Ptu)tosynthesisResearch 49: 263-268, 1996. C) 1996KluwerAcademicPublishers. Printedin the Netherlands. Regular paper

A comparative fluorescence kinetics study of Photosystem I monomers and trimers from Synechocystis PCC 6803 S a n d r a T u r c o n i 1,3, J o c h e n K r u i p 2, G e r d S c h w e i t z e r 1,4, M a t t h i a s R 6 g n e r 2,5 & A l f r e d R. H o l z w a r t h 1,*

1 Max-Planck-Institutfiir Strahlenchemie, Stiftstr. 34-36, D-45470 Mfilheim a. d. Ruhr, Germany; 2 Institutfiir Botanik, Universit~itM~inster, Schlossgarten 3, D-48149 MUnster, Germany; 3 Present address: Department of Biology, Darwin Building, University College London, Gower Street, London WC1E 6BT, UK; 4present address: Department of Chemistry, Institute for Molecular Dynamics and Spectroscopy, University of Leuven, Celestijnenlaan 20017, 3001 Heverlee, Belgium; 5Present address: Lehrstuhl Biochemie der Pflanzen, Ruhr-Universittit Bochum, D-44780 Bochum, Germany; *Author for correspondence Received 14 December1995;acceptedin revisedform30 July 1996

Key words: Photosystem I, time-resolved fluorescence, Photosystem I structure (Synechocystis), picosecond fluorescence, energy transfer kinetics

Abstract

Picosecond time-resolved fluorescence measurements have been performed as a function of emission wavelengths in order to investigate the possible functional differences between monomeric and trimeric Photosystem I (PS I) particles from a cyanobacterium Synechocystis. Applying global analysis, four kinetic components were found necessary to describe the fluorescecne decay for both monomers and trimers of PS I. The lifetimes and spectra of the respective components are quite similar, indicating that they can be attributed to identical processes in both the monomers and trimers. It is concluded that both forms of P S I are capable of efficient energy transfer and charge separation, in agreement with a physiological role of both forms. Small differences in the fluorescence decays are discussed in terms of a slightly higher ratio of red emitting pigments per reaction centre in trimers of PS I. A comparison to Synechococcus P S I particles reveals the higher red chlorophyll content of the latter.

Abbreviations: fl-DM-/~-dodecyl-maltoside;

Chl-chlorophyll; CMC-critical micellar concentration; DAS-decay-associated spectrum; DCM-4-dicyano-methylene-2-methyl-6-(-dimethyl-aminostyryl)-4h-pyran; FWHM-full-width at half-maximum; P700-primary electron donor of Photosystem I; PS-photosystem; R C reaction centre Introduction

Cyanobacterial Photosystem (PS) I is a pigmentprotein complex consisting of at least 12 polypeptide subunits, approximately 100 chlorophylls and additional carotenoids (Li et al. 1991; Golbeck 1992). While the constitution and the stoichiometry of the complex have been studied extensively by biochemical methods (Golbeck and Bryant 1991), its quaternary 'in vivo' structure in the thylakoid membrane as well as its three-dimensional structure are still not clarified

in detail. Whereas first reports on the crystallization of P S I complexes from thermophilic bacteria date back to 1987 (Witt et al. 1987; Ford et al. 1987), X-ray crystallographic data became available only very recently (Krauss et al. 1993). These results allowed to propose a preliminary three-dimensional structure of PS I, i.e. a trimeric organisation, but yielded no reliable information on the native organisation of the complexes in the thylakoid membrane. The quaternary structure of P S I has so far been investigated by means of electron microscopy (Lundell et al. 1985; Boekema et

