Fluorescence lifetimes in the bipartite model of the photosynthetic apparatus with alpha, beta heterogeneity in photosystem II

Recent studies of the lifetime of fluorescence after picosecond pulse excitation of photosynthetic organisms revealed relatively complex decay kinetics that indicated a sum of three exponential components with lifetimes spanning the range from about 0.1-2.5 ns. These fluorescence lifetime data were examined in the context of a simple photochemical model for photosystem II that was used previously to account for fluorescence yield data obtained during continuous illumination. The model, which consists of a single fluorescing species of antenna chlorophyll and a reaction center, shows that, in general, the decay kinetics after pulse excitation should consist of the sum of two exponential decays. The model also shows that in going from open to closed reaction centers the lifetime of fluorescence may increase much more than the yield of fluorescence and surprisingly long fluorescence lifetimes can be obtained. However, conditions can be stated where fluorescence will decay essentially as a single component and with lifetime changes that are proportional to the yield changes. A heterogeneity was also introduced to distinguish photosystem II_α units, which can transfer excitation energy among themselves but not the photosystem I, and photosystem II_β units, which can transfer energy to photosystem I but not to other photosystem II units. It is proposed that the rather complex fluorescence lifetime data can be accounted for in large part by the simple photochemical model with the α, β heterogeneity in photosystem II.     

Antenna size dependence of fluorescence decay in the core antenna of photosystem I: estimates of charge separation and energy transfer rates.

We have examined the photophysics of energy migration and trapping in photosystem I by investigating the spectral and temporal properties of the fluorescence from the core antenna chlorophylls as a function of the antenna size. Time-correlated single photon counting was used to determine the fluorescence lifetimes in the isolated P700 chlorophyll a-protein complex and in a mutant of Chlamydomonas reinhardtii that lacks the photosystem II reaction center complex. The fluorescence decay in both types of sample is dominated by a fast (15-45 psec) component that is attributed to the lifetime of excitations in the photosystem I core antenna. These excitations decay primarily by an efficient photochemical quenching on P700. The measured lifetimes show a linear relationship to the core antenna size. A linear dependence of the excitation lifetime on antenna size was predicted previously in a lattice model for excitation migration and trapping in arrays of photosynthetic pigments [Pearlstein, R.M. (1982) Photochem. Photobiol. 35, 835-844]. Based on this model, our data predict a time constant for photochemical charge separation in the photosystem I reaction center of 2.8 +/- 0.7 or 3.4 +/- 0.7 psec, assuming monomeric or dimeric P700, respectively. The predicted average single-step transfer time for excitation transfer between core antenna pigments is 0.21 +/- 0.04 psec. Under these conditions, excitation migration in photosystem I is near the diffusion limit, with each excitation making an average of 2.4 visits to the reaction center before photoconversion.     

Chlorophyll b can serve as the major pigment in functional photosystem II complexes of cyanobacteria

An Arabidopsis thaliana chlorophyll(ide) a oxygenase gene (cao), which is responsible for chlorophyll b synthesis from chlorophyll a, was introduced and expressed in a photosystem I-less strain of the cyanobacterium Synechocystis sp. PCC 6803. In this strain, most chlorophyll is associated with the photosystem II complex. In line with observations by Satoh et al. [Satoh, S., Ikeuchi, M., Mimuro, M. & Tanaka, A. (2001) J. Biol. Chem. 276, 4293-4297], chlorophyll b was made but accounted for less than 10% of total chlorophyll. However, when lhcb encoding light-harvesting complex (LHC)II from pea was present in the same strain (lhcb(+)/cao(+)), chlorophyll b accumulated in the cell to levels exceeding those of chlorophyll a, although LHCII did not accumulate. In the lhcb(+)/cao(+) strain, the total amount of chlorophyll, the number of chlorophylls per photosystem II center, and the oxygen-evolving activity on a per-chlorophyll basis were similar to those in the photosystem I-less strain. Furthermore, the chlorophyll a/b ratio of photosystem II core particles (retaining CP47 and CP43) and of whole cells of the lhcb(+)/cao(+) strain was essentially identical, and PS II activity could be obtained efficiently by chlorophyll b excitation. These data indicate that chlorophyll b functionally substitutes for chlorophyll a in photosystem II. Therefore, the availability of chlorophylls, rather than their binding specificity, may determine which chlorophyll is incorporated at many positions of photosystem II. We propose that the transient presence of a LHCII/chlorophyll(ide) a oxygenase complex in the lhcb(+)/cao(+) strain leads to a high abundance of available chlorophyll b that is subsequently incorporated into photosystem II complexes. The apparent LHCII requirement for high chlorophyll(ide) a oxygenase activity may be instrumental to limit the occurrence of chlorophyll b in plants to LHC proteins.     

