Comparison of the Excited-State Dynamics of Five- and Six-Chlorophyll Photosystem II Reaction Center Complexes F. T. H. den Hartog, † F. Vacha, †,‡,§ A. J. Lock, † J. Barber, § J. P. Dekker, | and S. Vo 1 lker* ,†,| Center for the Study of Excited States of Molecules, Huygens and Gorlaeus Laboratories, UniVersity of Leiden, P.O. Box 9504, 2300 RA Leiden, The Netherlands, Faculty of Biological Sciences, UniVersity of South Bohemia, Institute of Plant Molecular Biology, Academy of Sciences of the Czech Republic, BranisoVska 31, Ceske BudejoVice, 370 05, Czech Republic, Photosynthesis Research Group, Department of Biochemistry, Imperial College of Science, Technology and Medicine, London SW7 2AY, United Kingdom, and Department of Physics and Astronomy, Institute of Molecular Biological Sciences, Free UniVersity, De Boelelaan 1081, 1081 HV Amsterdam, The Netherlands ReceiVed: June 26, 1998; In Final Form: September 2, 1998 Spectral hole-burning experiments have been performed at liquid He temperature on the Q y -band of isolated reaction center complexes of photosystem II (PS II RC) containing five (RC-5) and six (RC-6) chlorophyll a (Chl a) molecules. The aim was to investigate the nature of the redmost shoulder in the absorption spectrum of RC-5 and to identify distributions of “trap” pigments. The “effective” homogeneous line width Γ′ hom was measured at 682 nm as a function of temperature between 1.2 and 4.2 K. It follows a T 1.3(0.1 power law in both complexes and extrapolates to the fluorescence lifetime-limited value, τ fl ) (4 ( 1) ns, for T f 0. These results indicate that the redmost absorbing pigments act as “traps” for the excitation energy. The spectral distribution of these traps was reconstructed from the hole depth measured as a function of excitation wavelength λ exc and compared to that of RC-6 previously obtained by us (Groot, M. L.; et al. J. Phys. Chem. 1996, 100, 11488). The maximum of the RC-5 trap distribution lies at (682.9 ( 0.2) nm. We discovered a second distribution of fluorescing pigments centered at (673.4 ( 0.5) nm in both RC-5 and RC-6. The dependence of Γ′ hom on the delay time t d between burning and probing, for the red- and blue-absorbing pigments, is constant for t d e 1 s and increases linearly with log t d for longer delay times. The molecules absorbing at ∼674 nm, which can be chemically removed, are not free Chl a but are bound to a protein with the same mass as that of the RC complex. 1. Introduction The reaction center (RC) of photosystem II (PS II) in green plants is the pigment-protein complex where the primary charge-separation process occurs that eventually leads to the oxidation of water and the evolution of oxygen. The isolated PS II RC, as first prepared from spinach by Nanba and Satoh, 1 consists of the D1 and D2 proteins, the R- and -subunits of cytochrome b559, and the psbI gene protein. It binds six chlorophyll a (Chl a) and one or two -carotenes per two pheophytin a (Pheo a) molecules. 2 The plastoquinone is lost during isolation. It is assumed that the RC has a “core” comprising four of the six Chl a molecules and the two pheophytins and that two Chl a molecules are located at the periphery. Recently, a PS II reaction center containing five Chl a per two Pheo a molecules was isolated using immobilized metal affinity chromatography. 3 It was concluded from time-resolved absorption-difference spectroscopy that a peripheral Chl a absorbing at about 670 nm had been removed. 3 The absorption spectrum at 4.2 K of this RC-5 preparation is characterized by a shoulder at ∼684 nm. 4 This band is not well resolved in the 4.2 K absorption spectrum of the RC containing six chlorophyll a molecules (RC-6), but it is revealed in the second derivative of the spectrum. 5,6 The nature of the 684 nm shoulder is still a matter of debate, and several possibilities have been considered. From hole- burning spectroscopy on RC-6, it was suggested that it may be due to “linker” chlorophyll molecules serving to shuttle energy from the nearest CP47 antenna complex to the RC. 7,8 Other groups proposed that the 684 nm shoulder represents the low- energy exciton band of the RC primary electron donor P680 5 or that the 680 and 684 nm features are the result of “heterogeneity” of the absorption band of the primary electron donor. 9,10 It was also attributed to a red-absorbing accessory pigment acting as a “trap” for the excitation energy at low temperature. 11,12 From low-temperature fluorescence and triplet- minus-singlet (T-S) absorption-difference spectroscopy on RC- 5, it has recently been proposed that this shoulder originates, partly, from the PS II RC primary donor and, partly, from red- absorbing “traps” in the RC. 4 Since RC-5 has a much more pronounced shoulder at ∼684 nm than RC-6, it is a well-suited system to obtain more information on the nature of these red- absorbing pigments. Hole-burning (HB) has proven to be a sensitive method to determine spectral distributions of pigments characterized by their decay time. 12,13 More in particular, it revealed the existence of “trap”-pigments with a fluorescence lifetime of ∼4 ns and an absorption maximum at ∼682 nm in the red wing of the Q y -absorption band of RC-6 at low temperature. 12 HB, therefore, * To whom correspondence should be addressed. † University of Leiden. ‡ University of South Bohemia. §Imperial College of Science, Technology and Medicine. | Free University. 9174 J. Phys. Chem. B 1998, 102, 9174-9180 10.1021/jp982791l CCC: $15.00 © 1998 American Chemical Society Published on Web 10/16/1998