264 al. 1987, 1989; Ford and Holzenburg 1988; Kruip et al. 1994), non-denaturatingelectrophoresis (Takahashi and Katoh 1982; Ford 1987), and HPLC (Boekema et al. 1987; Witt et al. 1987; R6gner et al. 1990a, 1990b; Kruip et al. 1994). As a result of these studies, oligomeric structures ofPS I have been detected in various cyanobacteria like e.g. Synechococcus sp. (Boekema et al. 1987, 1989), Prochlorothrix hollandica (van der Staay et al. 1993), Phormidium sp. (Ford and Holzenburg 1988), Mastigocladus laminosus (Almog et al. 1991) and in Synechocystis PCC 6803 (R6gner et al. 1990b). Mostly monomeric and trimeric organisation of P S I complexes was found, but dimers were reported as well (R6gner et al. 1990a). However, the question of the in vivo quaternary organisation of PS I is still a matter of debate, although much biochemical evidence has been accumulated (see R6gner et al. 1990a; Hladik and Sofrova 1991) suggesting at least the trimeric form as a native one. In cyanobacterial membranes electron microscopy also indicates the existence of P S I trimers (Hefti et al. 1992; Kruip et al. 1994; Peterman et al. 1994). A recent hypothesis claims that this trimeric form is in dynamic equilibrium with the monomeric form, possibly triggered by the state transitions (Kruip et al. 1994; Peterman et al. 1994) Time-resolved fluorescence is a very sensitive method for probing the functionality of a photosystem by monitoring processes such as energy transfer or charge separation in the antenna and the reaction centre, respectively. It is now well known that the PS I antenna in both cyanobacteria and higher plants is spectrally highly heterogeneous. This has been demonstrated in a number of steady-state (Gobets et al. 1994; Vanderlee et al. 1993; Wittmershaus et al. 1992; Gobets et al. 1994) as well as time-resolved (Turconi et al. 1993; Werst et al. 1992; Hastings et al. 1994a, 1994b; Evans et al. 1990; Sparrow et al. 1990) spectroscopic studies. A salient feature of the PS I complex is the existence of a small group of pigments absorbing and emitting at wavelengths substantially longer than the RC (P700) itself (Holzwarth et al. 1993; Trinkunas and Holzwarth 1994; Trinkunas et al. 1996). In a very recent study comparing stationary absorption and fluorescence of P S I monomers and trimers (Gobets et al. 1994) no differences were observed between the aggregation states with the exception of an increased absorption of some red pigments in the trimers. Monomeric and trimeric PS 1 particles from cyanobacteria had been well characterised in their biochemical composition (R6gner et al. 1990b), structure (Witt et al. 1990)

and stationary polarised absorption and fluorescence (Gobets et al. 1994) and thus represent a good model system to examine eventual differences between the two organisational forms by time-resolved techniques. The aim of this work is to compare the picosecond fluorescence kinetics of pure P S I monomers and trimers in order to probe whether there exist any functional differences which can be correlated with the different tertiary organisation. In addition, we wish to compare the time-resolved spectra with previous data on PSI cores (Turconi et al. 1993) in order to test for a lifetime component tentatively suggested to be due to aggregation.

Materials and methods

Pure monomeric and trimeric PS I complexes from

Synechocystis PCC 6803 (R6gner et al. 1990b) were isolated essentially as described (Kruip et al. 1994; R~gner et al. 1990b). The antenna size for both monomers and trimers was determined to be 85 410 Chl/P700 using the light-induced photooxidation method (R6gner et al. 1990b). Purity and homogeneity of the particles has been checked by PAGE and size exclusion chromatography according to (Kruip et al. 1994; R6gner et al. 1990b). For measurements, the samples were diluted to approx. 8 #g Chl/ml in a buffer containing 20 mM MES, pH 6.5, 10 mM MgC12, 10 mM CaCI2, 0.5 M mannitol and 0.03%/3-dodecylmaltoside. For measurements 10 mM ascorbate as well as 10 #M phenazine methosulfate were freshly added. The samples were pumped at a flow rate of 12 ml/min through the measuring cuvette (1.5 × 1.5 mm2). Both the sample reservoir and the cuvette were thermostated at 282 4- 2 K. Corrected steady-state fluorescence spectra were recorded on a Fluorolog spectrofluorimeter (Spex Industries) (Holzwarth et al. 1978). Stationary spectra were taken before and after each set of time-resolved measurements in order to check for the stability of the samples. Picosecond time-resolved fluorescence was measured by the single-photon-timing technique, using a synchronously pumped, cavity dumped dye laser as described previously (Wendler and Holzwarth 1987) with a repetition rate of 800 kI-Iz and DCM as the laser dye. The overall system prompt-response was typically (32 4- 2) ps (FWHM). The emission decay was recorded every 7 nm between 680 nm and 736 nm and analyzed both by single-decay and by global analysis methods (Wendler and Holzwarth 1987). The global

