The effect of outer antenna complexes on the photochemical trapping rate in barley thylakoid Photosystem II

We have investigated the previous suggestions in the literature that the outer antenna of Photosystem II of barley does not influence the effective photosystem primary photochemical trapping rate. It is shown by steady state fluorescence measurements at the F₀ fluorescence level of wild type and the chlorina f2 mutant, using the chlorophyll b fluorescence as a marker, that the outer antenna is thermally equilibrated with the core pigments, at room temperature, under conditions of photochemical trapping. This is in contrast with the conclusions of the earlier studies in which it was suggested that energy was transferred rapidly and irreversibly from the outer antenna to the Photosystem II core. Furthermore, the effective trapping time, determined by single photon counting, time-resolved measurements, was shown to increase from 0.17±0.017 ns in the chlorina Photosystem II core to a value within the range 0.42±0.036-0.47±0.044 ns for the wild-type Photosystem II with the outer antenna system. This 2.5-2.8-fold increase in the effective trapping time is, however, significantly less than that expected for a thermalised system. The data can be explained in terms of the outer antenna increasing the primary charge separation rate by about 50%.     

Determination of the excitation migration time in Photosystem II consequences for the membrane organization and charge separation parameters

The fluorescence decay kinetics of Photosystem II (PSII) membranes from spinach with open reaction centers (RCs), were compared after exciting at 420 and 484 nm. These wavelengths lead to preferential excitation of chlorophyll (Chl) a and Chl b, respectively, which causes different initial excited-state populations in the inner and outer antenna system. The non-exponential fluorescence decay appears to be 4.3+/-1.8 ps slower upon 484 nm excitation for preparations that contain on average 2.45 LHCII (light-harvesting complex II) trimers per reaction center. Using a recently introduced coarse-grained model it can be concluded that the average migration time of an electronic excitation towards the RC contributes approximately 23% to the overall average trapping time. The migration time appears to be approximately two times faster than expected based on previous ultrafast transient absorption and fluorescence measurements. It is concluded that excitation energy transfer in PSII follows specific energy transfer pathways that require an optimized organization of the antenna complexes with respect to each other. Within the context of the coarse-grained model it can be calculated that the rate of primary charge separation of the RC is (5.5+/-0.4 ps)(-1), the rate of secondary charge separation is (137+/-5 ps)(-1) and the drop in free energy upon primary charge separation is 826+/-30 cm(-1). These parameters are in rather good agreement with recently published results on isolated core complexes [Y. Miloslavina, M. Szczepaniak, M.G. Muller, J. Sander, M. Nowaczyk, M. R?gner, A.R. Holzwarth, Charge separation kinetics in intact Photosystem II core particles is trap-limited. A picosecond fluorescence study, Biochemistry 45 (2006) 2436-2442].     

Slow exciton trapping in Photosystem II: A possible physiological role

Photosystem II, which has a primary photochemical charge separation time of about 300 ps, is the slowest trapping of all photosystems. On the basis of an analysis of data from the literature this is shown to be due to a number of partly independent factors: a shallow energy funnel in the antenna, an energetically shallow trap, exciton dynamics which are partly trap limited and a large antenna. It is argued that the first three of these properties of Photosystem II can be understood in terms of protective mechanisms against photoinhibition. These protective mechanisms, based on the generation of non photochemical quenching states mostly in the peripheral antenna, are able to decrease pheophytin reduction under conditions in which the primary quinone, Q_A, is already reduced, due to the slow trapping properties. The shallow antenna funnel is important in allowing quenching state-protective mechanisms in the peripheral antenna.Abbreviations chl chlorophyll - PS I Photosystem I - PS II Photosystem II - Q_A the primary quinone acceptor - RC reaction centre - RT room temperature     

Picosecond time-resolved fluorescence study of chlorophyll organisation and excitation energy distribution in chloroplasts from wild-type barley and a mutant lacking chlorophyll b.

