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Nonradiative dissipation of excitation energy is the major photoprotective mechanism in plants. The formation of zeaxanthin in the antenna of photosystem II has been shown to correlate with the onset of nonphotochemical quenching in vivo. We have used recombinant CP29 protein, over-expressed in Escherichia coli and refolded in vitro with purified pigments, to obtain a protein indistinguishable from the native complex extracted from thylakoids, binding either violaxanthin or zeaxanthin together with lutein. These recombinant proteins and the native CP29 were used to measure steady-state chlorophyll fluorescence emission and fluorescence decay kinetics. We found that the presence of zeaxanthin bound to CP29 induces a approximately 35% decrease in fluorescence yield with respect to the control proteins (the native and zeaxanthin-free reconstituted proteins). Fluorescence decay kinetics showed that four components are always present but lifetimes (tau) as well as relative fluorescence quantum yields (rfqy) of the two long-lived components (tau3 and tau4) are modified by the presence of zeaxanthin. The most relevant changes are observed in the rfqy of tau3 and in the average lifetime ( approximately 2.4 ns with zeaxanthin and 3.2-3.4 ns in the control proteins). When studied in vitro, no significant effect of acidic pH (5.2-5.3) is observed on chlorophyll A fluorescence yield or kinetics. The data presented show that recombinant CP29 is able to bind zeaxanthin and this protein-bound zeaxanthin induces a significant quenching effect.

A photosystem I preparation from maize, containing its full antenna complement (PSI-200) and in which detergent effects on chlorophyll coupling are almost completely absent, has been studied by time-resolved fluorescence techniques with approximately 5 ps resolution at 280 and 170 K in the wavelength interval of 690-780 nm. The data have been analyzed in terms of both the decay-associated spectra (DAS) and the time-resolved emission spectra (TRES). As in a previous room temperature study [Turconi, S., Weber, N., Schweitzer, D., Strotmann, H., and Holzwarth, A. R. (1994) Biochim. Biophys. Acta 1187, 324-334], the 280 K decay is well described by three DAS components in the 11-130 ps time range, the fastest of which displays both positive and negative amplitudes characteristic of excitation transfer from the bulk to the red antenna forms. Both the 57 and 130 ps components have all positive amplitudes and describe complex decay and equilibration processes involving the red forms. At 170 K, four major components in the 10-715 ps time range are required to describe the decay. The fastest represents bulk to red form transfer processes, while the 55, 216, and 715 ps decays, with all positive amplitudes, have maxima near 720, 730, and 740 nm, respectively, in accord with previous steady-state fluorescence measurements. The width and asymmetry of these DAS indicate that they are spectrally complex and represent decay and equilibration processes involving the red forms. Spectral evolution during the fluorescence decay process was analyzed in terms of the TRES. The red shifting of the TRES was analyzed in terms of the first central spectral moment (mean spectral energy) which is biexponential at both temperatures. The slower component, which describes equilibration between the red forms, leads to spectral red shifting during the entire fluorescence decay process, and the mean lifetimes of the spectral moments at 280 and 170 K (86 and 291 ps, respectively) are similar to the mean lifetimes of the fluorescence decays (119 and 384 ps, respectively). Thus, both spectral evolution and the trapping-associated fluorescence decay occur on a similar time scale, and both processes display a very similar temperature sensitivity. On the basis of these data, it is concluded that trapping in PSI-200 is to a large extent rate-limited by excitation diffusion in the antenna and in particular by the slow "uphill" transfer from the low-energy forms to the bulk and/or inner core chlorophyll molecules.

