And P700-Induced Quenching of the Fluorescence at 760 Nm in Trimeric

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Biochimica et Biophysica Acta 1706 (2005) 53–67 http://www.elsevier.com/locate/bba

P700+- and 3P700-induced quenching of the fluorescence at 760 nm in trimeric Photosystem I complexes from the cyanobacterium Arthrospira platensis

Eberhard Schloddera,*, Marianne C¸ etina, Martin Byrdina,b, Irina V. Terekhovac, Navassard V. Karapetyanc

aMax-Volmer-Laboratorium fu¨r Biophysikalische Chemie, Technische Universita¨t Berlin, Strasse des 17 Juni, 135, 10623 Berlin, Germany bService de Bioe´nerge´tique and CNRS URA 2096, DBJC, CEA Saclay, 91191 Gif-sur-Yvette Cedex, France cA.N. Bakh Institute of Biochemistry, Russian Academy of Sciences, Leninsky Prospect, 33, 119071 Moscow, Russia

Received 30 March 2004; received in revised form 27 August 2004; accepted 27 August 2004 Available online 11 September 2004

Abstract

The 5 K absorption spectrum of Photosystem I (PS I) trimers from Arthrospira platensis (old name: Spirulina platensis) exhibits long-wavelength antenna (exciton) states absorbing at 707 nm (called C707) and at 740 nm (called C740). The lowest energy state (C740) fluoresces around 760 nm (F760) at low temperature. The analysis of the spectral properties (peak position and line width) of the lowest energy transition (C740) as a function of temperature within the linear electron–phonon approximation indicates a large optical reorganization energy of ~110 cm1 and a broad inhomogeneous site distribution characterized by a line width of ~115 cm1. Linear dichroism (LD) measurements indicate that the transition dipole moment of the red-most state is virtually parallel to the membrane plane. The relative fluorescence yield at 760 nm of PS I with P700 oxidized increases only slightly when the temperature is lowered to 77 K, whereas in the presence of reduced P700 the fluorescence yield increases nearly 40-fold at 77 K as compared to that at room temperature (RT). A fluorescence induction effect could not be resolved at RT. At 77 K the fluorescence yield of PS I trimers frozen in the dark in the + presence of sodium ascorbate decreases during illumination by about a factor of 5 due to the irreversible formation of P700 FA/B in about + 60% of the centers and the reversible accumulation of the longer-lived state P700 FX. The quenching efficiency of different functionally relevant intermediate states of the photochemistry in PS I has been studied. The redox state of the acceptors beyond A0 does not affect F760. Direct kinetic evidence is presented that the fluorescence at 760 nm is strongly quenched not only by P700+ but also by 3P700. Similar kinetics were observed for flash-induced absorbance changes attributed to the decay of 3P700 or P700+, respectively, and flash-induced fluorescence changes at 760 nm measured under identical conditions. A nonlinear relationship between the variable fluorescence around 760 nm and the [P700red]/[P700total] ratio was derived from titration curves of the absorbance change at 826 nm and the variable fluorescence at 760 nm as a function of the redox potential imposed on the

Abbreviations: A0, primary electron acceptor in PS I; A1, secondary electron acceptor in PS I (a phylloquinone); Chl a, chlorophyll a; Chl aV, C-13 epimer of Chl a; C708 (C740), Chl absorbing at 708 (740) nm; CAPS, 3-(cyclohexylaminol-1-propanesulfonic acid); h-DM, n-dodecyl-h-d-maltoside; DPIP, 2,6-

dichlorophenolindophenol; Em, midpoint potential; FeS, iron–sulfur cluster; FX,FA and FB, three [4Fe–4S] clusters in PS I; F760, fluorescence with an emission maximum at 760 nm; LHC I, light-harvesting complex of Photosystem I; P700 (P700+), primary electron donor of PS I in the reduced (oxidized) 3 state; P700, P700 in the excited triplet state; PA and PB, the two chlorophylls constituting P700 coordinated by subunit PsaA and PsaB, respectively; PMS, phenazine methosulfate; PS I (PS II), Photosystem I (Photosystem II); RT, room temperature; t1/2, half-life; TMPD, N,N,NV,NV-tetramethyl-p- phenylenediamine dihydrochloride; TS, triplet-minus-singlet * Corresponding author. Tel.: +49 30 3142 2688; fax: +49 30 3142 1122. E-mail address: [email protected] (E. Schlodder).

0005-2728/$ - see front matter D 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.bbabio.2004.08.009 54 E. Schlodder et al. / Biochimica et Biophysica Acta 1706 (2005) 53–67 sample solution at room temperature before freezing. The result indicates that the energy exchange between the antennae of different monomers within a PS I trimer stimulates quenching of F760 by P700+. D 2004 Elsevier B.V. All rights reserved.

Keywords: Photosystem I; P700; Energy transfer; Fluorescence quenching; Transient absorbance spectroscopy; Long-wavelength antenna chlorophyll; Arthrospira platensis

