Fluorescence lifetime heterogeneity in aggregates of LHCII revealed by time-resolved microscopy.

Two-photon excitation, time-resolved fluorescence microscopy was used to investigate the fluorescence quenching mechanisms in aggregates of light-harvesting chlorophyll a/b pigment protein complexes of photosystem II from green plants (LHCII). Time-gated microscopy images show the presence of large heterogeneity in fluorescence lifetimes not only for different LHCII aggregates, but also within a single aggregate. Thus, the fluorescence decay traces obtained from macroscopic measurements reflect an average over a large distribution of local fluorescence kinetics. This opens the possibility to resolve spatially different structural/functional units in chloroplasts and other heterogeneous photosynthetic systems in vivo, and gives the opportunity to investigate individually the excited states dynamics of each unit. We show that the lifetime distribution is sensitive to the concentration of quenchers contained in the system. Triplets, which are generated at high pulse repetition rates of excitation (>1 MHz), preferentially quench domains with initially shorter fluorescence lifetimes. This proves our previous prediction from singlet-singlet annihilation investigations (Barzda, V., V. Gulbinas, R. Kananavicius, V. Cervinskas, H. van Amerongen, R. van Grondelle, and L. Valkunas. 2001. Biophys. J. 80:2409-2421) that shorter fluorescence lifetimes originate from larger domains in LHCII aggregates. We found that singlet-singlet annihilation has a strong effect in time-resolved fluorescence microscopy of connective systems and has to be taken into consideration. Despite that, clear differences in fluorescence decays can be detected that can also qualitatively be understood.

Peridinin chlorophyll a protein: relating structure and steady-state spectroscopy.

Peridinin chlorophyll a protein (PCP) from Amphidinium carterae has been studied using absorbance (OD), linear dichroism (LD), circular dichroism (CD), fluorescence emission, fluorescence anisotropy, fluorescence line narrowing (FLN), and triplet-minus-singlet spectroscopy (T-S) at different temperatures (4-293 K). Monomeric PCP binds eight peridinins and two Chls a. The trimeric structure of PCP, resolved at 2 A [Hofmann et al. (1996) Science 27, 1788-1791], allows modeling of the Chl a-protein and Chl a-Chl a interactions. The FLN spectrum shows that Chl a is not or is very weakly hydrogen-bonded and that the central magnesium of the emitting Chl a is monoligated. Simulation of the temperature dependence of the absorption spectra indicates that the Huang-Rhys factor, characterizing the electron-phonon coupling strength, has a value of approximately 1. The width of the inhomogeneous distribution function is estimated to be 160 cm(-)(1). LD experiments show that the two Chls a in PCP are essentially isoenergetic at room temperature and that a substantial amount of PCP is in a trimeric form. From a comparison of the measured and simulated CD, it is concluded that the interaction energy between the two Chls a within one monomer is very weak, <10 cm(-)(1). In contrast, the Chls a appear to be strongly coupled to the peridinins. The 65 cm(-)(1) band that is visible in the low-frequency region of the FLN spectrum might indicate a Chl a-peridinin vibrational mode. The efficiency of Chl a to peridinin triplet excitation energy transfer is approximately 100%. On the basis of T-S, CD, LD, and OD spectra, a tentative assignment of the peridinin absorption bands has been made.

Förster excitation energy transfer in peridinin-chlorophyll-a-protein.

Time-resolved fluorescence anisotropy spectroscopy has been used to study the chlorophyll a (Chl a) to Chl a excitation energy transfer in the water-soluble peridinin-chlorophyll a-protein (PCP) of the dinoflagellate Amphidinium carterae. Monomeric PCP binds eight peridinins and two Chl a. The trimeric structure of PCP, resolved at 2 A (, Science. 272:1788-1791), allows accurate calculations of energy transfer times by use of the Förster equation. The anisotropy decay time constants of 6.8 +/- 0.8 ps (tau(1)) and 350 +/- 15 ps (tau(2)) are respectively assigned to intra- and intermonomeric excitation equilibration times. Using the ratio tau(1)/tau(2) and the amplitude of the anisotropy, the best fit of the experimental data is achieved when the Q(y) transition dipole moment is rotated by 2-7 degrees with respect to the y axis in the plane of the Chl a molecule. In contrast to the conclusion of, Biochemistry. 23:1564-1571) that the refractive index (n) in the Förster equation should be equal to that of the solvent, n can be estimated to be 1.6 +/- 0.1, which is larger than that of the solvent (water). Based on our observations we predict that the relatively slow intermonomeric energy transfer in vivo is overruled by faster energy transfer from a PCP monomer to, e.g., the light-harvesting a/c complex.

Decreasing the chlorophyll a/b ratio in reconstituted LHCII: structural and functional consequences.

Trimeric (bT) and monomeric (bM) light-harvesting complex II (LHCII) with a chlorophyll a/b ratio of 0.03 were reconstituted from the apoprotein overexpressed in Escherichia coli. Chlorophyll/xanthophyll and chlorophyll/protein ratios of bT complexes and 'native' LHCII are rather similar, namely, 0.28 vs 0. 27 and 10.5 +/- 1.5 vs 12, respectively, indicating the replacement of most chlorophyll a molecules with chlorophyll b, leaving one chlorophyll a per trimeric complex. The LD spectrum of the bT complexes strongly suggests that the chlorophyll b molecules adopt orientations similar to those of the chlorophylls a that they replace. The circular dichroism (CD) spectra of bM and bT complexes indicate structural arrangements resembling those of 'native' LHCII. Thermolysin digestion patterns demonstrate that bT complexes are folded and organized like 'native' trimeric LHCII. Surprisingly, in the bT complexes at 77 K, half of the excitations that are created on either chlorophyll b or xanthophyll are transferred to chlorophyll a. No or very limited triplet transfer from chlorophyll b to xanthophyll appears to take place. However, the efficiency of triplet transfer from chlorophyll a to xanthophyll is close to 100%, even higher than in 'native' LHCII at 77 K. It is concluded from the triplet-minus-singlet and CD results that the single chlorophyll a molecule that on the average is present in each bT complex binds preferably next to a xanthophyll molecule at the interface between the monomers.

Energy transfer in LHCII monomers at 77K studied by sub-picosecond transient absorption spectroscopy.

Energy transfer from chlorophyll b (Chl b) to chlorophyll a (Chl a) in monomeric preparations of light-harvesting complex II (LHCII) from spinach was studied at 77 K using pump-probe experiments. Sub-picosecond excitation pulses centered at 650 nm were used to excite preferentially Chl b and difference absorption spectra were detected from 630 to 700 nm. Two distinct Chl b to Chl a transfer times, approximately 200 fs and 3 ps, were found. A clearly distinguishable energy transfer process between Chl a molecules occurred with a time constant of 18 ps. The LHCII monomer data are compared to previously obtained LHCII trimer data, and both data sets are fitted simultaneously using a global analysis fitting routine. Both sets could be described with the following time constants: 140 fs, 600 fs, 8 ps, 20 ps, and 2.9 ns. In both monomers and trimers 50% of the Chl b to Chl a transfer is ultrafast (<200 fs). However, for monomers this transfer occurs to Chl a molecules that absorb significantly more toward shorter wavelengths than for trimers. Part of the transfer from Chl b to Chl a that occurs with a time constant of 600 fs in trimers is slowed down to several picoseconds in monomers. However, it is argued that observed differences between monomers and trimers should be ascribed to the loss of some Chl a upon monomerization or a shift of the absorption maximum of one or several Chl a molecules. It is concluded that Chl b to Chl a transfer occurs only within monomeric subunits of the trimers and not between different subunits.