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Figure 1. Corrected steady-stateemissionspectraof PSI monomers (top) and trimers (bottom) isolated from Synechocystis PCC 6803

at room temperature (24°C, solid line) and at 77 K (dotted line). Excitation wavelengthwas 435 nm,

analysis yielded decay-associated spectra (DAS) for each data set.

Results Figure 1 shows the steady-state fluorescence spectra of monomers and trimers of P S I recorded at both room temperature (24 o C) and at 77 K. At room temperature the maxima are located at 680 4- 1 nm for both types of particles. Also a broad shoulder at 720-730 nm is present. This long wavelength fluorescence dominates the low temperature emission spectra peaking at around 725 nm, followed by a broad shoulder around 790 nm. As reported in (Gobets et al. 1994) the 77 K fluorescence spectrum of monomeric P S I shows a weak and relatively broad band around 680 nm which is essentially absent with trimeric particles. Stationary absorption spectra for trimers (not shown) revealed a slightly increased absorption around 710 nm in agreement with a previous report (Hladik and Sofrova 1991). Picosecond fluorescence kinetics were measured by single-photon counting. Applying global analysis on the decay data we found four lifetimes (7-1 ... 7"4) for both monomeric and trimeric PS 1.1 Figures 2a and b show the fluorescence DAS of monomeric and trimeric particles, respectively. The single-decay analysis (data not shown) of the fluorescence decays showed

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Figure 2. Correcteddecay-associatedfluorescencespectra(DAS)of PSI monomers(top)and trimers(bottom)isolatedfromSynechocystis PCC 6803. Excitation wavelength 670 nrn, T = 9°C. Relative amplitudes are shown, normalizedto the stationary emissionspectram.

that these lifetimes were indeed independent of the detection wavelength. The DAS are dominated by the spectra of the two fastest components (rl ~ 5 ps and r2~ 25 ps). The amplitude of the rl component shows a clear change from positive to negative value around 710 nm for both monomers and trimers. This indicates an energy transfer component (see (Turconi et al. 1993)). The value for the lifetime of the 7"3 component is higher in trimers as compared to monomers (90 ps vs. 220 ps). However, the amplitude of this component is fairly low, which reduces the accuracy of that lifetime. Overall, the amplitude of this as well as of the component r4 (3.3 ns or 4.0 ns) are somewhat higher in the trimers as compared to the monomers.

Discussion The stationary fluorescence spectra at 77 K of the monomeric and trimeric forms of P S I (Figure 1) are quite similar in all features to the ones reported previously (Gobets et al. 1994). To our knowledge, no stationary fluorescence spectra of monomers and trimers of P S I at room temperature were published until now. In the room temperature spectra no significant differences between the two particle types could be detected. Comparing these data with the steady-state fluorescence spectra of phycobilisome-depleted thylakoid

266 membranes from Synechocystis wild type and from a PS II-lacking mutant (Wittmershaus et al. 1992) the spectra lead to the conclusion that the particles are not significantly affected by the isolation procedure in any way. The global analysis of the fluorescence decay data shows that four kinetic components are necessary and sufficient to describe the fluorescence decay kinetics of both the monomers and trimers of PS I. Except for the

A comparative fluorescence kinetics study of Photosystem I monomers and trimers from Synechocystis PCC 6803.

Picosecond time-resolved fluorescence measurements have been performed as a function of emission wavelengths in order to investigate the possible func...
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