Two-Photon Excited Fluorescence from Higher Electronic States of Chlorophylls in Photosynthetic Antenna Complexes: A New Approach to Detect Strong Excitonic Chlorophyll a/b Coupling

Abstract

Stepwise two-photon excitation of chlorophyll a and b in the higher plant main light-harvesting complex (LHC II) and the minor complex CP29 (as well as in organic solution) with 100-fs pulses in the Q(y) region results in a weak blue fluorescence. The dependence of the spectral shape of the blue fluorescence on excitation wavelength offers a new approach to elucidate the long-standing problem of the origin of spectral "chlorophyll forms" in pigment-protein complexes, in particular the characterization of chlorophyll a/b-heterodimers. As a first result we present evidence for the existence of strong chlorophyll a/b-interactions (excitonically coupled transitions at 650 and 680 nm) in LHC II at ambient temperature. In comparison with LHC II, the experiments with CP29 provide further evidence that the lowest energy chlorophyll a transition (at approximately 680 nm) is not excitonically coupled to chlorophyll b.

Elucidation of structure–function relationships in photosynthetic light-harvesting antenna complexes by non-linear polarization spectroscopy in the frequency domain (NLPF)

“…These results are 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, 2006b). In LHC II as well as in CP29 an ultra-weak (quantum yield of less than 10 −4 ) "blue" fluorescence can be observed following stepwise resonant twophoton excitation with ∼100 fs laser pulses in the Chl a/b Q y region Leupold et al, 2006b).…”

“…However, two "blue" fluorescence maxima were observed for CP29, one at about 450 nm (upon Chl a excitation at 680 nm) and one at about 475 nm (upon Chl b excitation at 650 nm). This observation also supports the notion of two differently coupled Chls b in CP29: one, absorbing at 640 nm, is strongly coupled to Chl a, the other one absorbing at 650 is not excitonically coupled (Pieper et al, 2000;Voigt et al, 2002;Leupold et al, 2002;Leupold et al, 2006b).…”

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). …”

A comparison of the three isoforms of the light-harvesting complex II using transient absorption and time-resolved fluorescence measurements

“…The lowest energy state(s) absorbing at around 680 nm (Pieper et al 1999) become(s) populated during the first few hundreds of fs. This suggests some exciton delocalization and relatively strong interactions between one or two pairs of chlorophylls a and b, as pointed out very recently by Novoderezhkin et al (2004) and by Leupold et al (2002). Only a minor fraction ($10%) of the Chl b excitations is transferred to Chls a on a picosecond regime.…”