The fluorescence emission of individual photosystem I complexes from Synechocystis PCC 6803 in protonated and deuterated buffer shows zero-phonon lines as well as broad intensity distributions. The number and the line width of the zero phonon lines depend strongly on the solvent (H(2)O/D(2)O). The spectral diffusion rate of the whole fluorescence emission from photosystem I is significantly reduced upon deuteration of the solvent. This leads to a substantial increase of well-resolved zero-phonon lines. Since the chlorophyll a chromophores lack exchangeable protons, these observed changes in the spectral diffusion have to be assigned to exchangeable protons at the amino acids and structural water molecules in the chromophore binding pocket.

Strong anticorrelation between the fluorescence emission of different emitters is observed by employing single-molecule fluorescence spectroscopy on photosystem I at cryogenic temperatures. This anticorrelation demonstrates a time-dependent interaction between pigments participating in the exciton transfer chain, implying that uniquely defined energy transfer pathways within the complex do not exist. Fluctuations of the chromophores themselves or their immediate protein surroundings induce changes in their site energy, and, as a consequence, these fluctuations change the coupling within the excitation transfer pathways. The time scales of the site energy fluctuations of the individual emitters do not meet the time scales of the observed correlated emission behavior. Therefore, the emitters must be fed individually by energetically higher lying states, causing the observed intensity variations. This phenomenon is shown for photosystem I pigmentprotein complexes from 2 different cyanobacteria (Thermosynechococcus elongatus and Synechocystis sp. PCC 6803) with strongly different spectral properties underlining the general character of the findings. The variability of energy transfer pathways might play a key role in the extreme robustness of light-harvesting systems in general.exciton transfer ͉ FRET ͉ light harvesting ͉ photosynthesis ͉ single-molecule T he complex structural dynamics of proteins as a result of their combination of properties resembling, in part, the crystalline, the glassy, and the liquid state of matter is a prerequisite for protein function (1, 2). The picture of transitions between hierarchically ordered minima in a multidimensional configuration energy landscape (3) has emerged from the pioneering experiments of Frauenfelder et al. (4) as well as molecular dynamics simulations (5, 6). Local minima correspond to conformational substates (CS) separated by energy barriers into distinct tiers. Protein-embedded chromophore cofactors are ideal reporters for transitions between CS because of the susceptibility of their electronic transition energies on the specific conformation of their protein environment (2). The changes of the electronic transition energies (site energies) of individual chromophores are accessible by single-molecule spectroscopy (SMS) (7) on the single-protein level (8)(9)(10)(11)(12)(13)(14).The transition rates between different CS in proteins are spread out over an extremely wide range of time scales. As a consequence, for room temperature single-molecule experiments on pigment-protein complexes only transitions between CS of higher tiers occurring slower than the presently achievable time resolution of Ϸ0.1 s Ϫ1 can be monitored directly (14, 15). There, the individual spectra approximately resemble ensembleaveraged spectra with characteristic fluctuations in their width and specific position (13). Lowering the temperature slows down the transitions between CS in high tiers beyond realistic durations of the experiment, and transitions involving lower tiers move into the exper...

Single-molecule spectroscopy at low temperatures was used to elucidate spectral properties, heterogeneities, and dynamics of the red-shifted chlorophyll a (Chl a) molecules responsible for the fluorescence from photosystem I (PSI). Emission spectra of single PSI complexes from the cyanobacterium Synechocystis PCC 6803 show zero-phonon lines (ZPLs) as well as broad intensity distributions without ZPLs. ZPLs are found most frequently on the blue side of the broad intensity distributions. The abundance of ZPLs decreases almost linearly at longer wavelengths. The distribution of ZPLs indicates the existence of at least two pools with maxima at 699 and 710 nm. The pool with the maximum at 710 nm is assigned to chlorophylls absorbing around 706 nm (C706), whereas the pool with the maximum at 699 nm (F699) can be assigned to chlorophylls absorbing at 692, 695, or 699 nm. The broad distributions dominating the red side of the spectra are made up of a low number of emitters assigned to the red-most pool C714. The properties of F699 show close relation to those of F698 in Synechococcus PCC 7002 and C708 in Thermosynechococcus elongatus. Furthermore, a high similarity is found between the C714 pool in Synechocystis PCC 6803 and C708 in Synechococcus PCC 7002 as well as C719 in T. elongatus.

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