Stepwise Two-photon Excited Fluorescence from Higher Excited States of Chlorophylls in Photosynthetic Antenna Complexes

Abstract

Stepwise two-photon excited fluorescence (TPEF) spectra of the photosynthetic antenna complexes PCP, CP47, CP29, and lightharvesting complex II (LHC II) were measured. TPEF emitted from higher excited states of chlorophyll (Chl) a and b was elicited via consecutive absorption of two photons in the Chl a/b Q y range induced by tunable 100-fs laser pulses. Global analyses of the TPEF line shapes with a model function for monomeric Chl a in a proteinaceous environment allow distinction between contributions from monomeric Chls a and b, strongly excitonically coupled Chls a, and Chl a/b heterodimers/-oligomers. The analyses indicate that the longest wavelength-absorbing Chl species in the Q y region of LHC II is a Chl a homodimer with additional contributions from adjacent Chl b. Likewise, in CP47 a spectral form at ϳ680 nm (that is, however, not the red-most species) is also due to strongly coupled Chls a. In contrast to LHC II, the red-most Chl subband of CP29 is due to a monomeric Chl a. The two Chls b in CP29 exhibit marked differences: a Chl b absorbing at ϳ650 nm is not excitonically coupled to other Chls. Based on this finding, the refractive index of its microenvironment can be determined to be 1.48. The second Chl b in CP29 (absorbing at ϳ640 nm) is strongly coupled to Chl a. Implications of the findings with respect to excitation energy transfer pathways and rates are discussed. Moreover, the results will be related to most recent structural analyses.Higher plants possess a complex light-harvesting antenna system for both photosystems, I and II. In photosystem II, the trimeric light-harvesting complex (LHC II), 2 harboring almost 50% of the total chlorophyll (Chl) a and b in the thylakoid membranes, comprises the most peripheral antenna. The monomeric minor antenna complexes CP29, CP26, and CP24 (lhcb4 -6 gene products) are connected to LHC II and are located proximal to the core antenna complexes, CP43 and CP47, which surround the reaction center; for a recent review see Ref.1. In contrast to LHC II, for which very recently high resolution x-ray structural analyses have been obtained (2, 3), no such high resolution structures of the minor complexes are available yet. Their structures are assumed to be comparable with that of the monomeric subunits of LHC II but with lower Chl content: According to present knowledge CP29 binds six Chls a and two Chls b (4, 5).In addition, currently no high resolution structure for CP47 is available. However, x-ray structural analyses of three-dimensional crystals of oxygen evolving PS II core complexes from the thermophilic cyanobacteria Thermosynechococcus elongatus (6 -8) and T. vulcanus (9) at medium resolution have been obtained. According to the most recent analysis at 3.0 Å resolution (8), 16 Chl a molecules are bound to cyanobacterial CP47. Purified CP47 from spinach was found to bind 15 Ϯ 1 Chls a and 3-4 ␤-carotenes (10), which is in good agreement with the recent structural analyses of its cyanobacterial homologues (6 -8). The amino acid sequences of the CP47 apo...

Elucidation of structure–function relationships in plant major light-harvesting complex (LHC II) by nonlinear spectroscopy

“…A Chl b molecule absorbing at about 640 nm was found to be strongly coupled with Chl(s) a absorbing at about 670 nm in CP29 . These results were found to be consistent with other spectroscopic studies at RT as well as cryogenic temperatures (e.g., Pieper et al 2000;Leupold et al 2002;Leupold et al 2006).…”

“…The other Chl b (absorbing at about 650 nm) is not excitonically coupled (cf. Pieper et al 2000;Voigt et al 2002;Leupold et al 2002;Leupold et al 2006). …”

“…For both, LHC II as well as CP29 (as well as several other LHCs) an ultra-weak (quantum yield of less than 10 -4 ) ''blue'' fluorescence was observed following stepwise resonant two-photon excitation with *100 fs laser pulses in the Chl a/b Q y absorption region Leupold et al 2006).…”

“…The (usually) extremely weak emission from the higher excited state can be detected for isolated Chls in solution as well as for Chls in pigmentprotein complexes Leupold et al 2006).…”

Ultrafast resonance energy transfer from a site-specifically attached fluorescent chromophore reveals the folding of the N-terminal domain of CP29

“…The spectroscopic properties of each pigment in each binding site have been determined with the help of mutation analysis followed by in vitro reconstitution [5][6][7][8] and by applying different spectroscopic techniques [9][10][11][12]. Several time-resolved studies have been performed on the complex, allowing to elucidate the energy transfer rates and pathways between Chls and between carotenoids and Chls [4,[13][14][15][16][17][18].…”