which was predominantly inhomogeneously broadened with a width (fwhm) Γinh of ∼85 cm-1. The ZPH action spectrum of monomeric LHC II obtained in the present study is located at 679.6 nm and thus

Monomeric Light-Harvesting Complex II of Green Plants

Figure 9. (left) Schematic representation of the arrangement of Chl molecules at the stromal (top) and lumenal side (bottom) of trimeric LHC II and their nomenclature following ref 11. The Chl molecules are shown in blue and green for Chl b and Chl a, respectively. Clusters of strongly coupled Chl molecules are encircled by blue and red lines. Chl a612 which carries the lowest excitonic state at room temperature according to refs 8 and 10 is shown in purple, while Chl a604 which may correspond to the lowest state at 679.6 nm at 4.5 K is shown in red. (right) Location of individual inhomogeneously broadened zerophonon absorption bands (bottom) within the 4.5 K absorption spectra of trimeric (upper red line) and monomeric LHC II (upper black line). The normalized absorption bands assigned in this study are shown at the bottom by black and red lines for monomeric and trimeric LHC II, respectively. The blue line is the absorption band of the fluorescing state of trimeric LHC II at 4.5 K. Each band is labeled by its peak wavelength.

shifted by ∼1.2 nm to the red compared to the trimeric case. It also exhibits a broader width Γinh of ∼110 cm-1. In addition, the ZPH action spectrum has a more pronounced non-Gaussian tailing toward the red than observed before for trimeric LHC II. The intensity of this non-Gaussian contribution accounts for less than 10% of the action spectrum. One possible reason for the non-Gaussian tailing is that the absorption peaks of the lowest energy states of Lhcb 1, 2, and 3 are most likely different30 with Lhcb3 exhibiting the red-most fluorescence origin at 77 K.13 Thus, the lowest state of Lhcb3 could be farther red-shifted upon monomerization than those of the other two Lhcb proteins. In the case of CP43, two quasi-degenerate lowenergy states leading to a non-Gaussian action spectrum were distinguished on the basis of a comparison of transient and persistent SHB.56,57 Transient SHB experiments on trimeric LHC II, however, did not yield results similar to CP43 (Pieper et al., unpublished) so that the contributions of the energetically inequivalent but uncorrelated low-energy states of the Lhcb 1, 2, and 3 proteins are most likely additive in monomeric LHC II. This situation precludes a further unambiguous deconvolution of the ZPH action spectrum. In addition, a small contribution of nonspecifically retrimerized complexes cannot be fully ruled out. Both the broadening and the slight red-shift of the entire ZPH action spectrum are phenomenologically consistent with a broadening of the 4.2 K absorption and fluorescence spectra as well as with a red-shift of the fluorescence peak of LHC II monomers (see Figure 1). These findings indicate that the protein environment of the chromophore carrying the 680 nm state is slightly disturbed upon monomerization. The broader inhomogeneous width and non-Gaussian shape of the ZPH action spectrum suggest an increased heterogeneity in monomeric LHC II, which can be readily explained by a higher degree of conformational freedom in the vicinity of the monomer-monomer interface and that of the PG molecule removed by lipolysis. This alteration of pigment-protein interaction must be taken into account when simulating spectroscopic data of monomeric

J. Phys. Chem. B, Vol. 113, No. 31, 2009 10877 LHC II. Note that the non-Gaussian tailing toward the red of the ZPH action spectrum does not indicate a large-scale aggregation, because narrow ZPH can be burned within this spectral region unlike in LHC II aggregates.45 The position of the inhomogeneously broadened zero-phonon absorption bands of the ∼680 nm Qy states of monomeric and trimeric LHC II are compared in Figure 9. In trimeric LHC II, only one low-energy Qy state located at ∼680 nm (see blue profile in Figure 9) was assigned as the fluorescent state at 4.2 K;30 i.e., fluorescence does not originate from the whole distribution of the low-energy states represented by the 678.4 nm ZPH action spectrum (see red profile at 678.4 nm in Figure 9). Such an assignment is plausible only in the case of remaining slow EET among the low-energy excited Qy states constituting the action spectrum, which leads to a population of the ∼680 nm state from energetically slightly higher levels within the