Excitation Wavelength-Dependent Electron−Phonon and Electron

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J. Phys. Chem. B 2008, 112, 110-118

Excitation Wavelength-Dependent Electron-Phonon and Electron-Vibrational Coupling in the CP29 Antenna Complex of Green Plants Margus Ra1 tsep,† Jo1 rg Pieper,*,§ Klaus-Dieter Irrgang,⊥ and Arvi Freiberg*,†,‡ Institute of Physics, and Institute of Molecular and Cell Biology, UniVersity of Tartu, Riia 142, 51014 Tartu, Estonia, Max-Volmer-Laboratories for Biophysical Chemistry, Technical UniVersity Berlin, PC14, Strasse des 17. Juni 135, 10623 Berlin, Germany, and Department of Life Science and Technology, Laboratory of Biochemistry, UniVersity of Applied Sciences, Forum Seestrasse, Seestrasse 64, 13347 Berlin, Germany ReceiVed: July 3, 2007; In Final Form: September 23, 2007

Electron-phonon and electron-vibrational coupling strengths of a weakly (excitonically) coupled chlorophyll a S1 f S0 transition of the CP29 antenna complex of plant photosystem II were studied by difference fluorescence-line-narrowing spectroscopy at 4.5 K. A strong, almost linear increase of the electron-phonon coupling strength toward longer wavelengths was observed, with Huang-Rhys factors Sph increasing from 0.41 ( 0.05 at 680 nm to about 0.66 ( 0.07 at 688 nm. The former and latter wavelengths are located close to the peak and on the red edge of the inhomogeneous site distribution function, respectively. The experimentally obtained wavelength dependence of Sph may originate either from an alteration of the electron-phonon coupling strength by the local environment of the fluorescing chromophore and/or from the presence of two isoforms of CP29, which are characterized by different coupling strengths to the protein environment. The one-phonon profile peaks at ωm ) 22 cm-1 and is described by an asymmetric function composed of a Gaussian lowenergy wing and a Lorentzian high-energy tail with half-widths at half-maximum of 10 ( 1 and 60 ( 10 cm-1, respectively. Thirty-nine individual vibrational modes between 90 and 1665 cm-1 were resolved, and their Huang-Rhys factors were determined, which fall in the range between 0.0004 and 0.032. The broad feature present in the overlap region of phonon and vibrational modes at about 90 cm-1 is characterized by S ) 0.048. An integral value of vibrational coupling strengths Svib ) 0.36 ( 0.05 was determined, which is similar to that observed earlier for the trimeric LHC II complex.

1. Introduction Photosynthesis is initiated by light absorption in antenna protein complexes and subsequent excitation energy transfer (EET) to reaction center complexes. There, the primary charge separation takes place upon the formation of a cation anion radical pair P+•I- (see, e.g., refs 1 and 2). In plants, the largest among the minor chlorophyll (Chl) a/b binding antenna complexes is CP29, also referred to as Lhcb4. It has been shown to bind six Chl a, two Chl b, and two to three xanthophyll molecules.3-5 Despite the lack of direct information from electron or X-ray crystallography, a first structural model for CP29 has been proposed6 which is based on the high sequence homology among the family of light-harvesting complex II (LHC II) proteins and the finding that most of the Chls present in CP29 acquire similar binding sites as in LHC II.7-9 The transmembrane folding pattern of CP29 is also expected to be similar to that of the LHC II complex. In contrast to LHC II, however, CP29 does not form trimers. Early spectroscopic studies of both native5,10 and reconstituted CP293 involved temperature-dependent absorption and fluorescence as well as linear and circular dichroism spectroscopy. The * Authors to whom correspondence should be addressed. Tel.: +3727374612 (A.F.); 49-30-31427782 (J.P.). Fax: +3727383033 (A.F.); 49-30-31421122 (J.P.). E-mail: [email protected] (A.F.); [email protected] (J.P.). † Institute of Physics, University of Tartu. ‡ Institute of Molecular and Cell Biology, University of Tartu. § Technical University Berlin. ⊥ University for Applied Sciences.