Picosecond time-resolved fluorescence spectroscopy has been used to investigate the fluorescence emission from wild-type barley chloroplasts and from chloroplasts of the barley mutant, chlorina f-2, which lacks the light-harvesting chlorophyll a/b-protein complex. Cation-controlled regulation of the distribution of excitation energy was studied in isolated chloroplasts at the Fo and Fm levels. It was found that: (a) The fluorescence decay curves were distinctly non-exponential, even at low excitation intensities (less than 2 x 10(14) photons . cm(-2). (b) The fluorescence decay curves could, however, be described by a dual exponential decay law. The wild-type barley chloroplasts gave a short-lived fluorescence component of approximately 140 ps and a long-lived component of 600 ps (Fo) or 1300 ps (Fm) in the presence of Mg2+; in comparison, the mutant barley yielded a short-lived fluorescence component of approx. 50 ps and a long-lived component of 194 ps (Fo) and 424 ps (Fm). (c) The absence of the light-harvesting chlorophyll a/b-protein complex in the mutant results in a low fluorescence quantum yield which is unaffected by the cation composition of the medium. (d) The fluorescence yield changes seen in steady-state experiments on closing Photosystem II reaction centres (Fm/Fo) or on the addition of MgCl2 (+Mg2+/-Mg2+) were in overall agreement with those calculated from the time-resolved fluorescence measurements. The results suggest that the short-lived fluorescence component is partly attributable to the chlorophyll a antenna of Photosystem I, and, in part, to those light-harvesting-Photosystem II pigment combinations which are strongly coupled to the Photosystem I antenna chlorophyll. The long-lived fluorescence component can be ascribed to the light-harvesting-Photosystem II pigment combinations not coupled with the antenna of Photosystem I. In the case of the mutant, the two components appear to be the separate emissions from the Photosystem I and Photosystem II antenna chlorophylls.     

Effect of photosystem II reaction center closure on nanosecond fluorescence relaxation kinetics

The fluorescence decay of chlorophyll in spinach thylakoids was measured as a function of the degree of closure of Photosystem II reaction centers, which was set for the flowed sample by varying either the preillumination by actinic light or the exposure of the sample to the exciting pulsed laser light. Three exponential kinetic components originating in Photosystem II were fitted to the decays; a fourth component arising from Photosystem I was determined to be negligible at the emission wavelength of 685 nm at which the fluorescence decays were measured. Both the lifetimes and the amplitudes of the components vary with reaction center closure. A fast (170–330 ps) component reflects the trapping kinetics of open Photosystem II reaction centers capable of reducing the plastoquinone pool; its amplitude decreases gradually with trap closure, which is incompatible with the concept of photosynthetic unit connectivity where excitation energy which encounters a closed trap can find a different, possibly open one. For a connected system, the amplitude of the fast fluorescence component is expected to remain constant. The slow component (1.7–3.0 ns) is virtually absent when the reaction centers are open, and its growth is attributable to the appearance of closed centers. The middle component (0.4–1.7 ns) with approximately constant amplitude may originate from centers that are not functionally linked to the plastoquinone pool. To explain the continuous increase in the lifetimes of all three components upon reaction center closure, we propose that the transmembrane electric field generated by photosynthetic turnover modulates the trapping kinetics in Photosystem II and thereby affects the excited state lifetime in the antenna in the trap-limited case.Abbreviations DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea - HEPES 4-(2-hydroxyethyl)-1-piperazineethane sulfonic acid - PQ plastoquinone - PSI and PSII Photosystem I and II - Q_A and Q_B primary and secondary quinone acceptor of PSII     