The organization and interaction of chlorophylls (Chl) and the kinetics of the energy transfer in the core antenna of photosystem I (PSI) trimeric and monomeric complexes, isolated from Spirulina platensis with Triton X-100 have been studied by stationary and time-resolved fluorescence. At 295 K both complexes show an unusually intense long-wavelength emission band with prominent peaks at 730 nm (trimers) or 715 nm (monomers), whose intensity is independent of the redox state of P700. A broad band extending from 710 to 740 nm in the absorption and fluorescence excitation spectra of trimers also indicates the existence of the longwave Chls at 295 K. The 77 K fluorescence emission of PSI trimers frozen after addition of dithionite under illumination (P700 and the PSI acceptor side reduced) shows an intense band at 760 (F760) and a smaller one at 725 nm (F725); when P700 is oxidized, the intensity of F760 decreases about 15 times. In the 77 K spectrum of monomers only F725 is present in the longwave region, and its intensity does not depend on the redox state of P700. Bands of Chls with maxima near 680, 710, and 738 nm were found in the 77 K excitation spectrum of trimers, and bands near 680 and 710 nm were seen in the spectrum of monomers. Five spectrally different red Chl forms in PSI trimers and three red Chl in monomers have been resolved by deconvolution of their 77 K absorption spectra. The difference absorption spectrum, trimers-minus-monomers, shows that the appearance of the 735 nm band in trimers is accompanied by a decrease of 708, 698, and 688 nm bands present in monomers. The reversible changes of F760 intensity of Spirulina membranes as a result of their salt treatment confirm the idea that the most longwave Chl form originates from an interaction of Chls bound to different monomeric PSI subunits forming the trimer. The time-resolved fluorescence spectra of PSI trimers and monomers, measured at 287 K in the region 680-770 nm, are substantially different, although a set of similar lifetimes (9, approximately 30, approximately 66, and 1400-2200 ps) was necessary for a good fit. No effect of P700 redox state was observed on the fluorescence kinetics of both complexes at 287 K.

Trimeric complexes of photosystem I (PSI) of Spirulina contain the extreme longwave chlorophyll (Chl) form absorbing at 735 nm (Ch1735) and emitting at 760 nm (F760). The intensity of F760 is maximal when P700 is reduced and decreases when P700 is oxidized (P700+). Ch1735 originates from interaction of Chl molecules, located on the surface of the monomeric subunits forming trimer: (i) deconvolution of 77 K absorption spectrum of PSI trimers shows that each monomer contains two molecules of Ch1735; (ii) transfer of PSI trimers into monomers in cyanobacterial membranes at „high salt“ is accompanied by decrease of F760 (Ch1735 disaggregation), while trimerization of monomers at „low salt“ results the recovery of F760 (Ch1735 formation); (iii) incubation of PSI monomers in liposomes causes reconstitution of trimers containing Ch1735. Illumination of PSI complexes with intense light bleaches bulk Chls but does not effect P700. The non-linear relationship between the kinetics of P700 photooxidation and F760 quenching in PSI trimers at 77 K is interpreted as energy exchange (via Ch1735) between antennae of monomers and quenching of the excess energy by P700+. The energy exchange between monomers promotes dissipation of the overexcitation into heat. Thus both PSI trimers and monomers contribute in electron transport whereas PSI trimers in addition protect complex against photodestruction.

The energy transfer and trapping processes in photosystem I (PSI) can be monitored conveniently by time-resolved fluorescence. The typical kinetics of PSI particles shows an energy transfer component, reflecting the equilibration of excitations over the antenna, and an all-positive component, which has been ascribed to the trapping process by the reaction center (trapping component). However, in recent work multiple positive amplitude components have been resolved (1). These multiple positive components have been explained by sample heterogeneity (e.g. different antenna size, aggregation states), giving rise to a different trapping kinetics for each subpopulation.

In contrast to higher plants, cyanobacteria have no light-harvesting complex (LHC) containing chlorophyll (Chl), and whole Chl in cyanobacterial membranes is localized in core complexes of photosystem I (PSI) and PSII [1]. Cyanobacteria are highly enriched with PSI (PSI/PSII ratio is about 5-10); PSI complex in cyanobacteria is organized as monomer and trimer [2,3]. The antenna size (Ch1/P700) of both complexes is about 100 indicating that each monomeric subunit within the trimer contains P700. The equilibrium between PSI trimeric and monomeric complexes in isolated membranes is regulated by changing of electrostatic interaction at various salt conditions [4].

The structure of a cyanobacterial photosystem (PS) I complex has recently been determined to high resolution (1). This now opens the possibility to understand the functioning and the spectral properties of PS I complexes in terms of structural parameters. Pertinent questions that need to be addressed are the particular role of the special “red pigment” pools for the excitation dynamics and charge separation kinetics, the locations of these red pigments in the antenna, the overall excitation dynamics, i.e. trap- or diffusion limit, the influence of the aggregation state on the dynamics and the spectral red pools, and finally the quenching by P700+. Time-resolved kinetics studies, in particular time-resolved fluorescence as well as femtosecond transient absorption methods are suitable to provide more insight into these questions. In the following we report here on studies on PS I particles from the cyanobacterium Spirulina platensis. Additional data are provided in the contribution by Dorra et al. (see these Proceedings, (6)).