Isolation of a photosystem II reaction center consisting of D-1 and D-2 polypeptides and cytochrome b-559

A photosystem II reaction center complex consisting of D-1 and D-2 polypeptides and cytochrome b-559 was isolated from spinach grana thylakoids, treated with 4% (wt/vol) Triton X-100, by ion-exchange chromatography using DEAE-Toyopearl 650S. The isolated complex appears to contain five chlorophyll a, two pheophytin a, one β-carotene, and one or two cytochrome b-559 heme(s) (molar ratio) and exhibits a reversible absorbance change attributable to the photochemical accumulation of reduced pheophytin typical for the intermediary electron acceptor of photosystem II reaction center. These results strongly suggest that the site of primary charge separation in photosystem II is located on the heterodimer composed of D-1 and D-2 subunits.     

Lateral redistribution of cytochrome b6/f complexes along thylakoid membranes upon state transitions.

The cytochrome b6/f complex operates in photosynthetic electron transfer either in linear electron flow from photosystem II to photosystem I or in cyclic flow around photosystem I. Using membrane fractionation and immunocytochemistry, we show a change in lateral distribution of cytochrome b6/f complexes along the thylakoid membranes during state transitions. This change is seen in maize as well as in the green algae Chlamydomonas reinhardtii. When either of the two organisms were adapted to state II in vivo, the proportion of cytochrome b6/f complexes found in the photosystem I-enriched stroma lamellae regions was significantly larger than after adaptation to state I. A similar observation was made upon state I to state II transitions done in vitro by illuminating, in the presence of ATP, broken maize chloroplasts prepared from dark-adapted leaves. This reorganization of the electron-transfer chain is concurrent with the change in light-energy distribution between the two photosystems, which requires lateral displacement of light-harvesting complex II. That the changes in lateral distribution of both cytochrome b6/f and light-harvesting II complexes seen upon state transition in vitro similarly required addition of exogenous ATP, suggests that the change in cytochrome b6/f organization also depends on kinase activity. The increased concentration of cytochrome b6/f complexes in the vicinity of photosystem I in state II is discussed in terms of an increase in cyclic electron flow, thus favoring ATP production. Because transition to state II can be triggered in vivo by ATP depletion, we conclude that state transitions should be regarded not only as a light-adaptation mechanism but also as a rerouting of photosynthetic electron flow, enabling photosynthetic organisms to adapt to changes in the cell demand for ATP.     