low-temperature absorption exhibits two discernible bands at 638 and 650 nm attributed to Chl b molecules and a relatively broad Chl a band peaking at 674 nm. This provides the possibility to study Chl b f Chl a excitation energy transfer selectively for each of the two Chl b bands of CP29. Lowtemperature time-resolved pump-probe spectroscopy of CP29 at 77 K11 revealed that excitation energy is transferred from the Chl b band at 638 nm to a Chl a molecule absorbing at ∼676 nm in about 350 fs. EET from the second Chl b band at 650 nm to Chl a at ∼670 nm was found to be slower with a kinetic component of 2.2 ps. A fast 280 fs and a slower 10-13 ps time constant were observed for equilibration among the Chl a molecules. Similarly rapid EET kinetics have been obtained for Chl b f Chl a EET at room temperature12 and for carotenoid to Chl EET.13,14 A detailed understanding of the ultrafast dynamics of EET requires information on the energy level structure of the excited electronic states, which is governed by pigment-pigment and pigment-protein interactions on the nanoscale level,15,16 but obscured by inhomogeneous broadening of the spectra. In this regard, spectral hole-burning (SHB) studies performed at 4.2 K have established that the lowest Qy-state of CP29 absorbs at 678.2 nm and carries an inhomogeneous width of ∼120 cm-1.17 Its absorbance was shown to correspond to that of just one Chl a molecule out of six in CP29. The latter finding along with the weak satellite hole structure produced by hole-burning within the Qy absorption band indicated that this particular Chl a molecule is weakly coupled to the other chlorophylls. Therefore, it appears that the wavefunction of the lowest-energy state is

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∆FLN Spectra of the CP29 Antenna Complex highly localized on a single Chl a molecule. Further studies using nonlinear polarization spectroscopy and stepwise twophoton excited fluorescence techniques confirmed the nature of the lowest Qy-state of CP29 as being widely localized on a single Chl a molecule.18,19 Less detailed information is available about electron-phonon and electron-vibrational (vibronic) coupling in CP29. So far, the hole-burning data17 indicated that the phonon sideband of the 678.2 nm Qy-state peaks at ωm ∼ 20 cm-1 and that the electron-phonon coupling is weak with a Huang-Rhys factor Sph in the order of 0.5. A number of S0 f S1 vibrational frequencies were observed, in general agreement with those of Chl a embedded in amorphous matrixes.20 However, as pointed out in the case of the closely related trimeric LHC II complex, a reliable characterization of electronphonon coupling requires a combination of hole-burning and site-selective fluorescence spectroscopy.21 Considerable uncertainty is also present in the literature concerning the vibronic coupling strengths. The vibronic Franck-Condon factors determined by Gillie et al.22 for PSI-200 complexes using SHB spectroscopy and those reported later by Peterman et al.23 from fluorescence-line-narrowing (FLN) spectra of trimeric LHC II differ by 1-2 orders of magnitude. (Do not be confused by the intermittent use of Huang-Rhys and Franck-Condon factors. The vibronic Franck-Condon factor is equal to Sj exp(-Sj), which for small (250 cm-1,20 so the origin of the lowfrequency modes must be different. Various possibilities argued about in the literature involve partly localized vibrations of the protein or intermolecular, e.g., Chl-Chl, vibrations. Among the best-known interpretations of the latter kind is the so-called marker mode of bacterial reaction centers (115-135 cm-1), assigned to the intermolecular vibration of a pair of bacteriochlorophyll molecules.50 Similar explanations were given later for features observed in the 50-100 cm-1 range in the spectra of plant PS II reaction centers,51 in CP47 antenna complexes,52 as well as in bacterial antenna complexes.34 The mode at 90 cm-1 observed in CP29 could be of similar intermolecular nature. Despite the precise origin of the low-frequency vibrations, however, they should be classified as pseudolocal rather than local modes, because of strong overlap with the continuous one-phonon spectrum. The relatively large breadth of these modes evident in Figure 3 supports this conclusion. Nature of the Lowest-Energy Emitting State(s). As discussed above, using ∆FLN spectroscopy it is straightforward to determine Sph factors at various positions within an inhomogeneously broadened fluorescence band. A genuine question now arises about the possible origin of the observed systematic rise of Sph in CP29 from 0.41 to 0.66 with increasing excitation wavelength from 678 to 688 nm. Variances of Sph within an inhomogeneous ensemble of chromophores have been observed.53 Therefore, the simplest possibility to explain our observations is to assume a direct correlation of the electronphonon coupling strength with the electronic transition energy within the SDF, i.e., within the inhomogeneously broadened absorption profile of the lowest Qy-state of CP29. Alternatively, the experimental wavelength dependence could stem from the heterogeneous mix of two antenna complexes with slightly shifted inhomogeneous distributions of their Qy origin states, which also possess different coupling strength to the protein environment. If these two SDF are not coupled by excitation energy transfer, Sph of each of these distributions would dominate the electron-phonon coupling strength measured at the blue and red side of the CP29 fluorescence origin