1. Introduction heterogeneity at low temperature. In one fraction of the PS I complexes, an irreversible charge separation due to the + + Photosystem I (PS I) is a pigment–protein complex stable formation of P700 FA and P700 FB takes place, located in the thylakoid membranes of cyanobacteria, algae whereas in the other fraction, forward electron transfer to the and plants that mediate light-induced electron transfer from terminal iron–sulfur clusters is completely blocked at plastocyanin or cytochrome c6 on the lumenal side to cryogenic temperatures [11,12]. In this fraction, the charge ferredoxin on the stromal side (for a review see Refs. [1,2] separation is reversible at low temperature and can be + and references therein). PS I of higher plants and algae attributed to the formation and decay of P700 A1 and + + (named PS I-200) consists of the PS I core complex and the P700 FX. The charge recombination of P700 A1 occurs peripheral light-harvesting complex (LHC I). Cyanobacteria with t1/2 ~200 As. This half-life was found to be almost lacking LHC I contain only the PS I core. The PS I core temperature independent [12]. The charge recombination + complex coordinates all of the redox cofactors and the core between FX and P700 takes place in the millisecond time antenna of ~100 chlorophyll a (Chl a) and ~20 h-carotene range at low temperature. molecules. The PS I core complexes in cyanobacteria are When the forward electron transfer to A1 is prevented by organized preferentially as trimers [3–6], whereas PS I in prereduction or removal of A1, the primary radical pair, + higher plants and algae is present only as a monomer [7]. P700 A0 , can decay by three pathways: (i) by charge Structural characterization of green plant PS I by electron recombination to the singlet ground state, (ii) by an microscopy [8], as well as the recent X-ray structure of plant activated back reaction to the excited singlet state of P, PS I at 4.4 2 resolution [9] indicate that LHC I (Lhca1–4) is and (iii) after singlet–triplet mixing in the radical pair, by 3 only attached in a half-moon shape to the core complex at charge recombination to P700Ao. The lowest excited triplet the side of the subunits PsaF/PsaJ of the core and thus does state of P700, 3P700, is formed at 5 K with high yield and not cause structural hindrances for trimerization. decays in the millisecond time range (t1/2c0.7 ms (65%) A high-resolution X-ray structure is available for trimeric and 7 ms (35%)). PS I core complexes from the cyanobacterium Thermosy- The function of the antenna is to harvest solar energy and nechococcus elongatus [10]. The structure exhibits 12 to transfer this energy to the reaction center, where the protein subunits, 96 Chls, 22 carotenoids (Car), 2 phyllo- excitation energy is converted into a charge separated state. quinones, 3 iron–sulfur [4Fe–4S] clusters, 4 lipids, about One striking feature of the light-harvesting antenna of PS I 200 water molecules and a metal ion (presumably Ca2+) for is the presence of long-wavelength (red, low-energy) Chls each PS I monomer. that absorb at energies lower than that of the primary The two large subunits, PsaA and PsaB, each consisting electron donor P700 (for reviews see Refs. [13–16]). Plants of 11 transmembrane helices, coordinate most of the and some algae contain their most-red Chls in the LHC I, antenna pigments and the following redox cofactors which give rise to the emission around 735 nm at low involved in the electron-transfer process: the primary temperature, whereas long-wavelength Chls in the PS I core electron donor P700 (a heterodimer of chlorophyll a (PB) of plants and algae absorb around 705 nm and emit at 720 and aV (PA), the primary acceptor A0 (a Chl a monomer), nm. The existence of red Chls in the core antenna absorbing the secondary acceptor A1 (a phylloquinone), and FX (a even further to the red is unique to cyanobacteria. The [4Fe–4S] iron–sulfur–cluster). The terminal electron accept- content and the spectral characteristics of long-wavelength ors FA and FB (two [4Fe–4S] iron–sulfur clusters) are both Chl a antenna states are species-dependent. PS I trimers coordinated by subunit PsaC, one of the three extrinsic usually contain more red Chls than monomers. The 5 K subunits located on the stromal side. absorption spectrum of PS I trimers of T. elongatus exhibits After absorption of light by an antenna pigment, the Chl a antenna states absorbing at 708 and 720 nm [17,18], excitation energy is efficiently trapped via charge separation whereas PS I trimers of Synechocystis sp. PCC 6803 contain in the reaction center. P700 in the lowest excited singlet red Chls peaking at 708 and 714 nm [19–22]. The most-red state donates an electron to the primary acceptor A0. Charge Chl a antenna state absorbing at 740 nm (C740) and stabilization is achieved by subsequent electron transfer to emitting at 760 nm (F760) at low temperature is present in the secondary acceptor A1, then further to FX and finally to PS I trimers of Arthrospira platensis [5,23,24].The the terminal electron acceptors FA and FB. An interesting intensity of the fluorescence at 760 nm (F760) in PS I aspect of the electron transfer in PS I is the reported trimers of A. platensis is highly dependent on the redox state E. Schlodder et al. / Biochimica et Biophysica Acta 1706 (2005) 53–67 55 of P700 [4,23]. The maximum F760 level was observed for excitation energy (especially at low temperature) thus PS I trimers treated with dithionite and slowly frozen under affecting the efficiency of charge separation [17]. At room illumination with strong light to keep the PS I acceptor side temperature, the quantum yield of photochemistry, i.e. P700 reduced. The F760 steady-state intensity was lower if PS I oxidation, was independent of the wavelength of the trimers were frozen after incubation with dithionite in the excitation, even at wavelengths of up to 760 nm as a result dark. If only P700 was reduced as a result of dark incubation of uphill energy migration to bulk Chls and then to P700 of the sample with ascorbate, only ~1/3 of the maximum [17,25,26]. The extension of the spectral range for light intensity of F760 was observed. The reason of these data harvesting to longer wavelengths increases the efficiency of was not clear because the redox state of the PS I cofactors the antenna system. It has been suggested that the utilization was not determined in these experiments [4]. The 760 nm of far-red light for photochemistry is the result of the fluorescence is diminished by more than a factor of 10 upon adaptation of the cyanobacterial dense suspension to low oxidation of P700 at cryogenic temperatures. A decrease of light conditions [31]. Red Chls may also be involved in the the fluorescence from the most-red Chl a antenna state by protection of PS I complex against excess excitation light P700+ formation was also observed for PS I trimers of T. energy [32]. elongatus at low temperature [18], but the fluorescence was Since the long-wavelength chlorophylls have very not as strongly quenched by P700+ as in PS I trimers from pronounced effects on energy transfer and trapping in PS Arthrospira. Note that the fluorescence intensity around 735 I, the purpose of this paper is to characterize the spectral nm (F735) of the PS I–LHC I complex from higher plants at properties of the red-most chlorophylls (C740) in PS I low temperature seems to be independent of the P700 redox trimers of A. platensis and to analyze the intensity of the state. This might be explained by the large distance of z60 fluorescence at 760 nm (F760) as a function of different 2 between P700 and the red Chl form responsible for F735 functionally relevant intermediate states of the photochem- being localized on the Lhca1/Lhca4 heterodimer (LHC I- istry in PS I. Direct kinetic evidence is presented that the 730) [7,8]. fluorescence at 760 nm is quenched not only by P700+ but The excited-state dynamics of PS I complexes have been also by 3P700. The position of the most-red chlorophylls studied intensively by time-resolved absorption and fluo- with respect to P700 is discussed. rescence spectroscopy. In accordance with the difference in the low temperature yield of 760 nm fluorescence in PS I trimers with P700 and P700+, respectively, the lifetime of 2. Materials and methods the fluorescence decay at 760 nm was found to be significantly shorter in PS I trimers with P700 oxidized 2.1. Preparation of PS I complexes than that in PS I trimers with P700 reduced [25,26]. The Ffrster overlap integral between F760 and the absorption Cells of the cyanobacterium A. platensis were grown on spectrum of P700+ is sufficiently large (about ~1/3 of the Zarook medium and the thylakoid membranes were overlap between two isoenergetic chlorophylls with a Stokes prepared as described [4]. PS I trimers and monomers have shift of 130 cm1) to explain the quenching by direct been isolated from the membranes treated with 1% n- resonance energy transfer from the long-wavelength Chls to dodecyl-h-d-maltoside (h-DM) (detergent: Chl ~15) at 4 8C P700. From the decay rate of the fluorescence at 760 nm a for 30 min using the column chromatography with DEAE- distance between C740 and P700 of ~42 2 has been Toyopearl. Isolated complexes were stored in 50 mM Tris– reported [13,26,27]. HCl buffer (pH 8) containing 0.04% dodecyl-maltoside at It has been proposed that the long-wavelength antenna 40 8C until use. Chlorophyll concentrations were deter- states may result from strongly coupled Chls [5,20,28–30]. mined according to Porra et al. [33]. The Chl/P700 ratio for Evidence for this has been obtained especially for C708. It the PS I complexes was calculated from the flash-induced has been shown that C740 in PS I trimers of A. platensis absorption change at 703 nm due to the photooxidation of emerges upon trimerization of isolated monomeric PS I P700, using a differential molar extinction coefficient of complexes [24], indicating that about three Chls of each 83,000 M1 cm1 [34]. For monomeric and trimeric PS I monomer located at the periphery near the trimerization core complexes, the Chl/P700 ratio has been determined to domain are red-shifted due to interactions and/or changes in be 102F8. pigment organization induced by the trimerization. On the other hand, the difference between monomers and trimers 2.2. Measurement of steady-state optical spectra from T. elongatus has been ascribed to a lower number of long-wavelength pigments in the monomers due to the lack For the measurement of absorbance spectra, the concen- of the PsaL subunit [17]. trated samples were diluted to a final Chl concentration of The function of the red Chls in photosynthesis may be about 10–15 AM with a buffer containing 20 mM Tricine different dependent on their location in the antenna system (N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine, and therefore on the distance between the red Chls and P700 pH=7.5), 25 mM MgCl2, 100 mM KCl, 0.02% h-DM and [13]. Long-wavelength Chls compete with P700 for the redox mediators as indicated in the figure captions. For 56 E. Schlodder et al. / Biochimica et Biophysica Acta 1706 (2005) 53–67 measurements at cryogenic temperatures, glycerol was orientation function that reflects how well the PS I added to a final concentration of 60–65% (v/v). For low- complexes are oriented as a function of the compression temperature measurements, the cuvette was placed in a factor n [35]. Aiso is given by 2 AO+A8. The AO and A8 continuous flow helium cryostat (Oxford CF 1204) or a spectra were recorded with a spectral resolution of 1 nm on variable temperature liquid nitrogen bath cryostat (Oxford the Cary-1E-UV/VIS spectrophotometer using a film polar- DN1704). Absorption spectra were recorded with a spectral izer (Spindler and Hoyer model 10K). resolution of 1 nm on a Cary-1E-UV/VIS spectrophotometer using a home-built cryostat holder. Baselines for the 2.3. Transient absorption spectroscopy absorption spectra were measured separately as a function of temperature and subtracted from the sample spectra at the Flash-induced absorbance changes in the nano- to same temperature. Measuring and baseline samples were millisecond time range were performed with the set-ups identical except that PS I was omitted in the baseline described previously [12]. The sample was excited either samples. with flashes of 3 ns pulse duration (FWHM) at 532 nm from For the light-minus-dark absorbance difference spectrum a frequency-doubled Nd/YAG laser (Quantel YG 441) or at 5 K, PS I complexes were diluted as described above and with flashes from a Xe-flash lamp (pulse duration 15 As) cooled in the dark to 5 K in the presence of 5 mM sodium whose emission was filtered by a colored glass. + ascorbate and 3 AM PMS. Under these conditions, P700 is The charge recombination between P700 and A1 /FX at fully reduced and all electron acceptors are oxidized prior low temperature in the microsecond to millisecond range to excitation. Spectra were recorded with data intervals of was measured under conditions of prereduced iron–sulfur 0.1 nm, a scan speed of 20 nm/min and a spectral clusters FA and FB. PS I complexes were suspended in a bandwidth of 1 nm on the Cary 1E UV/VIS-spectropho- buffer containing 100 mM CAPS (pH 10), 25 mM MgCl2, tometer. The difference spectra were obtained by subtract- 0.02% h-DM, 65% (v/v) glycerol and 10 mM sodium ing the absorbance spectrum of the dark-adapted state from dithionite. Subsequently, the sample was frozen in the dark. that after illumination by 30 saturating flashes from a Xe- The decay of the triplet state of P700 has been followed flash lamp. by absorbance changes in the QY-region. PS I core For linear dichroism (LD) measurements, the samples complexes were diluted to 10–15 AM Chl in 100 mM were oriented by embedding the PS I complexes in a gelatin CAPS (pH 10), 10 mM MgCl2, 10 mM CaCl2 and 0.02% h- gel, which subsequently was squeezed by a compression DM. Glycerol was added to a final concentration of 65% (v/ factor n in the x-direction. The gel expands in the z- v). 10–20 mM dithionite was added to this solution under direction with the same factor n maintaining the y- argon. The samples were then illuminated at 270 K for 3 dimension constant. The measuring light beam is propagat- min with a 250 W focussed tungsten lamp filtered by water ing parallel to the y-axis. The composition of the gel was and additional heat absorbing filters. This procedure leads to 60% (v/v) glycerol, 20 mM Tricine pH=7.5, 25 mM MgCl2, the prereduction of the secondary electron acceptor A1, 100 mM KCl, 0.02% h-DM, 6.3% (w/v) gelatin (Dr. whereby further electron transfer to A1 is blocked. The + Oetker), 5 mM sodium ascorbate and 3 AM PMS. The primary radical pair P700 A0 formed under these con- mixture was heated to about 55 8C to dissolve the gelatin. ditions recombines with a high yield to the triplet state of Subsequently, the mixture was cooled to about 30 8C and P700. During freezing to cryogenic temperatures, the the PS I complexes were added. Then the mixture was intensity of the illumination was reduced by a factor of 10 poured into the gel press and kept in the refrigerator for 40 which was sufficient to keep A1 in the reduced state. min. After that time, the gel was squeezed by about a factor Absorbance changes which are connected to the pro- of n=2. For nN2.2 rupturing of the gel was observed. The cesses discussed above were measured with a home-built LD of the sample is defined as the difference in the flash photometer. The measuring light of a 250 W tungsten absorption of light polarized along the z- and x-axis, Az–Ax. halogen lamp (Osram) passed through a monochromator The PS I complexes are disc-shaped with two long axis with 3 nm bandwidth placed between light source and parallel to the membrane plane and a short axis which is sample and a combination of interference filters and colored perpendicular to the membrane plane. Considering the shape glasses in front of the photomultiplier (EMI 9558BQ). The of the PS I complexes they will align with their (membrane) signals were digitized and averaged by a transient recorder planes parallel to the y–z-plane. In this case, the LD can be (Tektronix TDS 540). expressed as Absorbance changes at 826 nm in the nanosecond time ÀÁ 2 range were measured as described previously [36]. In the LD ¼ AO A ¼3A h0:5 3cos # 1 iUðÞn : ð1Þ ? iso microsecond and millisecond range, a 250 W tungsten AO and A8 is the absorption of light polarized parallel halogen lamp (Osram) was used as measuring light source. and perpendicular to the plane of the disc; # is the angle The measuring light was monitored by a Si-photodiode between the transition moment and the normal to the (SGD 444 from EG&G or OSD-100-5T from Centronic) membrane plane; hi denotes averaging over all values of # if with a load resistor of 1 kg coupled to the amplifier TEK different transition moments are involved and U(n)isan AM 502 from Tektronix. E. Schlodder et al. / Biochimica et Biophysica Acta 1706 (2005) 53–67 57