The role of the individual Lhcas in photosystem I excitation energy trapping

In this work, we have investigated the role of the individual antenna complexes and of the low-energy forms in excitation energy transfer and trapping in Photosystem I of higher plants. To this aim, a series of Photosystem I (sub)complexes with different antenna size/composition/absorption have been studied by picosecond fluorescence spectroscopy. The data show that Lhca3 and Lhca4, which harbor the most red forms, have similar emission spectra (λ_max = 715–720 nm) and transfer excitation energy to the core with a relative slow rate of ∼25/ns. Differently, the energy transfer from Lhca1 and Lhca2, the “blue” antenna complexes, occurs about four times faster. In contrast to what is often assumed, it is shown that energy transfer from the Lhca1/4 and the Lhca2/3 dimer to the core occurs on a faster timescale than energy equilibration within these dimers. Furthermore, it is shown that all four monomers contribute almost equally to the transfer to the core and that the red forms slow down the overall trapping rate by about two times. Combining all the data allows the construction of a comprehensive picture of the excitation-energy transfer routes and rates in Photosystem I.     

Light-harvesting chlorophyll a-b complex requirement for regulation of Photosystem II photochemistry by non-photochemical quenching

Recently, it has been suggested (Horton et al. 1992) that aggregation of the light-harvesting a-b complex (LHC II) in vitro reflects the processes which occur in vivo during fluorescence induction and related to the major non-photochemical quenching (qE). Therefore the requirement of this chlorophyll a-b containing protein complex to produce qN was investigated by comparison of two barley mutants either lacking (chlorina f2) or depressed (chlorina¹⁰⁴) in LHC II to the wild-type and pea leaves submitted to intermittent light (IL) and during their greening in continuous light. It was observed that qN was photoinduced in the absence of LHC II, i.e. in IL grown pea leaves and the barley mutants. Nevertheless, in these leaves qN had no (IL, peas) or little (barley mutants) inhibitory effect on the photochemical efficiency of Q_A reduction measured by flash dosage response curves of the chlorophyll fluorescence yield increase induced by a single turn-over flash During greening in continuous light of IL pea leaves, an inhibitory effect on Q_A photoreduction associated to qN developed as Photosystem II antenna size increased with LHC II synthesis. Utilizing data from the literature on connectivity between PS II units versus antenna size, the following hypothesis is put forward to explain the results summarized above. qN can occur in the core antenna or Reaction Center of a fraction of PS II units and these units will not exhibit variable fluorescence. Other PS II units are quenched indirectly through PS II-PS II exciton transfer which develops as the proportion of connected PS II units increases through LHC II synthesis.     

The effect of salt concentration on the fluorescence parameters of isolated chloroplasts

The previously reported effect of salt concentration on the fluorescence and other photochemical activities of Photosystem II is interpreted in terms of a change in the radiationless transition and the trapping probabilities. This is confirmed by quantitative comparison of the fluorescence and the photochemical activity. As a by-product of this analysis a method is devised to estimate the background fluorescence.

We did not eliminate the possibility that the radiationless transition constant may include a contribution of energy transfer from Photosystem II to Photosystem I.     

The photochemical trapping rate from red spectral states in PSI-LHCI is determined by thermal activation of energy transfer to bulk chlorophylls

The average fluorescence decay lifetimes, due to reaction centre photochemical trapping, were calculated for wavelengths in the 690- to 770-nm interval from the published fluorescence decay-associated emission spectra for Photosystem I (PSI)-light-harvesting complex of Photosystem I (LHCI) [Biochemistry 39 (2000) 6341] at 280 and 170 K. For 280 K, the overall trapping time at 690 nm is 81 ps and increases with wavelength to reach 103 ps at 770 nm. For 170 K, the 690-nm value is 115 ps, increasing to 458 ps at 770 nm. This underlines the presence of kinetically limiting processes in the PSI antenna (diffusion limited). The explanation of these nonconstant values for the overall trapping time band is sought in terms of thermally activated transfer from the red absorbing states to the "bulk" acceptor chlorophyll (chl) states in the framework of the Arrhenius-Eyring theory. It is shown that the wavelength-dependent "activation energies" come out in the range between 1.35 and 2.7 kcal mol(-1), increasing with the emission wavelength within the interval 710-770 nm. These values are in good agreement with the Arrhenius activation energy determined for the steady-state fluorescence yield over the range 130-280 K for PSI-LHCI. We conclude that the variable trapping time in PSI-LHCI can be accounted for entirely by thermally activated transfer from the low-energy chl states to the bulk acceptor states and therefore that the position of the various red states in the PSI antenna seems not to be of significant importance. The analysis shows that the bulk antenna acceptor states are on the low-energy side of the bulk antenna absorption band.     