action spectrum itself. Indeed, fluencedependent hole burning with λB < 675 nm consistently revealed a broad hole (hole A in Figure 6) which shifted from ∼680 to ∼678.4 nm with increasing fluence. This is in line with a higher burn efficiency for the 680 nm state, i.e., with a shorter lifetime of higher low-energy Qy states and concomitant slow EET to the 680 nm state. Consequently, three low-energy Qy states within the 678.4 nm ZPH distribution were assigned whose energetic splitting of ∼30 cm-1 was attributed to structural heterogeneity among the subunits of LHC II;30 i.e., one lowenergy state is located on each of the three subunits of the LHC II trimer. Although this interpretation has been questioned,58 later spectroscopic studies have indeed revealed different terminal state positions for the individual Lhcb1-3 proteins at 77 K;13,25 see below. A comparison of results obtained at 4.2 and 77 K is justified, because the position of the LHC II Qy states appears to be temperature-independent below ∼120 K.35 To conclude this section, we add that a very slow equilibration of excitation energy in the picosecond to nanosecond range is also visible in the spectral region at ∼680 nm in 77 K transient absorption spectra of native (trimeric) LHC II,24 which is also in agreement with the above model of the low-energy Qy states of LHC II. Upon monomerization of LHC II, however, intersubunit EET should be negligible and fluorescence should be emitted from all (three) low-energy states proposed by Pieper et al.30 for trimeric LHC II. In agreement with the expected scenario, the fluence dependence of hole A appears to be much smaller than for trimeric LHC II (see Figure 3 and the Results section), indicating the absence of downward EET among the low-energy states of the different subunits. Furthermore, hole A of the spectra shown in Figures 2 and 3 located at ∼679.6 nm represents the absorption band of the lowest fluorescing state(s) of monomeric LHC II. As such, it should mirror the fluorescence origin band found at 14689 cm-1 (680.8 nm) with a Stokes shift of 2Sωm between the respective peak positions (see Figure 1). With S ∼0.6 and ωm ) 22 cm-1 (cf. Results section), the Stokes shift roughly equals 26.4 cm-1, which is in very good agreement with the experimental value of 26 cm-1. Red-shifted states responsible for the non-Gaussian tailing of the ZPH action spectrum (see above) may add to the more pronounced asymmetry of the fluorescence spectrum. Thus, we conclude that in the case of monomeric LHC II all low-energy Qy states constituting the ZPH action spectrum contribute to the fluorescence spectrum at 4.5 K. Therefore, our results confirm those of ref 30, indicating the presence of three low-energy Qy states in trimeric LHC II, which are related by intersubunit EET. In the case of monomeric LHC II, however, the contributions of

10878 J. Phys. Chem. B, Vol. 113, No. 31, 2009 the three low-energy states to the action spectrum behave as individual terminal states for EET in their respective subunits and are no longer distinguishable by their fluence dependence in hole burned spectra. Thus, a structural heterogeneity among the three different Lhcb proteins is hidden within the action spectrum of monomeric LHC II. Pigment-Pigment Coupling Strengths. As to the nature of the lowest energy excitonic states of monomeric LHC II, it is instructive to estimate their contribution to the entire Chl a absorption. In Figure 5, the ZPH action spectrum (black diamonds) is shown along with the 4.5 K absorption spectrum of monomeric LHC II (full line). The intensity of the 679.6 nm profile was adjusted to account for the non-Gaussian tailing of absorption at wavelengths to the red of 680 nm. This approach is valid under the reasonable assumption that the contribution of the most intense absorption band at ∼676 nm is almost negligible for λ > 680 nm. Furthermore, it is reasonable to truncate integration of the absorption corresponding to Chl a molecules at ∼660 nm. The result is that the integrated intensity of the 679.6 nm state is responsible for ∼10% of the total Chl a absorption or ∼0.8 Chl a molecules per monomer. Therefore, within experimental uncertainty, the absorption intensity of the 679.6 nm state is that of one of the eight Chl a molecules bound per LHC II monomer. As similarly found for trimeric LHC II,30 the result suggests that the 679.6 nm state is widely localized on a single Chl a molecule. Another approach to gauge the extent of excitonic