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. Vo 1 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, 1996 X Spectral hole burning has been performed on the Q y -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 Q y -band, follows a T 1.3(0.1 power law between 1.2 and 4.2 K characteristic of glasses and extrapolates to Γ′ 0 ) (2πT 1 ) -1 for T f 0 with T 1 ) (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 λ exc experiments. Their linear electron-phonon coupling strength was found to be rather weak, S ) 0.73 ( 0.05. For λ exc < 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-D2- cytochrome 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 polypep- tides, and the psbI gene product. It contains about six chlorophylls a (Chl a), two pheophytins a (Pheo a), one or two -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 40- 100 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 proposed 18 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 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 tempera- tures, 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 steady- state fluorescence at 4 K observed at λ > 683 nm by Kwa et al., 20 on the other hand, was attributed to a long-wavelength- emitting 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 Q y -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 pigments 13,19,22 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- * To whom correspondence should be addressed. † Free University. ‡ University of Leiden. X Abstract published in AdVance ACS Abstracts, June 1, 1996. 11488 J. Phys. Chem. 1996, 100, 11488-11495 S0022-3654(96)00326-7 CCC: $12.00 © 1996 American Chemical Society