Long-wavelength absorbing antenna pigments and heterogeneous absorption bands concentrate excitons and increase absorption cross section

The light-harvesting apparatus of photosynthetic organisms is highly optimized with respect to efficient collection of excitation energy from photons of different wavelengths and with respect to a high quantum yield of the primary photochemistry. In many cases the primary donor is not an energetic trap as it absorbs hypsochromically compared to the most red-shifted antenna pigment present (long-wavelength antenna). The possible reasons for this as well as for the spectral heterogeneity which is generally found in antenna systems is examined on a theoretical basis using the approach of thermal equilibration of the excitation energy. The calculations show that long-wavelength antenna pigments and heterogeneous absorption bands lead to a concentration of excitons and an increased effective absorption cross section. The theoretically predicted trapping times agree remarkably well with experimental data from several organisms. It is shown that the kinetics of the energy transfer from a long-wavelength antenna pigment to a hypsochromically absorbing primary donor does not represent a major kinetic limitation. The development of long-wavelength antenna and spectrally heterogeneous absorption bands means an evolutionary advantage based on the chromatic adaptation of photosynthetic organelles to spectrally filtered light caused by self-absorption.Abbreviations LHC light-harvesting complex - P primary donor - PSI Photosystem I of green plants - PS II Photosystem II of green plants - RC reaction center - X primary acceptor     

Determination of the excitation migration time in Photosystem II: Consequences for the membrane organization and charge separation parameters

The fluorescence decay kinetics of Photosystem II (PSII) membranes from spinach with open reaction centers (RCs), were compared after exciting at 420 and 484 nm. These wavelengths lead to preferential excitation of chlorophyll (Chl) a and Chl b, respectively, which causes different initial excited-state populations in the inner and outer antenna system. The non-exponential fluorescence decay appears to be 4.3 ± 1.8 ps slower upon 484 nm excitation for preparations that contain on average 2.45 LHCII (light-harvesting complex II) trimers per reaction center. Using a recently introduced coarse-grained model it can be concluded that the average migration time of an electronic excitation towards the RC contributes ~ 23% to the overall average trapping time. The migration time appears to be approximately two times faster than expected based on previous ultrafast transient absorption and fluorescence measurements. It is concluded that excitation energy transfer in PSII follows specific energy transfer pathways that require an optimized organization of the antenna complexes with respect to each other. Within the context of the coarse-grained model it can be calculated that the rate of primary charge separation of the RC is (5.5 ± 0.4 ps)^− 1, the rate of secondary charge separation is (137 ± 5 ps)^− 1 and the drop in free energy upon primary charge separation is 826 ± 30 cm^− 1. These parameters are in rather good agreement with recently published results on isolated core complexes [Y. Miloslavina, M. Szczepaniak, M.G. Muller, J. Sander, M. Nowaczyk, M. Rögner, A.R. Holzwarth, Charge separation kinetics in intact Photosystem II core particles is trap-limited. A picosecond fluorescence study, Biochemistry 45 (2006) 2436-2442].     

