Energy transfer in reconstituted peridinin-chlorophyll-protein complexes: ensemble and single-molecule spectroscopy studies

Abstract

We combine ensemble and single-molecule spectroscopy to gain insight into the energy transfer between chlorophylls (Chls) in peridinin-chlorophyll-protein (PCP) complexes reconstituted with Chl a, Chl b, as well as both Chl a and Chl b. The main focus is the heterochlorophyllous system (Chl a/b-N-PCP), and reference information essential to interpret experimental observations is obtained from homochlorophyllous complexes. Energy transfer between Chls in Chl a/b-N-PCP takes place from Chl b to Chl a and also from Chl a to Chl b with comparable Förster energy transfer rates of 0.0324 and 0.0215 ps(-1), respectively. Monte Carlo simulations yield the ratio of 39:61 for the excitation distribution between Chl a and Chl b, which is larger than the equilibrium distribution of 34:66. An average Chl a/Chl b fluorescence intensity ratio of 66:34 is measured, however, for single Chl a/b-N-PCP complexes excited into the peridinin (Per) absorption. This difference is attributed to almost three times more efficient energy transfer from Per to Chl a than to Chl b. The results indicate also that due to bilateral energy transfer, the Chl system equilibrates only partially during the excited state lifetimes.

FIGURE 1

Pigment arrangement in the N-PCP reconstituted with (a) Chl a, (b) Chl b, and (c) both Chl a and Chl b. The sample reconstituted with both Chl a and Chl b contains all three types of complexes. Red and blue colors correspond to Chl a and Chl b, and Pers are colored in orange. The protein scaffold has been omitted for clarity.

FIGURE 2

Normalized room temperature fluorescence spectra of (a) (Chl a)₂-N-PCP and (Chl b)₂-N-PCP, and (b) the N-PCP reconstituted with both Chl a and Chl b. The excitation wavelength was 532 nm.

FIGURE 3

Room temperature fluorescence excitation spectra of reconstituted PCP samples. (a) N-PCP reconstituted with both Chl a and Chl b (solid line) and (Chl a)₂-N-PCP (dashed line) detected at 668 nm, and (b) N-PCP reconstituted with both Chl a and Chl b (solid line) and (Chl b)₂-N-PCP (dashed line) detected at 647 nm.

FIGURE 4

Comparison of absorption (dashed line) and fluorescence excitation (solid line) spectra of the N-PCP reconstituted with both Chl a and Chl b divided by respective spectra measured for (Chl a)₂-N-PCP.

FIGURE 5

Single-molecule spectra measured at room temperature for (a) (Chl a)₂-N-PCP, (b) (Chl b)₂-N-PCP, and (c) Chl a/b-N-PCP. The emission of both Chl molecules in the complex is shown by solid lines, and dashed lines are the spectra where only one Chl contributes to the fluorescence emission. The excitation power was 3 μW and 5 μW for homo- and heterochlorophyllous samples, respectively.

FIGURE 6

Distributions of emission wavelength measured for over 120 complexes of (a) homochlorophyllous (Chl a)₂-N-PCP (right-hand side shading) and (Chl b)₂-N-PCP (left-hand side shading) samples, and (b) heterochlorophyllous Chl a/b-N-PCP sample for complexes showing both Chl a and Chl b emissions. The values were extracted using subsequent fluorescence bleaching of the two energetically distinguishable Chl molecules. The excitation power was 3 μW and 5 μW for homo- and heterochlorophyllous samples, respectively.

FIGURE 7

(a) Distributions of fluorescence intensities measured for (Chl a)₂-N-PCP. Upper panel (lower panel) displays the intensity of two (one) emitting Chl a. (b) Experimentally determined distribution of the intensity ratio between emissions of two Chl a and of one Chl a in the complexes at room temperature. The excitation power was 3 μW.

FIGURE 8

(a) Distributions of fluorescence intensity measured for Chl a (upper panel) and Chl b (lower panel) in Chl a/b-N-PCP complexes. (b) Distribution of the Chl a/Chl b fluorescence intensity ratio obtained for the same complexes. The excitation power was 5 μW.

FIGURE 9

Survival times measured for single Chl a/b-N-PCP complexes. (a) Chl a fluorescence, and (b) Chl b fluorescence.

FIGURE 10

Schematic diagram of energy transfer pathways for a single Chl a/b-N-PCP complex excited at 532 nm. The numbering of Per follows the nomenclature of (40). The two Per (1 and 4) excited in each of the clusters transfer the excitation to Chl a (left) and Chl b (right) and after bilateral energy transfer between the Chl the recombination takes place. All the characteristic times are given.