2.4. Redox titration protect the detection system from the strong fluorescence emission stimulated by the laser flash, a gated avalanche To determine the oxidation midpoint potential Em of photodiode (RCA C 30872) coupled to the transient P700, the flash-induced absorbance change at 826 nm, recorder (Tektronix TDS 540) has been used in the associated with oxidation of P700, was measured as a microsecond time range. The dead time after the flash was function of the redox potential. PS I complexes were diluted about 100 As. Remaining artefacts caused by the strong to 20–30 AM Chl in 20 mM Tricine (pH 7.5), 100 mM KCl, actinic flash were measured separately and subtracted. 25 mM MgCl2, 0.02% h-DM. The redox potentials were In the millisecond time range, the photomultiplier (EMI adjusted by adding ferricyanide and ferrocyanide. After 9668-BQ) connected to the transient recorder (Tektronix each experiment, the potential was measured using a TDS 540) could be used without gating circuit because the combination Pt/Ag/AgCl electrode (Schott PT5900A), recovery of the detection system was sufficiently fast (about which was calibrated against the redox potential of a 500 As) to follow the fluorescence yield changes on the saturated solution of quinhydrone as a function of pH. A millisecond time scale. In both cases, a Schott red cut-off pH meter (Knick PHM82) was used to read out the redox filter RG715 (20 mm) and an interference filter (AL 762 nm potential. All redox potentials are given relative to the from Schott) were additionally located in front of the standard hydrogen electrode (NHE). detector.

2.5. Fluorescence and fluorescence induction at 760 nm 2.7. Data analysis

Fluorescence was excited with a He–Ne laser. The The time course of the flash-induced absorption or excitation wavelength was 633 nm. An electrically fluorescence changes was fitted by a (multi)exponential controlled mechanical shutter with a 10%-to-90% rise time decay using an algorithm that minimizes the sum of the of ~1 ms allowed to expose the sample to the excitation unweighted least squares. beam for a chosen time period. The sample was excited near the edge of the cuvette to avoid self-absorption and recorded at an angle of 908 to the excitation beam by a 3. Results and discussion photomultiplier (EMI 9668-BQ) connected to a transient recorder (Tektronix TDS 540), which was triggered by the 3.1. Absorption spectra of trimeric and monomeric PS I shutter driving electronics. The photomultiplier was complexes shielded by a long-pass filter with a cut-off wavelength of 715 nm (20 mm) and an interference filter (AL 762 nm Absorption and LD spectra of PS I core complexes from from Schott). For measurements at cryogenic temperatures, A. platensis have been examined in increments of 30 K the cuvette was placed in a liquid nitrogen bath cryostat throughout the temperature range from 5 to 300 K. Fig. 1 (Oxford DN1704) or in a continuous flow helium cryostat shows the absorption spectra of trimeric PS I complexes (Oxford CF1204), either frozen in the dark in the presence from A. platensis at different temperatures. The inset shows of 5 mM ascorbate or 10 mM sodium dithionite to keep an enlargement of the red region of the absorption spectra. P700 in the reduced state or frozen under illumination The low temperature spectra clearly exhibit various bands in without additions to keep P700 oxidized. For measurements of the fluorescence yield as a function of temperature, the intensity of the short (5 ms) exciting light pulses was set sufficiently low that the photochemical state of the sample was not significantly altered. For measurements of variable fluorescence (fluorescence induction) the exciting light plays the dual role of stimulating fluorescence and changing the photochemical state of the sample (e.g. + P700FA/BYP700 FA/B). The time scale for the induced fluorescence yield changes can be adjusted by the intensity of the exciting light.

2.6. Flash-induced fluorescence changes at 760 nm

Fluorescence was excited by the continuous light from a He–Ne laser. The actinic light source was the frequency- doubled Nd/YAG laser (Quantel YG 441). The recovery of Fig. 1. Absorption spectra of trimeric PS I from A. platensis at different the flash-induced fluorescence changes at 760 nm has been temperatures. The inset shows an enlargement of the red region of the measured on the microsecond to millisecond time scale. To absorption spectra. 58 E. Schlodder et al. / Biochimica et Biophysica Acta 1706 (2005) 53–67