Description of energy migration and trapping in photosystem I by a model with two distance scaling parameters

The energy transfer and trapping kinetics in the core antenna of Photosystem I are described in a new model in which the distance between the core antenna chlorophylls and P700 is proposed to be considerably longer than the distance between the chlorophylls within the antenna. Structurally, the model describes the Photosystem I core antenna as a regular sphere around P700, while energetically it consists of three levels representing the bulk antenna, P700 and the red-shifted antenna pigments absorbing at longer wavelength than P700, respectively. It is shown that the model explains experimental results obtained from the Photosystem I complex of the cyanobacterium Synechococcus sp. (A.R. Holzwarth, G. Schatz, H Brock, and E. Bittersman (1993) Biophys. J. 64: 1813–1826) quite well, and that no unrealistic charge separation rate and organization of the long-wavelength pigments has to be assumed. We suggest that excitation energy transfer and trapping in Photosystem I should be described as a ‘transfer-to-the-trap’-limited process     

Excitation energy transfer in thylakoid membranes from Chlamydomonas reinhardtii lacking chlorophyll b and with mutant Photosystem I

Energy trapping in Photosystem I (PS I) was studied by time-resolved fluorescence spectroscopy of PS II-deleted Chl b-minus thylakoid membranes isolated from site-directed mutants of Chlamydomonas reinhardtii with specific amino acid substitutions of a histidine ligand to P₇₀₀. In vivo the fluorescence of the PS I core antenna in mutant thylakoids with His-656 of PsaB replaced by asparagine, serine or phenylalanine is characterized by an increase in the lifetime of the fast decay component ascribed to the energy trapping in PS I (25 ps in wild type PS I with intact histidine-656, 50 ps in the mutant PS I with asparagine-656 and 70 ps in the mutant PS I with phenylalanine-656). Assuming that the excitation dynamics in the PS I antenna are trap-limited, the increase in the trapping time suggests a decrease in the primary charge separation rate. Western blot analysis showed that the mutants accumulate significantly less PS I than wild type. Spectroscopically, the mutations lead to a decrease in relative quantum yield of the trapping in the PS I core and increase in relative quantum yield of the fluorescence decay phase ascribed to uncoupled chlorophyll–protein complexes which suggests that improper assembly of PS I and LHC in the mutant thylakoids may result in energy uncoupling in PS I.     

Excitation spectra for photosystem I and photosystem II in chloroplasts and the spectral characteristics of the distributions of quanta between the two photosystems.

The parameters listed in the title were determined within the context of a model for the photochemical apparatus of photosynthesis. The fluorescence of variable yield at 750 nm at -196 degrees C is due to energy transfer from Photosystem II to Photosystem I. Fluorescence excitation spectra were measured at -196 degrees C at the minimum, FO, level and the maximum, FM, level of the emission at 750 nm. The difference spectrum, FM-FO, which represents the excitation spectrum for FV is presented as a pure Photosystem II excitation spectrum. This spectrum shows a maximum at 677 nm, attributable to the antenna chlorophyll a of Photosystem II units, with a shoulder at 670 nm and a smaller maximum at 650 nm, presumably due to chlorophyll a and chlorophyll b of the light-harvesting chlorophyll complex. Fluoresence at the FO level at 750 nm can be considered in two parts; one part due to the fraction of absorbed quanta, alpha, which excites Photosystem I more-or-less directly and another part due to energy transfer from Photosystem II to Photosystem I. The latter contribution can be estimated from the ratio of FO/FV measured at 692 nm and the extent of FV at 750 nm. According to this procedure the excitation spectrum of Photosystem I at -196 degrees C was determined by subtracting 1/3 of the excitation spectrum of FV at 750 nm from the excitation spectrum of FO at 750 nm. The spectrum shows a relatively sharp maximum at 681 nm due to the antenna chlorophyll a of Photosystem I units with probably some energy transfer from the light-harvesting chlorophyll complex. The wavelength dependence of alpha was determined from fluorescence measurements at 692 and 750 nm at -196 degrees C. Alpha is constant to within a few percent from 400 to 680 nm, the maximum deviation being at 515 nm where alpha shows a broad maximum increasing from 0.30 to 0.34. At wavelengths between 680 and 700 nm, alpha increases to unity as Photosystem I becomes the dominant absorber in the photochemical apparatus.     

