Fluorescence spectral dynamics of single LHCII trimers

Abstract

Single-molecule spectroscopy was employed to elucidate the fluorescence spectral heterogeneity and dynamics of individual, immobilized trimeric complexes of the main light-harvesting complex of plants in solution near room temperature. Rapid reversible spectral shifts between various emitting states, each of which was quasi-stable for seconds to tens of seconds, were observed for a fraction of the complexes. Most deviating states were characterized by the appearance of an additional, red-shifted emission band. Reversible shifts of up to 75 nm were detected. By combining modified Redfield theory with a disordered exciton model, fluorescence spectra with peaks between 670 nm and 705 nm could be explained by changes in the realization of the static disorder of the pigment-site energies. Spectral bands beyond this wavelength window suggest the presence of special protein conformations. We attribute the large red shifts to the mixing of an excitonic state with a charge-transfer state in two or more strongly coupled chlorophylls. Spectral bluing is explained by the formation of an energy trap before excitation energy equilibration is completed.

Figure 1

Fluorescence (FL) spectral time traces from single LHCII trimeric complexes at 5°C, acquired during continuous irradiation corresponding to a power of 1 μW at 630 nm. FL was binned into 1-s integration times. The distinct spectroscopic states are denoted by I, II and III, respectively, where I/III indicates alternation between states I and III. Spectra on top are the time averages of the respective spectral states. FL is expressed in counts per second (cps).

Figure 2

FLP distribution from ∼2000 individually measured LHCII trimers. On average, ∼100 consecutive spectra were acquired for every complex. Bins of 1 nm and 5 nm were used for the narrow and broad bars, respectively. Narrow bars denote the weighted time-dependent frequency of occurrence, whereas the broad bars describe the fraction of complexes that exhibited a peak in the specified wavelength window during the course of a spectral time trace. Broad bars comprise mainly the peak positions of the redder bands of double-band states (see Table 1). Standard errors of 10% and 20% were estimated on the narrow and broad bars, respectively.

Figure 3

Selection of deviating spectral profiles from single LHCII complexes (points connected by lines, red online). The rows correspond to the three groups in Table 1, respectively. Spectra are time averages of similar spectral profiles, with each average corresponding to one complex. The reference spectrum (solid lines) is a time- and population-averaged single-molecule spectrum. The sharp decrease of the left wing below 640 nm in I–L stems from the fluorescence filter cutoff. All spectra are normalized to facilitate comparison of different profiles. See text and Table 1 for further details.

Figure 4

Fluorescence profiles calculated for different realizations of the static disorder for a single LHCII complex at room temperature. The realizations were obtained by using different sets of the site energies (Q_y transition energies for 14 Chls), randomly taken from a Gaussian distribution with full width at half-maximum of 90 cm⁻¹. This disorder width was the same as for the Gaussian fit of the bulk spectrum.

Figure 5

Comparison of eight selected modeled (solid lines, blue online) and measured (points connected by lines, red online) deviating fluorescence spectral profiles, along with the calculated PR of the different pigments to the lowest exciton state (histograms to the right of corresponding spectra). All spectra are normalized and compared with the nondeviating reference spectrum (dashed lines). Measured spectra are averages of spectral profiles with similar shapes, and calculated spectra are averaged over a number of realizations. For all realizations, the disorder was 140 cm⁻¹. PR_n is averaged over a number of realizations of the static disorder within each group. See text for details.