1 the QY region, which can be assigned to different spectral 14,411 cm (693.9 nm) for a fit where all parameters were forms of the chlorophylls. Pigment–protein and/or pigment– free. With a higher number of Gaussian bands, the fits do pigment interactions are probably the reason for this spectral not converge to a unique solution. Treating the position of heterogeneity. Besides the broad main peak centered at the maximum and the line width for the three Gaussian about 678 nm and a shoulder around 672 nm on the short- bands as adjustable parameters (i.e. only the integrated wavelength side, a shoulder at 694 nm and distinct bands at intensity of each of the three red absorption bands was about 707 and 740 nm are resolved at 5 K on the long- fixed), we determined the best fit for each spectrum wavelength side of the main peak. Above 180 K, these long- throughout the whole temperature range. In the following, wavelength features resolved well at 5 K transform with we focus on the temperature dependence of the red-most increasing temperature into the long, featureless red tail band. The results of the decomposition are most reliable for which can be seen in the room temperature spectrum. this band, because the red-most band has the smallest The integrated absorption between 550 and 800 nm overlap with neighboring bands. The inset of Fig. 2 shows increases by about 10% as the temperature is lowered from the squared line width r2 (A) and the position of the 295 to 5 K, most of these changes occur between room maximum v˜ max (B) of the red-most band as a function of temperature and 150 K. This is probably caused by changes temperature. It can be seen that a blue shift of the peak 1 1 in sample volume (chlorophyll concentration) and optical position (v˜ max=740 nm at 5 KYv˜ maxc724 nm at room path length due to solvent contraction. Moreover, redistrib- temperature) and a broadening of the band (r=140 cm1 at ution of absorption can be noticed. The increase of 5KYr=240 cm1 at 295 K) occurs as the temperature is absorption with decreasing temperature is especially pro- raised from 5 to 295 K. This behavior is also clearly visible nounced in the red region above 705 nm at the expense of in the inset of Fig. 1. From the target analysis of time- the 688–705 nm region. resolved fluorescence data, the absorption and emission To analyze the spectral properties of the red-most maxima of the most-red pool at room temperature have been chlorophylls, we decomposed the red region (685–770 estimated to be at 715 and 730 nm, respectively [19]. In the nm) of the absorbance spectra into Gaussian bands and Franck–Condon approximation, the first and second determined the first moment (=position of the maximum for moment can be expressed as a symmetric band) and second moment (squared line width 2 1 hv˜ r ) of the long-wavelength bands as a function of temper- hv˜maxiðÞ¼hT v˜maxiðÞT ¼ 0 v˜ðÞ1 R coth ð2Þ ature. For the decomposition, the spectra were transformed 4 2kT to a wave number scale and the minimum number of and Gaussian bands was used to obtain excellent fits. Fig. 2 presents the decomposition of the red part of the 5 K hv˜ r2ðÞ¼T SR2v˜ coth þ r2 ð3Þ spectrum into three Gaussian bands with absorption maxima 2kT inh at 13,509 cm1 (740.2 nm), 14,157 cm1 (706.4 nm) and where v˜ is an effective frequency of the vibrational mode coupled to the electronic transition. S and R are the linear and quadratic coupling constants with S=(fD2)/(2hcv˜)(f is the force constant and D is the shift of the minimum of the potential energy curve for the excited state with respect to the ground state) and R ¼ ðÞv˜V=v˜ 2 (v˜ and v˜V are the effective frequencies of the vibrational mode in the ground and excited state, respectively) [37–39]. The solid line in Fig. 2, inset (A) represents a fit of the data using Eq. (2) with 1 1 2 v˜=(65F10) cm , rinh=(112F6) cm and SR =1.7F0.3. Assuming Rc1, a Stokes’ shift (=2Sv˜) of about 220 cm1 can be calculated. Rather high values for the inhomogeneous broadening and the Stokes’ shift have been found to be a common feature of the long-wavelength chlorophylls in PS I from different species [17,20,28,40,41]. In the vibronic coupling model, a shift of the peak maximum is related to the quadratic coupling (see Eq. (2)). With v˜VNv˜ a red shift of the absorption band is expected as Fig. 2. Decomposition of the red part of the 5 K absorption spectrum, which observed in the experiment (see Fig. 2, inset (B)). To explain has been transformed to a wave number scale, into three Gaussian bands the magnitude of the shift, the fit yields a value for the with absorption maxima at 13,509 cm1 (740.2 nm), 14,157 cm1 (706.4 nm) and 14,411 cm1 (693.9 nm). Inset: squared line width (A) and peak quadratic coupling constant R of about 4 which corresponds 1 position (B) of the red-most band located at 740 nm at 5 K as a function of to a frequency change from 65 cm in the ground state to the temperature. 130 cm1 in the excited state. This frequency change seems E. Schlodder et al. / Biochimica et Biophysica Acta 1706 (2005) 53–67 59 rather large and we tend to assume that there are other contributions to the temperature dependence of the peak position, e.g. contraction of the protein upon lowering the temperature and/or the involvement of internal charge transfer states, which might also contribute to the unusual strong broadening of the red transitions. The number of chlorophylls contributing to the red absorption has been estimated from the ratio of the integrated intensity of the long-wavelength absorption bands at 707 and 740 nm in the 5 K spectrum to the total integrated intensity of the QY-band and the number of Chls per PS I (see Materials and methods). In accordance with Gobets et al. [19], we calculated that about three Chls contribute to the red-most band peaking at 740 nm and about eight Chls contribute to the band with an absorption maximum at 707 nm. Fig. 3 shows the absorption spectra of monomeric PS I complexes from A. platensis at four temperatures. The inset shows an enlargement of the red region of the absorption spectra. The 77 K spectrum exhibits one distinct band at 707 nm on the long-wavelength side of the main peak. The area of the 707 nm band corresponds to the oscillator strength of about nine Chls. The 740 nm band observed for the trimeric PS I complexes is completely absent in the absorption Fig. 4. (A) LD of trimeric PS I from A. platensis at different temperatures. The spectra are multiplied by a factor of 5. LD spectra display the spectra of monomeric PS I. The almost same amount of difference in absorption of light polarized parallel (AO) and orthogonal Chls in monomeric and trimeric PS I contributing to the 707 (A8) to the plane constituted by the two long axis of the PS I complex. nm band rules out the suggestion that the absorption of three (B) Steady-state polarized absorption (AO and A8), LD and reduced LD of these Chls is shifted to 740 nm upon trimerization. From (=LD/Aisotropic) spectra of trimeric PS I from A. platensis at 5 K. The the temperature dependence of the line width an optical LD spectrum is multiplied by a factor of 5. reorganization energy of about 60 cm1 has been derived (not shown), which is roughly smaller by a factor of 2 other species [30,42,43]. At all temperatures, the maximum compared to the optical reorganization energy of the 740 nm of the LD spectra is observed at 683.4 nm, i.e. 5 nm red- band in trimeric PS I complexes. shifted compared to the absorption maximum. Fig. 4 shows Fig. 4A shows the LD spectra of trimeric PS I complexes that transitions at longer wavelengths (kN675 nm) lie from A. platensis. From the LD spectra, information can be preferentially parallel to the membrane plane (#N558). # obtained on the orientation of the transition dipole moments is the angle between the transition moment and the normal of the pigments relative to the membrane plane. The shape to the membrane plane. The maximal amplitude of the of the spectra is similar to LD spectra reported for PS I from observed LD (Fig. 4A and B) is about one-fifth of the total absorption (note that the LD spectra are multiplied by a factor of 5). On the short-wavelength side of the main peak, the LD is nearly zero, i.e. absorption due to transition moments with preferential orientation parallel to the membrane plane are quite well compensated by absorption due to transition moments oriented perpendicular to the membrane plane. In the range between 400 and 550 nm, the carotenoids give rise to strong positive LD with maxima at 505, 475 and 444 nm indicating that, on the average, the carotenoids are oriented rather parallel to the membrane (#N558) (not shown). Fig. 4B shows the absorption (AO and A8), LD and reduced LD (LD/Aiso) spectra at 5 K. On the long-wavelength side of the main peak in the LD spectrum, the shoulder at 695 nm and the two distinct bands at about 708 and 742 nm are even better resolved than in the Fig. 3. Absorption spectrum of monomeric PS I from A. platensis at absorbance spectrum. The LD/Aiso ratio is maximal for the c different temperatures. The inset shows an enlargement of the red region of red-most band at 740 nm (LD/Aiso 0.32). Under our the absorption spectra. conditions the orientation factor U(n) is about 0.43. Using 60 E. Schlodder et al. / Biochimica et Biophysica Acta 1706 (2005) 53–67

Eq. (1) (see Materials and methods), the angle # between of 5 mM ascorbate, whereas the other one was frozen under the 740 nm transition moment and the normal to the illumination in the presence of dithionite at pH 10. Under membrane plane can be calculated to be 668. To test the the latter conditions, most of the acceptors (FA,FB,FX and applicability of the orientation function U(n), we measured A1) are in the reduced state. The observed temperature LD spectra of different PS I core complexes (PS I trimers dependence was the same (see Fig. 5) indicating that the from T. elongatus and A. platensis, PS I monomers from A. redox state of the acceptors does not affect F760. However, platensis and C. reinhardtii) as a function of n (not shown). as an essential requirement the light intensity of the short The reduced LD at the position of the maximum of the LD (~5 ms) exciting light pulses has to be set sufficiently low so was similar for all the PS I complexes with the only that the photochemical state of the sample is not signifi- + exception of the PS I trimers from A. platensis where this cantly altered, especially the stable formation of P700 FA/B value was lower by about a factor of two. This is probably in the sample with ascorbate has to be avoided (see below). due to a lower degree of orientation. The reason for this is The increase in the fluorescence yield with decreasing not yet understood. Taking this into account, the mean angle temperature can be explained on the assumption that a between the red-most transitions around 740 nm and the significant part of the excitation is trapped on the energeti- normal to the plane is estimated to be N808, i.e. the cally lowest-lying antenna pigments C740, because uphill transition of the red-most state is virtually parallel to the energy transfer becomes improbable at low temperature. As membrane plane. a consequence, the yield of charge separation decreases [17]. At 80 K, the fluorescence at 760 nm of PS I trimers 3.2. Fluorescence around 760 nm (F760) with P700 reduced is mainly associated with long-lived fluorescence components with time constants between 1.3 At room temperature, the energy transfer from the long- and 3.3 ns [26,44]. If P700 is oxidized, the fluorescence at wavelength antenna pigments to P700 is very efficient. The 760 nm is effectively quenched at all temperatures (see Fig. fast trapping of the excitation energy in about 50 ps explains 5). P700+ exhibits a broad, flat absorption band that extends the low fluorescence yield of the PS I core complexes. Fig. 5 into the far red beyond 800 nm and provides spectral shows the temperature dependence of the fluorescence overlap with the emission bands of all the antenna pigments, intensity at 760 nm for trimeric PS I complexes with P700 so that excitation energy transfer from the excited long- in the reduced and oxidized states. Upon lowering the wavelength chlorophylls, C740*, to P700+ can occur by the temperature, the relative fluorescence yield at 760 nm inductive resonance mechanism (Ffrster transfer). The increases by a factor of about 40 for PS I with reduced P700, subsequent fast radiationless decay of (P700+)* probably as compared to a factor of ~4 for PS I with oxidized P700. constitutes the quenching mechanism (for a detailed The data indicate that the quenching efficiency of P700+ discussion of the quenching mechanism see Refs. [18,45]). relative to that of P700 increases with decreasing temper- This explains the much weaker increase of fluorescence ature. The temperature dependence for PS I complexes with intensity at 760 nm in the presence of oxidized P700 at low reduced P700 has been measured with two different temperatures. In the time-resolved measurements, this samples; one sample was frozen in the dark in the presence difference is reflected by an about 110 ps component dominating the decay kinetics of F760 in PS I with oxidized P700 as compared to the nanosecond decay components prevailing in samples with P700 reduced [26,44]. Taking into account that the observed 107 ps lifetime originates from two competing processes—the intrinsic nanosecond fluorescence decay as observed in open centers and direct quenching by P700+—a lifetime of about 115 ps results for the latter process [26,44].Ffrster theory allows to estimate a distance between the red-most pigments C740 and P700+ from this lifetime using the following equation [46]:

9ln10 j2J k d DA 4 DA ¼ 5 4 6 ð Þ 128p NL n s0RDA

in which kDA is the rate constant for resonance energy 2 transfer, JDA is the overlap integral, j is the orientational factor, RDA is the distance between donor D and acceptor A, n is the index of refraction and s0 is the radiative lifetime of Fig. 5. Temperature dependence of the relative fluorescence yield for 2 d 10 6 trimeric PS I samples from A. platensis with P700 oxidized (diamonds) and Chl a. We obtain a value of ~27 with J=2.5 10 cm / 2 with P700 reduced either by the addition of 5 mM ascorbate (squares) or 10 mol, n=1.3, j =0.67 (=mean value of all possible mutual mM dithionite (circles). orientations in three-dimensional space) and s0=15 ns. E. Schlodder et al. / Biochimica et Biophysica Acta 1706 (2005) 53–67 61

Assuming an optimized orientation j2=4 this value increases to 36 2. The value of 42 2 reported earlier was obtained with a value of 2 ns for s0 [26,44], i.e. the fluorescence lifetime was used for s0 instead of the intrinsic radiative lifetime. Leaving aside the exact value for this distance, the stronger quenching of F760 by P700+ compared to the quenching of the long-wavelength fluorescence from the most-red antenna states of other species gives evidence that the chlorophylls giving rise to the emission at 760 nm in trimeric PS I of A. platensis are closer to P700 than the red-most Chls in the other species. Since the emission at 760 nm in PS I trimers of A. platensis emerges upon trimerization of isolated monomeric PS I complexes [24], it has been suggested that the involved Chls are located near the monomer–monomer interfaces which would correspond to a distance to P700 of N30 2 [10].