The photochemical trapping rate from red spectral states in PSI–LHCI is determined by thermal activation of energy transfer to bulk chlorophylls

The average fluorescence decay lifetimes, due to reaction centre photochemical trapping, were calculated for wavelengths in the 690- to 770-nm interval from the published fluorescence decay-associated emission spectra for Photosystem I (PSI)–light-harvesting complex of Photosystem I (LHCI) [Biochemistry 39 (2000) 6341] at 280 and 170 K. For 280 K, the overall trapping time at 690 nm is 81 ps and increases with wavelength to reach 103 ps at 770 nm. For 170 K, the 690-nm value is 115 ps, increasing to 458 ps at 770 nm. This underlines the presence of kinetically limiting processes in the PSI antenna (diffusion limited). The explanation of these nonconstant values for the overall trapping time band is sought in terms of thermally activated transfer from the red absorbing states to the “bulk” acceptor chlorophyll (chl) states in the framework of the Arrhenius–Eyring theory. It is shown that the wavelength-dependent “activation energies” come out in the range between 1.35 and 2.7 kcal mol⁻¹, increasing with the emission wavelength within the interval 710–770 nm. These values are in good agreement with the Arrhenius activation energy determined for the steady-state fluorescence yield over the range 130–280 K for PSI–LHCI. We conclude that the variable trapping time in PSI–LHCI can be accounted for entirely by thermally activated transfer from the low-energy chl states to the bulk acceptor states and therefore that the position of the various red states in the PSI antenna seems not to be of significant importance. The analysis shows that the bulk antenna acceptor states are on the low-energy side of the bulk antenna absorption band.     

Chlorophyll triplet states associated with Photosystem I and Photosystem II in thylakoids of the green alga Chlamydomonas reinhardtii

The analysis of FDMR spectra, recorded at multiple emission wavelengths, by a global decomposition technique, has allowed us to characterise the triplet populations associated with Photosystem I and Photosystem II of thylakoids in the green alga Chlamydomonas reinhardtii. Three triplet populations are observed at fluorescence emissions characteristic of Photosystem II, and their zero field splitting parameters have been determined. These are similar to the zero field parameters for the three Photosystem II triplets previously reported for spinach thylakoids, suggesting that they have a widespread occurrence in nature. None of these triplets have the zero field splitting parameters characteristic of the Photosystem II recombination triplet observed only under reducing conditions. Because these triplets are generated under non-reducing redox conditions, when the recombination triplet is undetectable, it is suggested that they may be involved in the photoinhibition of Photosystem II. At emission wavelengths characteristic of Photosystem I, three triplet populations are observed, two of which are attributed to the P(700) recombination triplet frozen in two different conformations, based on the microwave-induced fluorescence emission spectra and the triplet minus singlet difference spectra. The third triplet population detected at Photosystem I emission wavelengths, which was previously unresolved, is proposed to originate from the antenna chlorophyll of the core or the unusually blue-shifted outer antenna complexes of this organism.     

Development of the Primary Photochemical Apparatus of Photosynthesis during Greening of Etiolated Bean Leaves

Seven-day-old dark-grown bean leaves were greened under continuous light. The amount of chlorophyll, the ratio of chlorophyll a to chlorophyll b, the O₂ evolving capacity and the primary photochemical activities of Photosystem I and Photosystem II were measured on the leaves after various times of greening. The primary photochemical activities were measured as the photo-oxidation of P₇₀₀, the photoreduction of C-550, and the photo-oxidation of cytochrome b₅₅₉ in intact leaves frozen to −196 C. The results indicate that the reaction centers of Photosystem I and Photosystem II begin to appear within the first few minutes and that Photosystem II reaction centers accumulate more rapidly than Photosystem I reaction centers during the first few hours of greening. The very early appearances of the primary photochemical activity of Photosystem II was also confirmed by light-induced fluorescence yield measurements at −196 C.     