Photosystem I reaction center from the thermophilic cyanobacterium Mastigocladus laminosus

Photosystem I reaction center was isolated from the cyanobacterium Mastigocladus laminosus. It contained four different subunits with molecular masses (as determined by sodium dodecyl sulfate gels) of about 70,000 (subunit I), 16,000 (subunit II), 11,000 (subunit III), and 10,000 (subunit IV) daltons. The purified reaction center contained about 100 chlorophyll a molecules per P₇₀₀; however, they could be readily depleted down to about 50 chlorophyll a per P₇₀₀ without loss in the photochemical activities. The reaction center was active in cytochrome c photooxidation, but the photooxidation of an acidic cytochrome, like the Euglena cytochrome 552, required the presence of cations. The purified reaction center was found to be similar in several respects to the photosystem I reaction centers from higher plants and, especially, to the one isolated from green algae. Subunit I appeared on sodium dodecyl sulfate gels in the same position and possessed the same shape of an apparent double band as the corresponding subunits I of green plants and of algae. Subunits I and II of photosystem I reaction centers from Mastigocladus, higher plants, and green algae showed immunological crossreactivity. This observation might serve as biochemical evidence for the common evolution of the photosystem I reaction centers. In higher plants and green algae subunit II is a product of cytoplasmic ribosomes and therefore, a high degree of homology should have been preserved upon transfer of its gene from the prokaryote to the nucleus of the eukaryotes.     

Excited-state dynamics in photosystem II: Insights from the x-ray crystal structure

The heart of oxygenic photosynthesis is photosystem II (PSII), a multisubunit protein complex that uses solar energy to drive the splitting of water and production of molecular oxygen. The effectiveness of the photochemical reaction center of PSII depends on the efficient transfer of excitation energy from the surrounding antenna chlorophylls. A kinetic model for PSII, based on the x-ray crystal structure coordinates of 37 antenna and reaction center pigment molecules, allows us to map the major energy transfer routes from the antenna chlorophylls to the reaction center chromophores. The model shows that energy transfer to the reaction center is slow compared with the rate of primary electron transport and depends on a few bridging chlorophyll molecules. This unexpected energetic isolation of the reaction center in PSII is similar to that found in the bacterial photosystem, conflicts with the established view of the photophysics of PSII, and may be a functional requirement for primary photochemistry in photosynthesis. In addition, the model predicts a value for the intrinsic photochemical rate constant that is 4 times that found in bacterial reaction centers.     

In vitro reconstitution of the photosystem I light-harvesting complex LHCI-730: heterodimerization is required for antenna pigment organization

Here we describe the in vitro reconstitution of photosystem I light-harvesting complexes with pigments and proteins (Lhca1 and Lhca4) obtained by overexpression of tomato Lhca genes in Escherichia coli. Using Lhca1 and Lhca4 individually for reconstitution results in monomeric pigment-proteins, whereas a combination thereof yields a dimeric complex. Interactions of the apoproteins is highly specific, as reconstitution of either of the two constituent proteins in combination with a light-harvesting protein of photosystem II does not result in dimerization. The reconstituted Lhca1/4, but not complexes obtained with either Lhca1 or Lhca4 alone, closely resembles the native LHCI-730 dimer from tomato leaves with regard to spectroscopic properties, pigment composition, and stoichiometry. Monomeric complexes of Lhca1 or Lhca4 possess lower pigment/protein ratios, indicating that interactions of the two subunits not only facilitates pigment reorganization but also recruitment of additional pigments. In addition to higher averages of chlorophyll a/b ratios in monomeric complexes than in LHCI-730, comparative fluorescence and CD spectra demonstrate that heterodimerization involves preferential ligation of more chlorophyll b.     

LIGHT-INDUCED OXIDATION OF A CHLOROPLAST B-TYPE CYTOCHROME AT -189 degrees C

The b-type cytochromes of chloroplasts have heretofore been viewed as photosynthetic electron carriers that probably occupy an intermediate position in a light-induced electron flow. The oxidation-reduction of such intermediate electron carriers, being removed from the primary photochemical reaction linked to photon capture by chlorophyll, would be expected to show a temperature dependence. Evidence has now been obtained that cytochrome b₅₅₉ is photooxidized at -189°C and that this photooxidation can be induced only by “short-wavelength” monochromatic light which activates the oxygen-evolving system in chloroplasts (photosystem II). In appears, therefore, that photooxidation of cytochrome b₅₅₉ is closely linked with photon capture by the chlorophyll pigments characteristic of photosystem II.     