3.3. Fluorescence induction

The change in fluorescence quantum yield upon changing the photochemical state of the sample (e.g. the light- induced oxidation of P700 in the case of PS I) can be sensitively monitored by the so-called fluorescence induction technique. The time scale for the light-induced fluorescence yield changes can be adjusted by the intensity of the exciting light. At room temperature, no change in fluorescence yield could be detected for trimeric PS I complexes from A. platensis upon illumination of a sample with initially reduced P700 prepared by dark incubation in the presence of 5 mM ascorbate (and 1 AM PMS) (not shown). This result indicates that the oxidized donor Fig. 6. Changes in fluorescence yield upon illumination (fluorescence (P700+) is as good a quencher as the reduced donor for induction curves) of trimeric PS I from A. platensis at 77 K with initially trimeric PS I complexes from A. platensis at room temper- reduced P700. The decrease of the fluorescence yield at 760 nm upon illumination is attributed to the light-induced photooxidation of P700. ature. This is different from trimeric PS I complexes of T. (A) Trace a: time course of F760 at 77 K for a sample with initially elongatus, where a ~12% increase in fluorescence quantum reduced P700 prepared by freezing in the dark in the presence of 5 mM yield upon P700 oxidation has been reported at room ascorbate+1 AM PMS. Trace b: time courses of F760 at 77 K of the temperature [18]. illuminated sample after 10 min of dark adaptation. Trace c: steady-state At low temperatures, a decrease of the fluorescence yield level of F760 reached in the light. Panel (B), same as panel A, except that the sample contains 100 mM CAPS (pH 10) and 10 mM dithionite. at 760 nm upon illumination is observed which is attributed to the light-induced photooxidation of P700. Fig. 6 shows time courses of F760 (trace a) at 77 K for samples with ascorbate (Fig. 6A) and 76% in the presence of dithionite initially reduced P700 either prepared by freezing in the (Fig. 6B)) and the steady-state level in the light given by the dark in the presence of 5 mM ascorbate+1 AM PMS (Fig. dashed lines. In both cases, a smaller reversible part of about 6A) or by freezing in the dark in the presence of 20 mM 10% could be resolved. dithionite (Fig. 6B). At t=0 the light was turned on. The This observation can be well explained by the reported steady-state level (trace c) finally reached in the light is heterogeneity of photochemistry at low temperature [11,12]. given by the dashed lines in Fig. 6A and B. At 77 K, the A detailed analysis at 77 K of trimeric PS I from T. fluorescence quantum yield at 760 nm decreases upon elongatus gave the following results: (a) the irreversible + + illumination to 22% (Fig. 6A) and 62% (Fig. 6B), formation of P700 FA or P700 FB occurs in about 35% of respectively. The fluorescence level of a sample with the PS I complexes. (b) In about 20% of the reaction centers + completely oxidized P700 corresponds to about 10%. Most the state P700 FX is formed, which recombines with of the fluorescence induction effect is irreversible. Trace b complex kinetics (t1/2=5–100 ms). (c) In about 45%, shows the changes of the fluorescence yield at 760 m upon flash-induced electron transfer is limited to the formation + the second illumination after 10 min of dark adaption. The and decay of the secondary pair P700 A1 . The charge + recovery in the dark is given by the difference between the recombination of P700 A1 occurs with a half-life of about initial amplitudes of these traces (32% in the presence of 170 As [12]. 62 E. Schlodder et al. / Biochimica et Biophysica Acta 1706 (2005) 53–67

+ + In order to determine the yield with which these different P700 FA or P700 FB at 77 K. In the presence of 20 mM fractions are formed in trimeric PS I complexes from A. dithionite at pH 10 the terminal iron–sulfur clusters should platensis used in this study, a sample frozen in the dark to be chemically reduced. However, the initial amplitudes of 77 K was exposed to a series of saturating flashes. Either 5 the absorbance change at 826 nm decreases by about 10% mM ascorbate+2 AM PMS or 20 mM dithionite at pH 10 (Fig. 7A, upper trace) indicating that FA and FB are not were added before freezing. The initial amplitudes of the completely reduced under these conditions. In both cases, absorbance change at 826 nm induced by a single flash out the fraction which can be trapped in stable charge-separated of the series are plotted as a function of the flash number in states cannot be increased significantly by further flashes or Fig. 7A. The observed decrease of the initial amplitude is continuous illumination. The last point in Fig. 7A (note the attributed to the irreversible formation of the states break of the x-axis) represents the initial amplitude after 100 + + P700 FA or P700 FB . This is supported by the spectrum flashes. Although the energy of the excitation flashes used of the flash-induced irreversible absorbance changes in these experiments was saturating; i.e. all PS I centers recorded with trimeric PS I complexes from A. platensis were excited by each flash, several successive flashes were at low temperature (Fig. 7B). To obtain this spectrum, the required to obtain the maximal decrease of the initial + + absorbance spectrum at 5 K was subtracted from that amplitude, i.e. to form P700 FA or P700 FB in the entire measured after illumination with 30 flashes. The light- fraction of PS I complexes capable of electron transfer to FA minus-dark difference spectrum is dominated by the or FB. In good approximation, the decrease of the relative characteristic bleaching of the low energy exciton band absorbance change as a function of the flash number can be around 707 nm which accompanies the oxidation of P700. described by the following equation In the presence of 5 mM ascorbate+2 AM PMS about n1 60% of the PS I complexes could be trapped in the states DAn ¼ ðÞDA1 DAr ðÞ1 U þ DAr ð5Þ