coupling is the evaluation of satellite hole structures. Briefly, in systems with strong pigment-pigment interaction, hole burning in any excitonic level evokes responses from the other exciton levels due to the delocalization of the excited state wave functions over the interacting chromophores. These responses appear as persistent satellite holes at the energetic positions of the related energy levels. Such effects have been observed, for example, for the special pair of Rhodopseudomonas Viridis59 and for the B800-less mutant of the bacterial LH2 antenna.60 The satellite hole structure produced by hole burning selectively anywhere within the Qy absorption band of monomeric LHC II is generally weak. Especially, no satellite holes are observed upon hole burning directly within the 679.6 nm state, which has been assigned as the lowest and fluorescent Qy state above. Shallow satellite holes become discernible only when nonselectively burning the broad hole A with λB < 676 nm, which corresponds to the inhomogeneously broadened absorption band of the 679.6 nm-state. As shown in Figure 6 for λB ) 660 nm, the satellite hole structure building on hole A encompasses one shallow hole at ∼648.8 nm in the Chl b region in the case of monomeric LHC II, while hole B is blue shifted to ∼648.2 nm and an additional hole C is found at ∼672.5 nm in the case of trimeric LHC II (see Figure 6). The positions of the inhomogeneously broadened zero-phonon absorption bands corresponding to the satellite holes observed for monomeric and trimeric LHC II are compared in Figure 9. As discussed in the Results section and ref 30, one possible reason for the appearance of hole B may be weak excitonic coupling between the Chl b and Chl a molecules carrying the 679.6 and 648.8 nm Qy states, respectively. Excitonic coupling between Chl a in the vicinity of ∼680 nm and Chl b is also in line with results of nonlinear polarization spectroscopy at room temperature for trimeric LHC II.61,62 Note in this regard that the term “weak coupling” refers to the intensity of the satellite hole structure and the degree of delocalization of the excitonic states responsible for production of satellite holes. As discussed in detail and illustrated by model calculations in ref 30, the actual delocalization of the excitonic

Pieper et al. wave functions and the excitonic splitting of the energy levels are small in the case of a Chl a-b heterodimer even for a relatively strong pairwise interaction energy of ∼100 cm-1, as suggested by the LHC II structure (e.g., ref 29) due to the relatively large energetic difference of the Qy levels of the interacting Chl molecules of ∼600 cm-1. The positions of the inhomogeneously broadened zero-phonon absorption bands of the ∼650 nm Qy states of monomeric and trimeric LHC II are also compared in Figure 9. The above observations of a pronounced localization of the 679.6 nm state and a possible weak excitonic coupling of the corresponding Chl a molecule to a Chl b can serve as benchmarks for a structural assignment of the lowest (fluorescing) Qy state of LHC II. On the basis of site-directed mutagenesis studies, Chl a612 (Chl a2 according to the nomenclature of ref 6) has been suggested to carry the lowest fluorescing Qy state of LHC II at room temperature.8,9 This Chl molecule is labeled in purple in Figure 9. According to the actual X-ray structures of LHC II,11,12 this Chl a molecule is part of a cluster of strongly coupled pigment molecules formed together with Chls a610 and a611. A similar assignment was obtained by simulations using a modified Redfield approach,28 which describe different 77 K spectroscopic data. The latter study finds the lowest Qy state of LHC II at ∼680 nm mainly localized at Chl a610 (∼50%), while the corresponding wave function also exhibits a significant delocalization over the neighboring Chls a611 and a612. In both cases, however, two satellite holes in the Chl a absorption range would be expected upon hole burning at ∼680 nm. Instead, hole A appears to be correlated with hole B in the Chl b absorption range (see above and the Results section). This apparent contradiction can be easily resolved taking into account the temperature dependence of the Chl a612 absorption band, which was shown to shift from ∼682 nm at room temperature to ∼676 nm at 4.2 K.10 Thus, it appears that the nature of the fluorescing Qy state at 4.2 K is essentially different from that at room temperature, and hole A at 679.6 nm cannot be identified