Monitoring fluorescence of individual chromophores in peridinin–chlorophyll–protein complex using single molecule spectroscopy

Abstract

Single molecule spectroscopy experiments are reported for native peridinin-chlorophyll a-protein (PCP) complexes, and three reconstituted light-harvesting systems, where an N-terminal construct of native PCP from Amphidinium carterae has been reconstituted with chlorophyll (Chl) mixtures: with Chl a, with Chl b and with both Chl a and Chl b. Using laser excitation into peridinin (Per) absorption band we take advantage of sub-picosecond energy transfer from Per to Chl that is order of magnitude faster than the Förster energy transfer between the Chl molecules to independently populate each Chl in the complex. The results indicate that reconstituted PCP complexes contain only two Chl molecules, so that they are spectroscopically equivalent to monomers of native-trimeric-PCP and do not aggregate further. Through removal of ensemble averaging we are able to observe for single reconstituted PCP complexes two clear steps in fluorescence intensity timetraces attributed to subsequent bleaching of the two Chl molecules. Importantly, the bleaching of the first Chl affects neither the energy nor the intensity of the emission of the second one. Since in strongly interacting systems Chl is a very efficient quencher of the fluorescence, this behavior implies that the two fluorescing Chls within a PCP monomer interact very weakly with each other which makes it possible to independently monitor the fluorescence of each individual chromophore in the complex. We apply this property, which distinguishes PCP from other light-harvesting systems, to measure the distribution of the energy splitting between two chemically identical Chl a molecules contained in the PCP monomer that reaches 280 cm(-1). In agreement with this interpretation, stepwise bleaching of fluorescence is also observed for native PCP complexes, which contain six Chls. Most PCP complexes reconstituted with both Chl a and Chl b show two emission lines, whose wavelengths correspond to the fluorescence of Chl a and Chl b. This is a clear proof that these two different chromophores are present in a single PCP monomer. Single molecule fluorescence studies of PCP complexes, both native and artificially reconstituted with chlorophyll mixtures, provide new and detailed information necessary to fully understand the energy transfer in this unique light-harvesting system.

Fluorescence imaging of hybrid nanostructures composed of natural photosynthetic complexes and reduced graphene oxide

“…1), which is in agreement with previous work [30]. Both the absorption and emission spectra of the PCP solution are identical to those previously published [24], indicating that the protein remains intact. Peridinins absorb light mainly from 350 nm to 550 nm, while chlorophylls absorb around 440 nm (Soret band) and 668 nm (Q Y band) [24].…”

“…Both the absorption and emission spectra of the PCP solution are identical to those previously published [24], indicating that the protein remains intact. Peridinins absorb light mainly from 350 nm to 550 nm, while chlorophylls absorb around 440 nm (Soret band) and 668 nm (Q Y band) [24]. The absorbed energy is rapidly (within a few picoseconds) transferred to the chlorophylls and emitted at a wavelength of 673 nm, as shown in Fig.…”

“…Due to sensitivity which enables the detection of photons emitted by individual molecules and also high acquisition peridinins, two chlorophylls a, and a lipid molecule, embedded in a protein capsule [25]. PCP is an unusual light-harvesting complex with a high peridinin:chlorophyll ratio (4:1) which results in a dominate contribution by the peridinins with regards to absorbing sunlight [24,25]. In experiments we used PCP complexes obtained by reconstituting the apo-protein with appropriate amounts of pigments, as described by Hofmann and coworkers [26].…”

“…Here we present the optical studies of the interactions between a photosynthetic complex, peridinin-chlorophyllprotein [24], and rGO. Both absorption, which for PCP extends over the whole visible range, and emission, which is characterized by high quantum yield (30%), make this natural light-harvesting complex a suitable probe.…”

Influence of Plasmon Excitations in Au Nanoparticles upon Fluorescence and Photostability of Photosynthetic Complexes

“…The absorption spectrum measured for Au nanoparticles is also shown in Figure 1, it features strong plasmon resonance at 540 nm, but the resonance is pretty broad, extending considerably towards the emission of the PCP complex. The fluorescence of the PCP is associated with Q y band of the weakly coupled [19,20] Chl a pair and appears at 673 nm. Due to solubility in water, high fluorescence quantum yield, and strong absorption in the spectral range similar to that of plasmon resonances in Au and Ag nanoparticles, the PCP complexes are very attractive biomolecules for studying interactions with metallic nanoparticles.…”