Q_y-Level Structure and Dynamics of Solubilized Light-Harvesting Complex II of Green Plants:  Pressure and Hole Burning Studies

Abstract

Nonphotochemical hole burning and pressure-dependent absorption and hole-burning results are presented for the isolated (disaggregated) chlorophyll a/b light-harvesting II trimer antenna complex of green plants. Analysis of the 4.2 K burn-fluence dependent hole spectra and zero-phonon hole action spectra indicates that the three lowest energy states (Q y ) lie at 677.1, 678.4 and 679.8 nm. Their combined absorption intensity is equivalent to that of three Chl a molecules. The inhomogeneous broadening of their absorption bands is 70 cm-1. It is argued that these states, separated by 30 cm-1, are associated with the lowest energy state of the trimer subunit with the 30 cm-1 separations due to the indigenous structural heterogeneity of protein complexes. The linear electron−phonon coupling of the 679.8 nm state is weak and characterized, in part, by a mean phonon frequency of ωm = 18 cm-1 and Huang−Rhys factor of S m = 0.8, values which yield the correct Stokes shift for fluorescence from the 679.8 nm state at 4.2 K. The temperature dependence of the zero-phonon hole (ZPH) width for that state is consistent with optical dynamics due to coupling with glasslike two-level systems of the protein. The ZPH width at 1.9 K is 0.037 cm-1. Satellite hole structure produced by burning in the above three states as well as their low linear pressures shift rates (about − 0.08 cm-1/MPa) indicate that the Chl a molecule of the subunit associated with them is weakly coupled to other Chl molecules. The linear pressure shift rates for the main Q y -absorption bands are also low. The shift rates appear to be dictated by protein−Chl interactions rather than excitonic couplings. Holes burned into the 650 nm absorption band reveal energy transfer times of 1 ps and ∼100 fs which are discussed in terms of time domain measurements of the Chl b → Chl a transfer rates (Connelly et al. J. Phys. Chem. B 1997, 101, 1902). The holewidths associated with burning into the 676 nm absorption band lead to Chl a → Chl a transfer times in the 6−10 ps range, in good agreement with the time domain values (Savikhin et al. Biophys. J. 1994, 66, 1597).

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

“…The first EADS is replaced by the second EADS in 130 fs and the amplitude at 650 nm of the latter is half of the former, which implies that half of the excitations flow from Chl b towards Chl a with a time constant of about 130 fs, in agreement with previous TA measurements on monomers and trimers of LHC II at 77 K (Bittner et al 1995;). The lowest energy states which absorb at around 680 nm (Pieper et al 1999) receive the excitation energy very fast, as can be deduced from the rapid rise of its bleaching/stimulated emission (PB/SE) in the first couple of hundreds of femtoseconds. Intermediate states located between 660 and 675 nm become populated too, in correspondence with the qualitative description of the kinetic traces (see above and Figure 4).…”

“…In view of the new crystal structures (Liu et al 2004;Standfuss et al 2005), this may only occur between the Chls 10 and 13, which form the pair of Chls b that exhibit the strongest coupling strength. 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).…”

“…Its location and fwhm is somewhat different in the three isoforms of LHC II, which implies that the absorption wavelength associated to the lowest energy states may vary between isoforms. This might explain why Pieper et al (1999) were able to burn three different holes in solubilized trimers of LHC II at 4.2 K in the red edge of the absorption spectrum. Figure 8 shows the normalized final EADS for the three isoforms after excitation at 662 nm and Table 4 shows the location and fwhm of the final EADS for all three of them.…”

Fluorescence hole-burning and site-selective studies of LHCII

“…Very often, additional holes covering different regions of the absorption spectrum are observed. These additional holes may reveal: (1) coupling of the electronic transitions in the pigment molecules to the vibrations of the protein environment (called electronphonon coupling; Gillie et al 1989a, Pieper et al 1999, Hayes et al 2000, Ihalainen et al 2003, (2) coupling of the electronic transitions to the vibrations in the pigment molecules (Gillie et al 1989b), (3) existence of the excitonic coupling between different pigment molecules (Reddy et al 1994), (4) excitation energy transfer from the higher to the low energy states (red Chls; Hayes et al 2000, Zazubovich et al 2002, Ihalainen et al 2003. Furthermore, analysis of the AHB spectra gives insight into spectral characteristics of individual molecules and their distributions (homogeneous and inhomogeneous broadening) as well as into the excited state dynamics (Reddy et al 1994, Groot et al 1996, Pieper et al 1999, Dědic et al 2004.…”

“…AHB was used to determine the ~ 680-nm energy level and inhomogeneous width (70-120 cm -1 ) of the lowest energy state responsible for the LHCII emission at low temperatures (Reddy et al 1994, Pieper et al 1999. Further, both AHB and FLN techniques were applied to investigate the electron-phonon coupling, which was concluded to be weak, and characterized by a mean phonon frequency ω m = 15-18 cm -1 and Huang-Rhys factor, S = 0.8-0.9 (Pieper et al 1999(Pieper et al , 2001).…”