Kinetics of excitation trapping in intact Photosystem I of Chlamydomonas reinhardtii and Arabidopsis thaliana

We measured picosecond time-resolved fluorescence of intact Photosystem I complexes from Chlamydomonas reinhardtii and Arabidopsis thaliana. The antenna system of C. reinhardtii contains about 30-60 chlorophylls more than that of A. thaliana, but lacks the so-called red chlorophylls, chlorophylls that absorb at longer wavelength than the primary electron donor. In C. reinhardtii, the main lifetimes of excitation trapping are about 27 and 68 ps. The overall lifetime of C. reinhardtii is considerably shorter than in A. thaliana. We conclude that the amount and energies of the red chlorophylls have a larger effect on excitation trapping time in Photosystem I than the antenna size.     

Long-lived primary radical pair state detected by time-resolved fluorescence and absorption spectroscopy in an isolated Photosystem two core

A Photosystem two (PS II) core preparation containing the chlorophyll a binding proteins CP 47, CP 43, D1 and D2, and the non-chlorophyll binding cytochrome-b559 and 33 kDA polypeptides, has been isolated from PS II-enriched membranes of peas using the non-ionic detergent heptylthioglucopyranoside and elevated ionic strengths. The primary radical pair state, P680⁺Pheo^-, was studied by time-resolved absorption and fluorescence spectroscopy, under conditions where quinone reduction and water-splitting activities were inhibited. Charge recombination of the primary radical pair in PS II cores was found to have lifetimes of 17.5 ns measured by fluorescence and 21 ns measured by transient decay kinetics under anaerobic conditions. Transient absorption spectroscopy demonstrated that the activity of the particles, based on primary radical pair formation, was in excess of 70% (depending on the choice of kinetic model), while time-resolved fluorescence spectroscopy indicated that the particles were 91% active. These estimates of activity were further supported by steady-state measurements which quantified the amount of photoreducible pheophytin. It is concluded that the PS II core preparation we have isolated is ideal for studying primary radical pair formation and recombination as demonstrated by the correlation of our absorption and fluorescence transient data, which is the first of its kind to be reported in the literature for isolated PS II core complexes from higher plants.Abbreviations CP 43 and CP 47 chlorophyll binding proteins of PS II having apparent molecular weights on SDS-PAGE of 43 kDa and 47 kDa, respectively - D1 and D2 polypeptides PS II reaction centre polypeptides encoded by the psbA and psbD genes, respectively - HPLC high performance liquid chromatography - PS II Photosystem two - SDS-PAGE sodium dodecyl sulphate-polyacrylamide gel electrophoresis - P680 primary electron donor of PS II - Pheo phenophytin a - SPC single photon counting - PBQ phenyl-p-benzoquinone - DPC 1,5-diphenylcarbazide AFRC Photosynthesis Research Group, Department of Biochemistry     

Chlorophyll triplet states associated with Photosystem I and Photosystem II in thylakoids of the green alga Chlamydomonas reinhardtii

The analysis of FDMR spectra, recorded at multiple emission wavelengths, by a global decomposition technique, has allowed us to characterise the triplet populations associated with Photosystem I and Photosystem II of thylakoids in the green alga Chlamydomonas reinhardtii. Three triplet populations are observed at fluorescence emissions characteristic of Photosystem II, and their zero field splitting parameters have been determined. These are similar to the zero field parameters for the three Photosystem II triplets previously reported for spinach thylakoids, suggesting that they have a widespread occurrence in nature. None of these triplets have the zero field splitting parameters characteristic of the Photosystem II recombination triplet observed only under reducing conditions. Because these triplets are generated under non-reducing redox conditions, when the recombination triplet is undetectable, it is suggested that they may be involved in the photoinhibition of Photosystem II. At emission wavelengths characteristic of Photosystem I, three triplet populations are observed, two of which are attributed to the P₇₀₀ recombination triplet frozen in two different conformations, based on the microwave-induced fluorescence emission spectra and the triplet minus singlet difference spectra. The third triplet population detected at Photosystem I emission wavelengths, which was previously unresolved, is proposed to originate from the antenna chlorophyll of the core or the unusually blue-shifted outer antenna complexes of this organism.