with the absorption band of Chl a612. Alternatively, the ∼679.6 nm state of monomeric LHC II should be localized on a Chl a molecule in close contact to a Chl b, which is weakly coupled to other Chl a molecules. A likely candidate for this Chl a molecule in light of the actual X-ray structures11,12 is Chl a604 (labeled in red in Figure 9), which is located in a cluster with several Chl b molecules. Especially, the Chl a604/Chl b606 dimer exhibits the highest heterodimeric dipolar coupling strength of ∼100 cm-1 within the LHC II subunit. Thus, it is reasonable to identify the 679.6 nm state with Chl a604 at low temperature (T < 120 K). See Figure 9 for the positions of the inhomogeneously broadened zero-phonon absorption bands of the corresponding Qy-states. Some further arguments support an assignment of the 679.6 nm state to Chl a604. While spectroscopic data for a mutant lacking Chl a604 itself are not available, the 4.2 K absorption difference spectrum of a Chl b60610 mutant shows a pronounced loss of intensity at ∼650 nm accompanied by weaker absorption changes at 680 nm, i.e., almost exactly the spectral position of hole A. This may be the result of a redistribution of oscillator strength within the Chl a604-b606 heterodimer. In addition, time-resolved absorption experiments at 77 K by Palacios et al.25 revealed a direct EET channel from 650 to 680 nm, which was not observed for a pump wavelength of 662 nm. As pointed out above, an assignment of the 679.6 nm state to Chl a604 is also in line with previous studies placing the fluorescent state of LHC II at Chl a612 at room temperature, because the absorption band of Chl a612 was shown to shift from ∼682

Monomeric Light-Harvesting Complex II of Green Plants nm at room temperature to ∼676 nm at 4.2 K. This shift is in agreement with the temperature dependence of the non-linenarrowed fluorescence spectrum of trimeric LHC II, which exhibits a similar blue-shift with decreasing temperature between room temperature and about 120 K.35 The fluorescence spectra of trimeric LHC II measured below 120 K could be consistently described in ref 35 using the low-temperature excited state positions and electron-phonon coupling parameters determined by hole burning in ref 30. In summary, we conclude that Chl a604 most likely carries the lowest and fluorescent excitonic state of monomeric LHC II at 679.6 nm for temperatures below 120 K, while the fluorescent state at room temperature switches to Chl a612. The close spacing of the absorption bands of two Chls in the low-energy region of LHC II raises the question for the physiological significance of this “spectral congestion” of two excitonic energy levels at 679.6 and ∼676 nm, respectively. In this regard, it is interesting to note that Duffy et al.63 discuss a possible quenching configuration of the Chl a604-b606 heterodimer, although a different spectral position is assumed. If this configuration can be adopted by a reversible structural change, the specific functional role of an ∼680 nm state localized on Chl a604 may lie in the dissipation of excess energy. Another interesting phenomenon is the disappearance of hole C located at ∼672.5 nm upon monomerization of LHC II, which is best seen comparing the spectra shown in Figure 6. This effect cannot be the result of intersubunit excitonic interactions, because the satellite hole C is located within the spectral range of Chl a, while relatively close center-to-center spacings of Chls of different subunits are only reported for Chl b.11,12 In addition, it is unlikely that hole C is a satellite hole of hole A at 679.6 nm, because it appears only at high fluence and, thus, its growth kinetics is not correlated with that of hole A. There are two possible explanations for the formation of hole C in the case of trimeric LHC II. First, as already discussed for hole B above, the structural changes produced when burning hole A may extend to a neighboring Chl a. Then, the disappearance of hole C for similar burn fluence could only be explained by a change in the efficiency of this “extended” structural change upon monomerization. An alternative, rather indirect, explanation is to associate hole C with a Chl a molecule located in a protein region disturbed by monomerization. This may concern molecules at the monomer-monomer interface (e.g., Chl a602, a603) or bound directly to the PG molecule (Chl a611), which is removed upon monomerization by lipolysis. In both cases, structural changes upon monomerization