Electron-Phonon Coupling and Vibronic Fine Structure of Light-Harvesting Complex II of Green Plants: Temperature Dependent Absorption and High-Resolution Fluorescence Spectroscopy Erwin J. G. Peterman,* ,² To ˜ nu Pullerits, ‡ Rienk van Grondelle, ² and Herbert van Amerongen ² Department of Physics and Astronomy and Institute for Molecular Biological Sciences, Vrije UniVersiteit, De Boelelaan 1081, 1081 HV Amsterdam, The Netherlands, and Department of Chemical Physics, Chemical Center, Lund UniVersity, Lund, Sweden ReceiVed: July 31, 1996; In Final Form: March 24, 1997 X Polarized, site-selected fluorescence was measured for light-harvesting complex II (LHCII), the major Chl a/b/xanthophyll binding light-harvesting complex of green plants. Upon selective excitation in the range of 679-682 nm at 4 K, separate zero-phonon lines and phonon wings could be observed, as well as sharp lines in the vibronic region of the emission: vibronic zero-phonon lines. The maximum of the phonon wing was located 22 cm -1 to the red of the zero-phonon line. Forty-eight vibrational modes could be identified, and their Franck-Condon factors were estimated. From the vibrational frequencies it is concluded that the Chl a responsible for the emission at 4 K is monoligated and accepts a hydrogen bond on the 13 1 -keto group. Also measured was the temperature dependence of the absorption spectrum of LHCII. Using the phonon wing obtained from the fluorescence measurements and an algorithm based on linear, harmonic Franck- Condon electron-phonon coupling and temperature independent inhomogeneous broadening, the temperature dependence of the low-energy part of the Q y absorption spectrum could be simulated very well up to 220 K. Above this temperature, the simulated and experimental results start to deviate. From the simulations it is concluded that inhomogeneous broadening of the long-wavelength band(s) (676 nm and above) is 120 ( 15 cm -1 below 220 K, whereas the Huang-Rhys factor of the protein phonons is 0.6 ( 0.1 (at 4 K). We have modeled the results from absorption, fluorescence, hole-burning, triplet-minus-singlet absorption, and fluorescence anisotropy measurements by one Gaussian inhomogeneous distribution function (peaking near 676 nm) with the spectroscopic properties of the lowest energy state(s) at 4 K. There was a significant discrepancy between the results from the simulations and the experiments. A much better agreement could be obtained by assuming either two Gaussian distributions (centered around 676 and 680 nm with an intensity ratio of 11:1) or a non-Gaussian distribution around 676 nm. Although we cannot discriminate between these two descriptions, both simulations have in common that at least nine separate electronic states per trimer are present in the 676 (and 680 nm) band. Introduction In photosynthetic light-harvesting complexes, a complicated interplay between different pigment molecules is responsible for the details of light absorption and efficient energy transfer to the reaction centers where photochemistry takes place. 1 For a good understanding of the light-harvesting processes, a detailed insight into the factors that determine the absorption spectrum of the pigment-protein complexes involved is essential. The absorption spectrum of a single pigment (“site”) in a protein at low temperature consists of two parts: 2,3 one that is only due to intramolecular (pure electronic and vibronic) transitions and another that reflects coupling of these transitions to the intermolecular vibrations of the protein environment (phonons). The former contributions give rise to Lorentzian-shaped zero- phonon lines (ZPLs), the widths of which are determined by the dephasing times of the excited states. The latter contribu- tions give rise to broad wings, the phonon wings (PWs) to the blue of the ZPLs. Both contributions can only be observed separately at low temperature; at temperatures above about 100 K the PW totally masks the zero-phonon contribution. The ratio of the intensity of pure electronic and electron-phonon transi- tions depends on the strength of electron-phonon coupling expressed in the Huang-Rhys factor (S). 2,3 The energy of the observed transitions not only depends on the type of pigment but also on interactions of the pigment with other pigments and the protein environment. Pigment-protein interactions can influence the electronic states of individual pigments via specific interactions like hydrogen bonding and ligation 4 or the polarizable Coulombic field it forms around the pigment. 5,6 Due to the glasslike disorder of the protein, the interactions with a pigment are inherently heterogeneous. 7,8 This heterogeneity leads to significant inhomogeneous broadening of the absorption bands of pigments bound to a protein. Also interactions between excited states of the pigments lead to shifting and splitting of corresponding energy levels and formation of so called excitonic states. These states have a delocalized character; i.e., the excitation is shared by several pigments. The extent of delocalization is largest when the energy differences between the levels of individual pigments and the dephasing are small compared to the interaction strength between the pigments. If on the other hand the interactions are relatively small, the excitations tend to be localized on the individual pigments. 1 The temperature dependence of the absorption spectrum of a pigment-protein complex is, to a large extent, determined * Author to whom correspondence is to be addressed. Tel: +31 20 4447941. Fax: +31 20 4447899. E-mail: erwinp@nat.vu.nl. ² Vrije Universiteit. ‡ Lund University. X Abstract published in AdVance ACS Abstracts, January 1, 1997. 4448 J. Phys. Chem. B 1997, 101, 4448-4457 S1089-5647(96)02338-3 CCC: $14.00 © 1997 American Chemical Society