Monitoring fluorescence of individual chromophores in peridininchlorophyll-protein complex using single molecule spectroscopy

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 (Chi) mixtures: with Chi a, with Chi b and with both Chi a and Chi b. Using laser excitation into peridinin (Per) absorption band we take advantage of sub-picosecond energy transfer from Per to Chi that is order of magnitude faster than the Forster energy transfer between the Chi molecules to independently populate each Chi in the complex. The results indicate that reconstituted PCP complexes contain only two Chi 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 Chi molecules. Importantly, the bleaching of the first Chi affects neither the energy nor the intensity of the emission of the second one. Since in strongly interacting systems Chi 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 Chi 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 Chi a and Chi b show two emission lines, whose wavelengths correspond to the fluorescence of Chi a and Chi 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. (C) 2007 Elsevier B.V. All rights reserved.