Localization of different photosystems in separate regions of chloroplast membranes

The stoichiometric amounts and the photoactivity kinetics of photosystem I (PSI) and of the α and β components of photosystem II (PSII_α and PSII_β) were compared in spinach chloroplast membrane (thylakoid) fractions derived from appressed and nonappressed regions. Stroma-exposed thylakoid fractions from the nonappressed regions were isolated by differential centrifugation following a mechanical press treatment of the chloroplasts. Thylakoid vesicles derived mainly from the appressed membranes of grana were isolated by the aqueous polymer two-phase partition method. Stroma-exposed thylakoids were found to have a chlorophyll a/chlorophyll b ratio of 6.0 and a PSII_β/PSI reaction center ratio of 0.3. Kinetic analysis of system II photoactivity revealed the absence of PSII_α from stroma-exposed thylakoids. The photoactivity of system I in stroma-exposed thylakoids showed a single kinetic component identical to that of unfractionated chloroplasts, suggesting that PSI does not receive excitation energy from the PSII-chlorophyll ab light-harvesting complex. Thus, stroma-exposed thylakoids are significantly enriched in both PSI and PSII_β. Inside-out vesicles from the appressed membranes of grana-partition regions had a chlorophyll a/chlorophyll b ratio of 2.0 and a PSII/PSI reaction center ratio of 10.0. The photoactivity of system II showed the membranes of the grana-partition regions to be significantly enriched in PSII_α. We conclude that PSII_α is exclusively located in the membranes of the grana partitions while PSII_β and PSI are located in stroma-exposed thylakoids. The low PSI reaction center (P700) content of vesicles derived from grana partitions and the kinetic homogeneity of the PSI complex suggest total exclusion of P700 as a functional component in the membrane of the grana-partition region.     

Shared thematic elements in photochemical reaction centers.

The structural, functional, and evolutionary relationships between photosystem II and the purple nonsulfur bacterial reaction center have been recognized for several years. These can be classified as "quinone type" (type II) photosystems because the terminal electron acceptor is a mobile quinone molecule. The analogous relationship between photosystem I and the green sulfur bacterial (and helicobacterial) reaction centers has only recently become clear. These can be classified as "iron-sulfur type" (type I) photosystems because the terminal electron acceptor consists of one or more bound iron-sulfur clusters. At a fundamental level, the quinone type and iron-sulfur type reaction centers share a common photochemical motif in the early process of charge separation, leading to the speculation that all photochemical reaction centers have a common evolutionary origin. This review summarizes the current state of knowledge in comparative reaction center biochemistry between prokaryotic bacteria, cyanobacteria, and green plants.     

Lithium dodecyl sulfate/polyacrylamide gel electrophoresis of thylakoid membranes at 4 degrees C: Characterizations of two additional chlorophyll a-protein complexes

Lithium dodecyl sulfate/polyacrylamide gel electrophoresis of Chlamydomonas reinhardtii thylakoid membranes at room temperature gave two chlorophyll-protein complexes, CP I and CP II, as had been reported previously. However, when the electrophoresis was performed at 4°C, there was an increase in the amount of chlorophyll associated with CP I and CP II, and in addition, three other chlorophyll-protein complexes appeared. Two of these complexes, designated CP III and CP IV, were characterized and found to be similar in their compositions. Each complex contains four to five molecules of chlorophyll a, one molecule of β-carotene, and one polypeptide chain. The apoprotein of CP III is polypeptide 5 (M_r 50,000) and that of CP IV is polypeptide 6 (M_r 47,000); the two polypeptides are structurally unrelated. Chlorophyll-protein complexes similar to C. reinhardtii CP III and CP IV were also detected in higher plants (e.g., Pisum sativum). The apoproteins of the higher plant complexes are immunochemically related to those of the C. reinhardtii complexes, as shown by crossed immunoelectrophoresis. Absorption spectra of CP III and CP IV at -196°C revealed a component at 682 nm. This observation, together with the previous results on photosystem II mutants [Chua, N.-H. & Bennoun, P. (1975) Proc. Natl. Acad. Sci. USA 72, 2175-2179], provides indirect evidence that CP III and CP IV may be involved in the primary photochemistry of photosystem II.     