band, whereas the linear dependence of Sph at intermediate wavelengths is due to a superposition of the transitions from different distributions. Although a former hole-burning study did not yield any indication for the presence of two separate SDF,17 there is recent evidence for two isoforms in CP29 from spinach, referred to as Lhcb4.1 and Lhcb4.x.54 A comparison of ∆FLN and FLN spectra of CP29 measured at the same excitation wavelength seems to bring up another supporting argument. Both types of the spectra are quite similar at 684 nm, i.e., at the red edge of SDF where maximum selectivity is reached for both FLN and ∆FLN. In contrast, at shorter wavelengths, e.g., at 680.0 nm, the ∆FLN spectrum appears more structured than the FLN spectrum. In the case of uncoupled but shifted distributions, the states from the “blue” SDF are selectively excited at its red edge, whereas transitions from the “red” SDF are excited mainly nonselectively. Then, the latter may undergo hole-burning with much smaller efficiency and would, consequently, contribute relatively less to the ∆FLN than to the FLN spectrum. Still, as an unambiguous assignment of the two species appears impossible at present, the issue of the lowest-energy states remains open awaiting future studies. 5. Summary A novel site-selective spectroscopic technique referred to as “difference fluorescence-line-narrowing” (∆FLN) spectroscopy has been used for a detailed characterization of electron-phonon and electron-vibrational coupling strengths of the lowest Qystate of the antenna complex CP29 of photosystem II of green plants at 4.5 K. ∆FLN spectra are obtained as the difference between FLN spectra recorded before and after hole-burning at the excitation wavelength. The main advantage of ∆FLN is that it readily yields the ZPL together with the phonon and vibrational structure building on it so that electron-phonon and electron-vibrational coupling strengths can be determined directly at any position within an inhomogeneously broadened fluorescence band. In the case of CP29, the electron-phonon coupling strength was found to depend strongly on the excitation wavelength with Huang-Rhys factors increasing almost linearly from 0.41 ( 0.05 at 680 nm to about 0.66 ( 0.07 at 688 nm. The simplest possible explanation of this effect is to assume a direct correlation of the electron-phonon coupling strength with the electronic transition energy within SDF. This means, the electron-phonon coupling strength could be altered by the local environment of the fluorescing chromophore. Alternatively, the experimental wavelength dependence may originate from the presence of two isoforms of CP29 with slightly shifted inhomogeneous distributions of their Qy origin states, which are characterized by different coupling strengths to the protein environment. A detailed simulation of the ∆FLN spectra of CP29 yielded parameters of the one-phonon profile which is related to the density of vibrational states of the protein phonons. The onephonon profile for the 680 nm excitation was found to peak at 22 cm-1 and is composed of a Gaussian with a half-width (hwhm) of 10 cm-1 at its low-energy side and a Lorentzian with a half-width (hwhm) of 65 cm-1 on its high-energy side. A slightly narrower one-phonon function with a Lorentzian hwhm of 55 cm-1 was obtained for 684 nm. These parameters have been shown to fit the apparently different line shapes of both the real- and the pseudo-PSB. As similarly found for trimeric LHC II, the latter nonresonant phonon sideband feature appears to be suppressed by the relatively narrow width of the SDF. This finding nicely demonstrates another advantage of