with DAn=absorbance change induced by the nth flash, DAr=remaining absorbance change after several (N50) flashes and U=quantum yield for the formation of long- lived states in that fraction of PS I complexes, which is capable of transferring an electron to FA and FB. The fit of the data obtained in the presence of 5 mM ascorbate and 2 AM PMS yields U=0.6 (see solid curve in Fig. 7A). In the remaining fraction of the PS I complexes a reversible charge separation followed by a charge recombination takes place at 77 K. The reversible absorbance changes observed at T=77 K exhibit a kinetic phase with t1/2i180 As(c60%) and slower phases with half-lives from about 1 ms up to 300 ms (c40%). Based on the absorbance difference spectra (not shown) the 180 As phase can be + attributed to the charge recombination of P700 A1 , whereas the millisecond phases reflect the charge recombination + between P700 and FX , respectively. Taken together, the irreversible fluorescence induction effect can be explained by the observation that at 77 K the + + charge-separated states P700 FA or P700 FB are irrever- sibly formed upon illumination in a fraction of the PS I complexes which corresponds to about 60% of the reaction centers if initially all cofactors are in the neutral ground state and to about 10% if the terminal iron–sulfur clusters are prereduced for the most part by the addition of dithionite at pH 10. The additional reversible decrease of F760 in the light can be attributed to the photoaccumulation of the + Fig. 7. (A) Relative initial amplitude of the flash-induced absorbance longer-lived charge separated state P700 FX . In both cases, change at 826 nm at 77 K of PS I trimers from A. platensis frozen in the the oxidized primary electron donor is the fluorescence dark either in the presence of 5 mM ascorbate+2 AM PMS (squares) or in quencher. Remarkably, the changes of the fluorescence yield the presence of 10 mM dithionite (circles). (B) Light-minus-dark at 760 nm do not depend linearly on the proportion of P700, absorbance difference spectrum of trimeric PS I complexes at 5 K frozen in the dark in the presence of 5 mM ascorbate+2 AM PMS. The absorbance which is oxidized. This can be explained by the connectivity spectrum at 5 K before illumination was subtracted from that measured after of the monomers within a trimer (see below): The excitation illumination with 30 flashes from a Xe flash lamp. energy trapped on a red pigment (C740) of a monomer with E. Schlodder et al. / Biochimica et Biophysica Acta 1706 (2005) 53–67 63 reduced P700 might be localized at a position (e.g. in the 760 nm of trimeric PS I complexes from A. platensis frozen trimerization domain) such that the direct Ffrster transfer to in the dark to 77 K in the presence of 10 mM dithionite. The the oxidized P700 of a neighboring monomer within the fluorescence is strongly quenched after the laser flash and trimer can also occur. Alternatively, the excitation energy recovers for the most part within 1 ms after the flash. Fig. might first migrate to the most-red state of a monomer with 8A shows the time course of the flash-induced absorbance oxidized P700. There it can be quenched efficiently by changes at 705 nm recorded under the same conditions. P700+. The absorbance changes at 705 nm can be attributed to the formation and decay of P700+. The solid line represents 3.4. Direct kinetic evidence for the quenching of 760 nm the fit. The fast decay phase with a half-life of about 180 As fluorescence by P700+ and 3P700 has a relative amplitude of about 65% and reflects the + charge recombination of P700 A1 . Slower decay phases In the next section we describe measurements of changes with an initial amplitude of about 35% are due to the charge + of the relative fluorescence yield at 760 nm upon excitation recombination between P700 and FX [12]. The recovery of by short laser flashes at low temperature. Fluorescence was the relative fluorescence yield exhibits very similar kinetics induced by the continuous light at 633 nm from a He–Ne (compare the time course of the upper trace in Fig. 8B with laser. The wavelength of the exciting laser pulse was 532 the trace in Fig. 8A) giving direct kinetic evidence that the nm with a pulse duration of about 3 ns. The energy of the fluorescence is quenched by the oxidized primary donor. laser flashes was non-saturating. Fig. 8B, upper trace, shows This conclusion is supported by the control experiment. If the time course of flash-induced fluorescence changes at P700 is oxidized before the flash, whereby the photochemistry is completely blocked, the flash-induced fluorescence changes at 760 nm are completely suppressed (see Fig. 8B, lower trace). It should be noted that the recovery of the flash-induced decrease of the fluorescence at 760 nm in trimeric PS I from A. platensis is somewhat faster than the decay of P700+ recorded by the absorbance changes at 705 nm. This difference is probably due to the energetic connectivity, i.e. the possibility of energy exchange between the antennae of different monomers within the PS I trimer (see next section). The same experimental approach has been used to study the quenching efficiency of the triplet state of P700. The triplet state of the primary electron donor, P700, is formed + by charge recombination of the radical pair P700 A0 , when the electron transfer to A1 is blocked (see Materials and methods). Fig. 9A shows the time course of the flash- induced absorbance changes measured under these conditions at 5 K. The decay kinetics and the difference spectrum of the absorbance changes (not shown, see Ref. [34]) give evidence that the flash-induced absorbance changes can be attributed to the formation and decay of 3P700. The solid line represents the best fit using two exponentials with half-lives of c0.75 (65%) and 5.2 ms (35%). The two phases reflect the different decay kinetics from the zero-field sublevels, which are uncoupled due to the slowing down of spin-lattice relaxation at 5 K. The flash-induced changes of the fluorescence at 760 nm in trimeric PS I from A. platensis measured under the same conditions are depicted in Fig. 9B. The flash-induced fluorescence decrease decays with almost the same kinetics Fig. 8. (A) Flash-induced absorbance change at 705 nm attributed to the as the flash-induced absorbance changes. The excellent formation and decay of P700+ at 77 K measured under conditions of kinetic match found between the decay of 3P700 and the prereduced terminal iron–sulfur clusters FA and FB (pH 10, 10 mM sodium flash-induced changes of the fluorescence at 760 nm dithionite). (B) Time course of flash-induced fluorescence changes at 760 demonstrates that the triplet state of P700 is also an efficient nm of trimeric PS I complexes from A. platensis measured under conditions quencher of 760 nm fluorescence. To our knowledge, this is of prereduced terminal iron–sulfur clusters FA and FB (pH 10, 10 mM sodium dithionite) (upper trace) or under conditions of preoxidized P700 the first direct experimental evidence for fluorescence (lower trace). quenching by 3P700. It should be noted that flash-induced 64 E. Schlodder et al. / Biochimica et Biophysica Acta 1706 (2005) 53–67

(3P700)*, respectively, constitutes most probably the quenching mechanism [18].

3.5. Energy transfer between the monomers within the trimeric PS I complex

In the following, we will address the question as to whether or not energy transfer takes place between the different monomers within the trimeric PS I complex. One experimental approach to study at this point are measurements of the variable fluorescence yield as a function of the fraction of reduced P700, [P700red]/[P700total] [48–50]. These experiments can be carried out with high sensitivity using trimeric PS I complexes from A. platensis due to the large variable fluorescence intensity at 760 nm at low temperature in this organism. A linear relationship is expected if the PS I trimer is organized in three separate photosynthetic units constituted by the three different monomers within the trimer. Previous reports are contra- dictory. Shubin et al. [51] measured redox titration curves of the variable and steady-state parts of F760 and light-induced absorbance changes at 702 nm for isolated Arthrospira membranes at 77 K. The P700 absorbance changes at room temperature and the F760 intensity at 77 K were found to depend in a similar way on increasing ferricyanide/ ferrocyanide ratios. Therefore, it was concluded that the F760 intensity is proportional to [P700red]/[P700total] ratio [51]. Later, a nonlinear relationship was derived from the comparison of the kinetics of the light-induced decrease of Fig. 9. (A) Flash-induced absorbance change at 705 nm attributed to the formation and decay of 3P700 at 5 K measured with PS I complexes fluorescence at 760 nm and light-induced oxidation of P700 containing the secondary electron acceptor A1 in the reduced state. At 5 K, [32]. To clarify this contradiction, we repeated the redox the decay kinetics of 3P700 are similar to that of the Chl a triplet state in titrations under more controlled conditions. c vitro (t1/2 0.75 ms (65%) and 5.2 ms (35%)). (B) Flash-induced changes Different ratios of oxidized to reduced P700 have been of the fluorescence at 760 nm in trimeric PS I from A. platensis measured adjusted by adding ferricyanide and ferrocyanide. The under the same conditions as described for panel A. Note that the flash- induced fluorescence decrease decays with almost the same kinetics as the correlated redox potential of the sample solution was flash-induced absorbance changes. measured at room temperature using a combination Pt/Ag/ AgCl electrode. The fraction of P700 in the reduced form changes of the fluorescence at 760 nm due to the formation was determined by measuring the magnitude of the flash- of chlorophyll triplet states in the antenna formed by induced absorbance change at 826 nm, associated with intersystem crossing have not been observed. Most prob- oxidation of P700. At room temperature, the dependence of ably, a fast triplet excitation transfer occurs from 3Chl a to the amplitude of the absorbance changes on the potential Car as has been observed for PS I complexes from T. could be satisfactorily fitted using the one-electron Nernst elongatus [47]. The kinetics of the triplet–triplet energy equation. The midpoint potential of P700+/P700 was transfer from Chl a to Car has been determined to occur determined to (430F8) mV in buffer solution which with half-lives of ~10 ns (~60%) and ~85 ns (~40%), contained 20 mM Tricine, pH 7.5, and 100 mM KCl [34]. which is too fast to be resolved with the set-up used. Redox titrations were also made with 65% glycerol in the Taken together, the results show that the long-wave- sample solution. A decrease of the midpoint potential of length fluorescence is quenched at low temperature sig- P700 by about 20 mV was observed in the presence of nificantly more strongly when the primary donor is either glycerol (not shown). To analyze the influence of temper- oxidized or in the triplet state than when it is reduced. Both ature on the level of reduction, we proceeded as follows. P700+ and 3P700 exhibit a broad, flat absorption that The potential of the sample solution in the cuvette was extends into the near infrared beyond 800 nm which adjusted by additions of ferricyanide and ferrocyanide, provides spectral overlap with the emission bands of all respectively. After measuring the potential, the cuvette was the C740 core-antenna pigments, so that P700+ and 3P700 placed in the cryostat and dark-adapted. The amplitude of can effectively act as an excitation energy acceptor. The the absorbance change at 826 nm induced by the first flash subsequent fast radiationless decay of (P700+)* and was recorded at room temperature. Subsequently, the sample E. Schlodder et al. / Biochimica et Biophysica Acta 1706 (2005) 53–67 65