A cDNA clone encoding a photosystem I protein with homology to photosystem II chlorophyll a/b-binding polypeptides.

We report here the isolation and nucleotide sequence of a complete cDNA clone encoding a photosystem I (PS I) polypeptide that is recognized by a monoclonal antibody made against photosystem II (PS II) chlorophyll a/b-binding (CAB) proteins. The deduced sequence of this PS I protein shows 30% overall identity to PS II CAB sequences, and two long segments within this protein show 50% and 65% identity to the corresponding segments in the PS II CAB polypeptides. Even though the sequence of this PS I CAB protein is substantially divergent from PS II CAB sequences, their hydropathy plots are very similar and suggest they all traverse the thylakoid membrane three times. A segment of the PS I CAB polypeptide shows similarity to the functionally analogous beta subunits of the antenna proteins of purple bacteria. In contrast, no homology was observed between these bacterial proteins and PS II CAB polypeptides.     

The PPH1 phosphatase is specifically involved in LHCII dephosphorylation and state transitions in Arabidopsis

The ability of plants to adapt to changing light conditions depends on a protein kinase network in the chloroplast that leads to the reversible phosphorylation of key proteins in the photosynthetic membrane. Phosphorylation regulates, in a process called state transition, a profound reorganization of the electron transfer chain and remodeling of the thylakoid membranes. Phosphorylation governs the association of the mobile part of the light-harvesting antenna LHCII with either photosystem I or photosystem II. Recent work has identified the redox-regulated protein kinase STN7 as a major actor in state transitions, but the nature of the corresponding phosphatases remained unknown. Here we identify a phosphatase of Arabidopsis thaliana, called PPH1, which is specifically required for the dephosphorylation of light-harvesting complex II (LHCII). We show that this single phosphatase is largely responsible for the dephosphorylation of Lhcb1 and Lhcb2 but not of the photosystem II core proteins. PPH1, which belongs to the family of monomeric PP2C type phosphatases, is a chloroplast protein and is mainly associated with the stroma lamellae of the thylakoid membranes. We demonstrate that loss of PPH1 leads to an increase in the antenna size of photosystem I and to a strong impairment of state transitions. Thus phosphorylation and dephosphorylation of LHCII appear to be specifically mediated by the kinase/phosphatase pair STN7 and PPH1. These two proteins emerge as key players in the adaptation of the photosynthetic apparatus to changes in light quality and quantity.     

Regulation of thylakoid protein phosphorylation at the substrate level: Reversible light-induced conformational changes expose the phosphorylation site of the light-harvesting complex II

Light-dependent activation of thylakoid protein phosphorylation regulates the energy distribution between photosystems I and II of oxygen-evolving photosynthetic eukaryotes as well as the turnover of photosystem II proteins. So far the only known effect of light on the phosphorylation process is the redox-dependent regulation of the membrane-bound protein kinase(s) activity via plastoquinol bound to the cytochrome bf complex and the redox state of thylakoid dithiols. By using a partially purified thylakoid protein kinase and isolated native chlorophyll (chl) a/b light-harvesting complex II (LHCII), as well as recombinant LHCII, we find that illumination of the chl-protein substrate exposes the phosphorylation site to the kinase. Light does not activate the phosphorylation of the LHCII apoprotein nor the recombinant pigment-reconstituted complex lacking the N-terminal domain that contains the phosphothreonine site. The suggested light-induced conformational change exposing the N-terminal domain of LHCII to the kinase is evidenced also by an increase in its accessibility to tryptic cleavage after light exposure. Light activates preferentially the trimeric form of LHCII, and the process is paralleled by chl fluorescence quenching. Both phenomena are slowly reversible in darkness. Light-induced exposure of the LHCII N-terminal domain to the endogenous protein kinase(s) and tryptic cleavage occurs also in thylakoid membranes. These results demonstrate that light may regulate thylakoid protein phosphorylation not only via the signal transduction chain connecting redox reactions to the protein kinase activation, but also by affecting the conformation of the chl-protein substrate.     