118 J. Phys. Chem. B, Vol. 112, No. 1, 2008 ∆FLN spectroscopy. In contrast to spectral hole-burning, the phonon wing of ∆FLN spectra is composed of both the realand the pseudophonon sideband contributions, thus permitting direct access to the parameters of the one-phonon profile from a resonantly excited spectral feature. A number of vibrational modes with frequencies between 90 and 1665 cm-1 could be resolved which build on a weakly (excitonically) coupled Chl a S1 f S0 transition of CP29. With the use of ∆FLN spectroscopy, the corresponding electronvibrational coupling strengths could be directly determined yielding individual Huang-Rhys factors in the range between 0.0004 and 0.032. These values are in good agreement with those found for PS I-200 complexes by Gillie et al.22 and with the integrated vibrational coupling strength of Svib ) 0.43 obtained by Pieper et al.24 for the LHC II trimer. Acknowledgment. The 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. Wess (TU Berlin) for their help in sample preparation. References and Notes (1) van Grondelle, R.; Dekker, J. P.; Gillbro, T.; Sundstro¨m, V. Biochim. Biophys. Acta 1994, 1187, 1. (2) Renger, G. In Concepts in Photobiology and Photomorphogenesis; Singhal, G. S., Renger, G., Sopory, K., Irrgang, K.-D., Govindjee, Eds.; Narosa Publishing House: New Delhi, India, 1999; p 52. (3) Giuffra, E.; Zucchelli, G.; Sandona, D.; Croce, R.; Cugini, D.; Garlaschi, F. M.; Bassi, R.; Jennings, R. C. Biochemistry 1997, 36, 12984. (4) Sandona, D.; Croce, R.; Pagano, A.; Crimi, M.; Bassi, R. Biochim. Biophys. Acta 1998, 1365, 207. (5) Pascal, A.; Gradinaru, C.; Wacker, U.; Peterman, E.; Calkoen, F.; Irrgang, K.-D.; Horton, P.; Renger, G.; van Grondelle, R.; Robert, B.; van Amerongen, H. Eur. J. Biochem. 1999, 262, 817. (6) Bassi, R.; Croce, R.; Cugini, D.; Sandona, D. Proc. Natl. Acad. Sci. U.S.A. 1999, 96, 10056. (7) Jansson, S. Biochim. Biophys. Acta 1994, 1184, 1. (8) Pesaresi, P.; Sandona, D.; Giuffra, E.; Bassi, R. FEBS Lett. 1997, 402, 151. (9) Pascal, A.; Wacker, U.; Irrgang, K.-D.; Horton, P.; Renger, G.; Robert, B. J. Biol. Chem. 2000, 275, 22031. (10) Zucchelli, G.; Dainese, P.; Jennings, R. C.; Breton, J.; Garlaschi, F. M.; Bassi, R. Biochemistry 1994, 33, 8982. (11) Gradinaru, C. C.; Pascal, A.; van Mourik, F.; Robert, B.; Horton, P.; van Grondelle, R.; van Amerongen, H. Biochemistry 1998, 37, 1143. (12) Croce, R.; Mu¨ller, M. G.; Bassi, R.; Holzwarth, A. R. Biophys. J. 2003, 84, 2508. (13) Gradinaru, C. C.; van Stokkum, I. H. M.; Pascal, A. A.; van Grondelle, R.; van Amerongen, H. J. Phys. Chem. B 2000, 104, 9330. (14) Croce, R.; Mu¨ller, M. G.; Caffarri, S.; Bassi, R.; Holzwarth, A. R. Biophys. J. 2003, 84, 2517. (15) Ku¨hn, O.; Renger, T.; May, V.; Voigt, J.; Pullerits, T.; Sundstro¨m, V. Trends Photochem. Photobiol. 1997, 4, 213. (16) Novoderezhkin, V. I.; Palacios, M. A.; van Amerongen, H.; van Grondelle, R. J. Phys. Chem. B 2004, 108, 10363. (17) Pieper, J.; Irrgang, K.-D.; Ra¨tsep, M.; Voigt, J.; Renger, G.; Small, G. J. Photochem. Photobiol. 2000, 71, 574. (18) Voigt, B.; Irrgang, K.-D.; Ehlert, J.; Beenken, W.; Renger, G.; Leupold, D.; Lokstein, H. Biochemistry 2002, 41, 3049. (19) Leupold, D.; Teuchner, K.; Ehlert, J.; Irrgang, K.-D.; Renger, G.; Lokstein, H. Biophys. J. 2002, 82, 1580. (20) Avarmaa, R. A.; Rebane, K. K. Spectrochim. Acta, Part A 1985, 41, 1365. (21) Pieper, J.; Voigt, J.; Renger, G.; Small, G. J. Chem. Phys. Lett. 1999, 310, 296. (22) Gillie, J. K.; Small, G. J.; Golbeck, J. H. J. Phys. Chem. 1989, 93, 1620.

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