was frozen in the dark to 77 K and again, the absorbance (F760–F760min)/(F760max–F760min). F760max is the maximal change induced by the first flash was measured. The relative fluorescence observed with 100% reduced P700, whereas amplitude of the absorbance change at 826 nm induced by F760min is the minimal fluorescence with 100% oxidized the first flash reflects directly the fraction of reduced P700. P700. The variable fluorescence titrates at apparently lower Fig. 10, squares, shows the relative amplitude of the potentials than P700. The variable fluorescence is half- absorbance change at 826 nm induced by the first flash at maximal at about 325 mV. Despite of a larger scattering of the 77 K as a function of the potential imposed on the sample data, the data points (circles in Fig. 10) roughly follow a solution at room temperature before freezing. Each data Nernst curve with Em=325 mV (see solid line). point was measured with a fresh sample. Taking together both titrations, the variable fluorescence Remarkably, the [P700red]/[P700total] ratio decreases on yield can be calculated as a function of the fraction of freezing to 77 K. For example: correlated with the measured reduced P700, [P700red]/[P700total](seeFig. 10,inset, redox potential of 370 mV at room temperature, the fraction dashed curve). In agreement with the previous report of of reduced P700 decreases from 0.8 at 295 K to 0.45 at 77 Karapetyan et al. [32], we obtain a nonlinear relationship. K. The temperature effect was completely reversible, i.e. This nonlinear relationship indicates clearly that energy upon warming the sample back to room temperature the transfer occurs between the different monomers within a fraction of reduced P700 was again 0.8. The apparent trimer. Thus, if P700 of one of the monomers within the PS I midpoint potential is shifted by about 45 mV to 365 mV trimer is oxidized, the excitation energy absorbed by when the sample temperature is lowered to 77 K. The solid antenna chlorophylls of a monomer with reduced P700 line represents a Nernst curve for one electron calculated for may migrate via long-wavelength absorbing C740 to a T=295 K with Em=365 mV. It should be noted that a monomer with oxidized P700 and will be quenched there. lowering of the midpoint potential of P700 for PS I cooled The nonlinear relationship between the F760 intensity and to 77 K is in agreement with a previous report by Se´tif and the fraction of reduced P700 also indicates that a low Mathis [52]. fraction of P700+ quenches more F760. As follows from the The fluorescence at 760 nm has also been measured as a inset of Fig. 10, about 30% of P700+ (i.e. one monomer function of the redox potential imposed on the sample within each trimer has P700+) causes the quenching of about solution at room temperature before freezing. The exper- 70% of F760, and 60% of P700+ quenches about 90% of imental procedure was as described above except that the F760 as a result of such energy exchange between chlorophyll concentration was lower. Fig. 10, dots, shows monomers within a trimer. This behavior can be explained the redox titration of the variable fluorescence F760var= by a model that takes into account only two different fluorescentstatesofthePSItrimerforthesakeof simplicity. The fluorescence yield at 760 nm is either low, when P700 is oxidized in one or more of the three monomers, or high, when P700 is reduced in each of three monomers within the trimer. The probability that the number of reduced P700 within PS I trimers equals exactly n with n=0,1,2,3, could be calculated as a function of the mean level of reduction (=[P700red]/[P700total]) by the binomial distribution. The dotted line in Fig. 10, inset, shows the probability of obtaining PS I trimers with P700 reduced in all three monomers (n=3). This calculated curve matches satisfactorily the experimentally obtained nonlinear relationship between the variable fluorescence and the mean level of reduction (measured curve). Deviations can be attributed to the oversimplified assumptions of our model described above. Fig. 10. Relative amplitude of the absorbance change at 826 nm induced by The observed exchange of excitation energy between the first flash (squares) and the variable F760 intensity (circles) of PS I different monomers within the PS I trimer supports a trimers from A. platensis at 77 K as a function of the redox potential localization of C740 pigments in the connecting domain of imposed on the sample solution at room temperature before freezing. F760 intensity was measured using short (5 ms) light pulses at 633 nm of low the trimeric unit. However, the distance between the intensity that causes no accumulation of P700+. A new sample for each trimerization axis and PB, on which the positive charge is point in the course of redox titration of F760 and the absorbance changes at mainly localized [53,54], is about 55 2. This is significantly 826 nm at 77 K was used. Inset: the dashed line shows the nonlinear larger than the value of 32 2 estimated for the distance dependence of the variable fluorescence at 760 nm on the [P700red]/ between C740 and P700+ using Ffrster theory (see above). [P700total] ratio which was derived from the titration curves of DA and F760. The dotted curve shows the probability of obtaining PS I trimers with However, it has to be kept in mind that this distance actually P700 reduced in all three monomers as a function of the mean level of refers to the nearest of all C740. Therefore, we assume that reduction (=[P700red]/[P700total]). the excitation energy transfer between the C740 chloro- 66 E. Schlodder et al. / Biochimica et Biophysica Acta 1706 (2005) 53–67 phylls of the different monomers is fast even at low [7] S. Jansson, The light-harvesting chlorophyll a/b-binding proteins, temperature compared to the quenching by energy transfer Biochim. Biophys. Acta 1184 (1994) 1–19. + [8] E.J. Boekema, P.E. Jensen, E. Schlodder, J.F.L. van Breemen, H. van to P700 . Possibly the nine C740 chlorophylls per trimer are Roon, H.V. Scheller, J.P. Dekker, Green plant photosystem I binds arranged in a ring-like structure around the trimerization light-harvesting complex I on one side of the complex, Biochemistry axis. 40 (2001) 1029–1036. [9] A. Ben-Shem, F. Frolow, N. Nelson, Crystal structure of plant photosystem I, Nature 426 (2003) 630–635. [10] P. Jordan, P. Fromme, H.T. Witt, O. Klukas, W. Saenger, N. Krauss, 4. Conclusions Three-dimensional structure of cyanobacterial photosystem I at 2.5 2 resolution, Nature 411 (2001) 909–917. The dependence of the fluorescence yield at 760 nm on [11] P. Setif, P. Mathis, T. V7nngard, Photosystem I photochemistry at low the redox state of P700 and various PS I electron acceptor temperature—heterogeneity in pathways for electron transfer to the cofactors has been investigated. It was kinetically resolved secondary acceptors and for recombination processes, Biochim. Biophys. Acta 767 (1984) 404–414. that F760 is quenched not only by P700+ but as well by 3 [12] E. Schlodder, K. Falkenberg, M. Gergeleit, K. Brettel, Temperature P700 comparing the kinetics of flash-induced fluorescence dependence of forward and reverse electron transfer from A1 , the and absorbance changes. Thus, fluorescence of the long- reduced secondary electron acceptor in photosystem I, Biochemistry wavelength chlorophylls is quenched effectively under all 37 (1998) 9466–9476. f conditions investigated so far: by P700 via photochemistry, [13] N.V. Karapetyan, A.R. Holzwarth, M. R gner, The photosystem I + 3 trimer of cyanobacteria: molecular organization, excitation dynamics by P700 under oxidizing conditions, and by P700 under and physiological significance, FEBS Lett. 460 (1999) 395–400. highly reducing conditions. Since excitation energy is [14] B. Gobets, R. van Grondelle, Energy transfer and trapping in channeled towards the long wavelength antenna states, photosystem I, Biochim. Biophys. Acta 1507 (2001) 80–99. excess energy can always be dissipated efficiently in PS I. [15] A.N. Melkozernov, Excitation energy transfer in photosystem I from The dependence of the fluorescence yield on the fraction oxygenic organisms, Photosynth. Res. 70 (2001) 129–153. [16] P. Fromme, E. Schlodder, S. Jansson, Structure and function of the of reduced P700 indicates that excitation energy absorbed antenna system in photosystem I, in: B. Green, W.W. Parson (Eds.), by antenna chlorophylls of a monomer with reduced P700 Light-harvesting Antennas in Photosynthesis, Kluwer Academic may migrate via long-wavelength absorbing C740 to a Publishers, Dordrecht, 2003, pp. 253–279. monomer with oxidized P700 and will be quenched there. [17] L.-O. P3lsson, C. Flemming, B. Gobets, R. van Grondelle, J.P. Dekker, E. Schlodder, Energy transfer and charge separation in photosystem I: P700 oxidation upon selective excitation of the long- wavelength antenna chlorophylls of Synechococcus elongatus, Bio- Acknowledgments phys. J. 74 (1998) 2611–2622. [18] M. Byrdin, I. Rimke, E. Schlodder, D. Stehlik, T.A. Roelofs, Decay We thank Heike Witt for critical reading of the manu- kinetics and quantum yields of fluorescence in photosystem I from script. This work was supported by the Deutsche For- Synechococcus elongatus with P700 reduced and oxidized state: are the kinetics of excited state decay trap-limited or transfer-limited? schungsgemeinschaft (Sonderforschungsbereich 498, Biophys. J. 79 (2000) 992–1007. TPA6) and the Russian Foundation of Basic Research [19] B. Gobets, I.H.M. van Stokkum, M. Rogner, J. Kruip, E. Schlodder, (grant 02-04-48348a). N.V. Karapetyan, J.P. Dekker, R. van Grondelle, Time-resolved fluorescence emission measurements of photosystem I particles of various cyanobacteria: a unified compartmental model, Biophys. J. 81 (2001) 407–424. References [20] M. R7tsep, T.W. Johnson, P.R. Chitnis, G.J. Small, The red-absorbing chlorophyll a antenna states of photosystem I: a hole-burning study of [1] K. Brettel, Electron transfer and arrangement of the redox cofactors in Synechocystis sp. PCC 6803 and its mutants, J. Phys. Chem. 104B photosystem I, Biochim. Biophys. Acta 1318 (1997) 322–373. (2000) 836–847. [2] BBA Special Issue: Type I photosynthetic reaction centres (Heathcote, [21] J.M. Hayes, S. Matsuzaki, M. R7tsep, G.J. Small, Red chlorophyll a P., Guest ed), Biochim. Biophys. Acta (2001) 1507. antenna states of photosystem I of the cyanobacterium Synechocystis [3] E.J. Boekema, J.P. Dekker, M.G. van Heel, M. Roegner, W. Saenger, sp. PCC 6803, J. Phys. Chem. 104B (2000) 5625–5633. I. Witt, H.T. Witt, Evidence for a trimeric organization of the [22] V. Zazubovich, S. Matsuzaki, T.W. Johnson, J.M. Hayes, P.R. Chitnis, photosystem I complex from the thermophilic cyanobacterium G.J. Small, Red antenna states of photosystem I from cyanobacterium Synechococcus sp., FEBS Lett. 217 (1987) 283–286. Synechococcus elongatus: a spectral hole-burning study, Chem. Phys. [4] V.V. Shubin, I.N. Bezsmertnaya, N.V. Karapetyan, Isolation from 275 (2002) 47–59. Spirulina membranes of two photosystem I-type complexes, one of [23] N.V. Karapetyan, D. Dorra, G. Schweitzer, I.N. Bezsmertnaya, A.R. which contains chlorophyll responsible for the 77 K fluorescence band Holzwarth, Fluorescence spectroscopy of the longwave chlorophylls at 760 nm, FEBS Lett. 309 (1992) 340–342. in trimeric and monomeric photosystem I core complexes from the [5] V.V. Shubin, V.L. Tsuprun, I.N. Bezsmertnaya, N.V. Karapetyan, cyanobacterium Spirulina platensis, Biochemistry 36 (1997) Trimeric forms of the photosystem I reaction center complex pre-exist 13830–13837. in the membranes of the cyanobacteriun Spirulina platensis, FEBS [24] J. Kruip, N.V. Karapetyan, I.V. Terekhova, M. Roegner, In vitro Lett. 334 (1993) 79–82. oligomerization of a membrane protein complex. Liposome-based [6] J. Kruip, D. Bald, E.J. Boekema, M. Rfgner, Evidence for the reconstitution of trimeric photosystem I from isolated monomers, existence of trimeric and monomeric photosystem I complexes in J. Biol. Chem. 274 (1999) 18181–18188. thylakoid membranes from cyanobacteria, Photosynth. Res. 40 (1994) [25] V.V. Shubin, I.N. Bezsmertnaya, N.V. Karapetyan, Efficient energy 279–286. transfer from the long-wavelength antenna chlorophylls to P700 in E. Schlodder et al. / Biochimica et Biophysica Acta 1706 (2005) 53–67 67