may lead to a spectral shift or a change in orientation of the respective Chl a molecule affecting the strength of excitonic interaction with other, so far unidentified, Chl a molecules. As to a possible change of orientation, CD spectra reveal a loss of structure upon monomerization mainly in the Chl b range at 77 K64 and at room temperature.16 In the spectral range of Chl a absorption, the changes in 77 K CD spectra are less pronounced and may rather be interpreted as spectral shifts than as a loss of structure. This would be in agreement with our above interpretation for the disappearance of hole C. An identification of this/these Chl a molecules would require detailed fluence-dependent hole burning experiments on trimeric LHC II, which are beyond the scope of this study. Nevertheless, the absence of hole C in hole burned spectra of monomeric LHC II permits a determination of the spectral shape of the corresponding absorption band because the monomer spectrum shown in Figure 6 gives a realistic shape of the antihole of hole A in the region of hole C. To obtain the difference spectrum, hole A of the monomer spectrum was normalized to match the shape and position of hole

J. Phys. Chem. B, Vol. 113, No. 31, 2009 10879 A of trimeric LHC II at its high-energy side to compensate the red-shift of the 680 nm state observed upon monomerization; see above. After applying this procedure, the antihole shape represented by the monomer spectrum matches the hole burning spectrum of trimeric LHC II very well on both the high- and low-energy sides of hole C (not shown). The difference spectrum shown at the bottom of Figure 6 (purple line) is a broad and asymmetric curve peaking at 672.5 nm, which represents the line shape of the absorption band associated with hole C. This curve can be fit according to eq 1 in a first approximation with the same parameters of inhomogeneous broadening and electron-phonon coupling, i.e., Γinh ) 110 cm-1 and S ) 0.6, as determined before for the 680 nm state (see Figure 6). The fit slightly overestimates the highenergy wing of the difference spectrum, because of interference with the pseudo-PSB of the ZPH at 660 nm. Therefore, it seems that the lineshapes of the 672.5 and 680 nm states are quite similar. Although a distinct assignment of hole C cannot be achieved on the basis of our hole burning data, the 672.5 nm state represented by this hole should be located on a Chl a site disturbed upon removal of PG during monomerization of LHC II. Conclusions Spectral hole burning at 4.5 nm has been employed to characterize the position and nature of excited energy Qy states of monomeric LHC II produced by a phospholipase treatment. In monomeric LHC II, the lowest energy state(s) is (are) located at 679.6 nm and populated by efficient excitation energy transfer from higher-energy Qy states. ZPH action spectroscopy establishes that the 679.6 nm state is predominantly inhomogeneously broadened with a width (fwhm) Γinh of ∼110 cm-1. Thus, monomerization of the LHC II trimer leads to a red-shift of the lowest energy states of ∼1.2 nm and a considerable increase of their inhomogeneous broadening. These findings indicate that the protein environment of the chromophore carrying the 679.6 nm state is slightly disturbed upon monomerization by lipolysis and appears to exhibit an enhanced protein heterogeneity due to a higher degree of (static) conformational freedom. Delta-FLN spectroscopy has been used to show that the absorption band of the lowest energy state(s) of monomeric LHC II located at ∼679.6 nm (14715 cm-1) is coupled to low-frequency protein vibrations with a Huang-Rhys factor S of ∼0.6 and a strongly asymmetric one-phonon profile with a mean phonon frequency of ωm ) 22 cm-1. The resulting Stokes shift 2Sωm roughly equals 26.4 cm-1, which readily explains the experimentally observed position of the fluorescence origin band at 680.8 nm (14689 cm-1). Thus, we conclude that the 679.6 nm state(s) is (are) the fluorescent state(s) of monomeric LHC II at 4.5 K. The integrated intensity of the 679.6 nm state(s) corresponds to ∼10% of the total Chl a absorption or ∼0.8 Chl a molecules per monomer. Therefore, within experimental uncertainty, the absorption intensity of the 679.6 nm state is close to that of one of the eight Chl a molecules bound per LHC II subunit. In addition, the satellite hole structure produced by hole burning within the 679.6 nm state is weak. As similarly found for trimeric LHC II, the result suggests that the 679.6 nm state is widely localized on a single Chl a molecule. However, a shallow satellite hole in the Chl b spectral range at 648.8 nm is observed upon nonselective hole burning within the lowest energy 679.6 nm state(s) regardless of the actual burn wavelength. The depth of the 648.8 nm hole scales roughly with that of the inhomogeneously broadened hole of the 679.6 nm state(s). This finding may indicate a weak excitonic coupling between the Chl a molecule carrying the lowest fluorescent state and a Chl b molecule. On the other hand, the structural changes