photosystem I complexes from Spirulina platensis, J. Photochem. fluorescence and absorption of Rhodopseudomonas viridis reaction Photobiol. 30B (1995) 153–160. centers, in: M.E. Michel-Beyerle (Ed.), Antennas and Reaction [26] D. Dorra, P. Fromme, N.V. Karapetyan, A.R. Holzwarth, Fluorescence Centers of Photosynthetic Bacteria: Structures, Interactions and kinetics of photosystem I: multiple fluorescence components, in: G. Dynamics, Springer Verlag, Berlin, 1984, pp. 131–137. Garab (Ed.), Photosynthesis: Mechanisms and Effects, vol. 1, Kluwer [40] A. Cometta, G. Zucchelli, N.V. Karapetyan, E. Engelman, F.M. Academic Publishers, Dordrecht, 1998, pp. 587–590. Garlashi, R.C. Jennings, Thermal behavior of long wavelength [27] N.V. Karapetyan, A.R. Holzwarth, R.J. Strasser, Grouping of pig- absorption transitions in Spirulina platensis photosystem I trimers, ment–protein complexes in cyanobacteria and plants stimulates de- Biophys. J. 79 (2000) 3235–3243. excitation of excess energy, Proc. 12th Photosynthesis Congress, [41] R.N. Frese, M.A. Palacios, A. Azzizi, I.H.M. van Stokkum, J. Kruip, Brisbane, 2001, pp. S9–005. M. Roegner, N.V. Karapetyan, E. Schlodder, R. van Grondelle, J.P. [28] B. Gobets, H. van Amerongen, R. Monshower, J. Kruip, M. Roegner, Dekker, Electric field effects on red chlorophylls, h-carotenes and R. van Grondelle, J.P. Dekker, Polarized site-selected fluorescence P700 in cyanobacterial photosystem I complexes, Biochim. Biophys. spectroscopy of isolated photosystem I particles, Biochim. Biophys. Acta 1554 (2002) 180–191. Acta 1188 (1994) 75–85. [42] P. Tapie, Y. Choquet, J. Breton, P. Delepelaire, F.-A. Wollmann, [29] E. Engelman, T. Tagliaube, N.V. Karapetyan, F.M. Garlaschi, Z. Orientation of Photosystem-I pigments—investigation by low temper- Zucchelli, R.C. Jennings, CD spectroscopy provides evidence for ature linear dichroism and polarized fluorescence emission, Biochim. excitonic interactions involving red-shifted chlorophyll forms in Biophys. Acta 767 (1984) 57–69. photosystem I, FEBS Lett. 499 (2001) 112–115. [43] J. van der Lee, D. Bald, S.L.S. Kwa, R. van Grondelle, J.P. Dekker, [30] M. Byrdin, P. Jordan, N. Kraug, P. Fromme, D. Stehlik, E. Schlodder, Steady-state polarized light spectroscopy of isolated Photosystem I Light harvesting in photosystem I: modelling based on the 2.5 2 complexes, Photosynth. Res. 35 (1994) 311–321. structure of photosystem I from Synechococcus elongatus, Biophys. J. [44] D. Dorra, Funktionelle Organisation des Photosystems I: Picosekun- 83 (2002) 433–457. den-aufgelfste Fluoreszenzuntersuchungen, Thesis, Heinrich-Heine- [31] H.-W. Trissl, Long-wavelength absorbing antenna pigments and Universit7tDqsseldorf (1998). heterogeneous absorption bands concentrate excitons and increase [45] H.-W. Trissl, Determination of the quenching efficiency of the absorption cross-section, Photosynth. Res 35 (1993) 247–263. oxidized primary donor in Photosystem I, P700+: implications for [32] N.V. Karapetyan, V.V. Shubin, R.J. Strasser, Energy exchange the trapping mechanism, Photosynth. Res. 54 (1997) 237–240. between the chlorophyll antennae of monomeric subunits within the [46] T. Ffrster, Delocalized excitation and excitation transfer, in: O. photosystem I trimeric complex of the cyanobacterium Spirulina, Sinanog˘lu (Ed.), Modern Quantum Chemistry, Academic Press, New Photosynth. Res. 61 (1999) 291–301. York, 1965, pp. 93–137. IIIB. [33] R.J. Porra, W.A. Thompson, P.E. Kriedermann, Determination of [47] E. Schlodder, A. Paul, M. C¸ etin, Triplet states in photosystem I accurate extinction coefficients and simultaneous equations for complexes from Synechococcus elongatus, In PS2001 Proc. 12th Int. assaying chlorophyll a and chlorophyll b extracted with 4 different Congress on Photosynthesis, CSIRO Publishing, Melbourne, Aus- solvents—verification of the concentration of chlorophyll standards tralia. S6-015. by atomic absorption spectroscopy, Biochim. Biophys. Acta 975 [48] G. Paillotin, Movement of excitations in photosynthetic domains of (1989) 384–394. Photosystem-2, J. Theor. Biol. 58 (1976) 237–252. [34] H. Witt, E. Bordignon, D. Carbonera, J.P. Dekker, N.V. Karapetyan, [49] R. Strasser, The grouping model of plant photosynthesis, in: G. C. Teutloff, A. Webber, W. Lubitz, E. Schlodder, Species specific Akoyunoglou (Ed.), Chloroplast Development, Elsevier, Amsterdam, differences of the spectroscopic properties of P700 investigated by 1978, pp. 513–524. site-directed mutagenesis of photosystem I from Chlamydomonas [50] G. Deinum, H. Kramer, T.J. Aartsma, F.A.M. Kleinherenbrink, J. reinhardtii, J. Biol. Chem. 278 (2003) 46760–46771. Amesz, Fluorescence quenching in Heliobacterium chlorum by [35] H. van Amerongen, H. Vasmel, R. Van Grondelle, Linear dichroism of reaction centers in the charge separated state, Biochim. Biophys. chlorosomes from Chloroflexus aurantiacus in compressed gels and Acta 1058 (1991) 339–344. electric fields, Biophys. J. 54 (1988) 65–76. [51] V.V. Shubin, S.D.S. Murthy, N.V. Karapetyan, P. Mohanty, Origin of [36] E. Schlodder, B. Meyer, pH-dependence of oxygen evolution and the 77 K variable fluorescence at 758 nm in the cyanobacterium

reduction kinetics of photooxidized chlorophyll aII (P680) in photo- Spirulina platensis, Biochim. Biophys. Acta 1060 (1991) 28–36. system II particles from Synecococcus sp., Biochim. Biophys. Acta [52] P. Se´tif, P. Mathis, The oxidation-reduction potential pf P-700 in 890 (1987) 23–31. chloroplast lamellae and sub-chloroplast particles, Arch. Biochem. [37] M.Z. Zgierski, Fluorescence-absorption energy-gap in stable anthra- Biophys. 204 (1980) 447–485. cene dimer spectra, J. Chem. Phys. 59 (1973) 1052–1053. [53] H. K7g, P. Fromme, H.T. Witt, W. Lubitz, Orientation and electronic [38] A. Di Pace, A. Cupane, M. Leone, E. Vitrano, L. Cordone, Protein structure of the primary electron donor radical cation in photosystem I: dynamics—vibrational coupling, spectral broadening mechanisms, a single crystal EPR and ENDOR study, J. Phys Chem., B 105 (2001) and anharmonicity effects in carbonmonoxy heme-proteins studied by 1225–1239. the temperature-dependence of the Soret band lineshape, Biophys. J. [54] L. Krabben, E. Schlodder, R. Jordan, D. Carbonera, G. Giacometti, L. 63 (1992) 475–484. Lee, A.N. Webber, W. Lubitz, Influence of the axial ligands on the [39] P.O.J. Scherer, S.F. Fischer, J.K.H. Hfrber, M.E. Michel-Beyerle, H. spectral properties of P700 of photosystem I: a study of site-directed Michel, On the temperature-dependence of the long wavelength mutants, Biochemistry 39 (2000) 13012–13025.