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produced by hole burning in the 679.6 nm state(s) may not be fully localized but extend into the vicinity of neighboring Chl b molecules. In any case, at 4.2 K, the lowest Qy state of LHC II appears to be widely localized on one Chl a molecule, which is possibly excitonically coupled to or at least located in the close neighborhood of a Chl b molecule. These characteristics are different from those expected for Chl a612, which has been associated with the fluorescent state at room temperature, but shifts to ∼676 nm at 4.2 K. Alternatively, the 679.6 nm state may be assigned to Chl a604 located in a cluster with several Chl b molecules. As a consequence, the low-energy-level structure of LHC II may be subject to a very complex temperature dependence. The reason for the very close energetic spacing of two different low-energy Qy states remains to be clarified. Acknowledgment. Estonian Science Foundation (Grant No. 7002) has supported this work. J.P. and K.-D.I. gratefully acknowledge support from Deutsche Forschungsgemeinschaft (SFB 429, TP A1 and TP A3, respectively). We are also grateful to S. Kussin and M. Weβ (TU Berlin) for their help in sample preparation. References and Notes (1) van Amerongen, H.; Croce, R. In Primary processes of photosynthesis: Basic principles and apparatus; Renger, G., Ed.; RSC Publ.: Cambridge, U.K., 2008; Vol. I, p 329. (2) Horton, P.; Ruban, A.; Walters, R. G. Annu. ReV. Plant Physiol. 1996, 47, 655. (3) Pascal, A. A.; Liu, Z. F.; Broess, K.; van Oort, B.; van Amerongen, H.; Wang, C.; Horton, P.; Robert, B.; Chang, W. R.; Ruban, A. Nature 2005, 436, 134. (4) Scho¨del, R.; Irrgang, K.-D.; Voigt, J.; Renger, G. Biophys. J. 1999, 75, 3143. (5) Allen, J. F.; Forsberg, J. Trends Plant Sci 2001, 6, 317. (6) Ku¨hlbrandt, W.; Wang, D. N.; Fujiyoshi, Y. Nature 1994, 367, 614. (7) Renger, T.; May, V. Phys. ReV. Lett. 2000, 84, 5228. (8) Remelli, R.; Varotto, C.; Sandona, D.; Croce, R.; Bassi, R. J. Biol. Chem. 1999, 274, 33510. (9) Rogl, H.; Ku¨hlbrandt, W. Biochemistry 1999, 38, 16214. (10) Rogl, H.; Scho¨del, R.; Lokstein, H.; Ku¨hlbrandt, W.; Schubert, A. Biochemistry 2002, 41, 2281. (11) Liu, Z.; Yan, H.; Wang, K.; Kuang, T.; Zhang, J.; Gui, L.; An, X.; Chang, W. Nature 2004, 428, 287. (12) Standfuss, J.; Lamborghini, M.; Ku¨hlbrandt, W.; van Scheltinga, A. C. T. EMBO J. 2005, 24, 919. (13) Standfuss, J.; Ku¨hlbrandt, W. J. Biol. Chem. 2004, 279 (35), 36884. (14) Irrgang, K.-D.; Slowik, D.; Miao, J.; Scharf, K.; Weβ, M.; Kussin, S. In Photosynthesis: Fundamental Aspects to Global PerspectiVes; van der Est, A., Bruce, D., Eds.; Alliance Communications Group: Lawrence, KS, 2005; pp 689-691. (15) Ruban, A. V.; Berera, R.; Ilioaia, C.; van Stokkum, I. H. M.; Kennis, J. T. M.; Pascal, A. A.; van Amerongen, H.; Robert, B.; Horton, P.; van Grondelle, R. Nature 2007, 450, 575. (16) Garab, G.; Cseh, Z.; Kova´cs, L.; Rajagopal, S.; Va´rkonyi, Z.; Wentworth, M.; Musta´rdy, L.; Der, A.; Ruban, A. V.; Papp, E.; Holzenburg, A.; Horton, P. Biochemistry 2002, 41, 15121. (17) Holm, J. K.; Va´rkonyi, Z.; Kova´cs, L.; Posselt, D.; Garab, G. Photosynth. Res. 2005, 86, 275. (18) Horton, P.; Wentworth, M.; Ruban, A. FEBS Lett. 2005, 579, 4201. (19) Lambrev, P. H.; Va´rkonyi, Z.; Krumova, S.; Kova´cs, L.; Miloslavina, Y.; Holzwarth, A. R.; Garab, G. Biochim. Biophys. Acta 2007, 1767, 847. (20) Du, M.; Xie, X.; Mets, L.; Fleming, G. R. J. Phys. Chem. 1994, 98, 4736. (21) Bittner, T.; Irrgang, K.-D.; Renger, G.; Wasilewski, M. R. J. Phys. Chem. 1994, 98, 11821. (22) 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. 1997, 101, 1902. (23) Kleima, F. J.; Gradinaru, C.; Calkoen, F.; van Stokkum, I. H. M.; van Grondelle, R.; van Amerongen, H. Biochemistry 1997, 36, 15262. (24) Gradinaru, C. C.; van Grondelle, R.; van Amerongen, H. J. Phys. Chem. B 2003, 107, 3938.

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