Live-cell imaging of photosystem II antenna dissociation during state transitions

Plants and green algae maintain efficient photosynthesis under changing light environments by adjusting their light-harvesting capacity. It has been suggested that energy redistribution is brought about by shuttling the light-harvesting antenna complex II (LHCII) between photosystem II (PSII) and photosystem I (PSI) (state transitions), but such molecular remodeling has never been demonstrated in vivo. Here, using chlorophyll fluorescence lifetime imaging microscopy, we visualized phospho-LHCII dissociation from PSII in live cells of the green alga Chlamydomonas reinhardtii. Induction of energy redistribution in wild-type cells led to an increase in, and spreading of, a 250-ps lifetime chlorophyll fluorescence component, which was not observed in the stt7 mutant incapable of state transitions. The 250-ps component was also the dominant component in a mutant containing the light-harvesting antenna complexes but no photosystems. The appearance of the 250-ps component was accompanied by activation of LHCII phosphorylation, supporting the visualization of phospho-LHCII dissociation. Possible implications of the unbound phospho-LHCII on energy dissipation are discussed.     

The pi-Cation Radical of Chlorophyll a

Chlorophyll a undergoes reversible one-electron oxidation in dichloromethane and butyronitrile. Removal of the electron by controlled potential electrolysis or by stoichiometric charge transfer to a known cation radical yields a radical (epr line width = 9 gauss, g = 2.0025 ± 0.0001) whose optical spectrum is bleached relative to that of chlorophyll. Upon electrophoresis this bleached species behaves as a cation. By comparison with the known properties of π-cation radicals of porphyrins and chlorins, the chlorophyll radical is also identified as a π-cation. Further correlation of optical and epr properties with published studies on photosynthesis leads to the conclusion that oxidized P700, the first photochemical product of photosystem I in green plants, contains a π-cation radical of the chlorin component of chlorophyll a. This radical is the likely source of the rapidly-decaying, narrow epr signal of photosynthesis.     

Impaired respiration discloses the physiological significance of state transitions in Chlamydomonas

State transitions correspond to a major regulation process for photosynthesis, whereby chlorophyll protein complexes responsible for light harvesting migrate between photosystem II and photosystem I in response to changes in the redox poise of the intersystem electron carriers. Here we disclose their physiological significance in Chlamydomonas reinhardtii using a genetic approach. Using single and double mutants defective for state transitions and/or mitochondrial respiration, we show that photosynthetic growth, and therefore biomass production, critically depends on state transitions in respiratory-defective conditions. When extra ATP cannot be provided by respiration, enhanced photosystem I turnover elicited by transition to state 2 is required for photosynthetic activity. Concomitant impairment of state transitions and respiration decreases the overall yield of photosynthesis, ultimately leading to reduced fitness. We thus provide experimental evidence that the combined energetic contributions of state transitions and respiration are required for efficient carbon assimilation in this alga.     

Quenching of chlorophyll fluorescence in the major light-harvesting complex of photosystem II: a systematic study of the effect of carotenoid structure

The role of carotenoids in quenching of chlorophyll fluorescence in the major light-harvesting complex of photosystem II has been studied with a view to understanding the molecular basis of the control of photoprotective nonradiative energy dissipation by the xanthophyll cycle in vivo. The control of chlorophyll fluorescence quenching in the isolated complex has been investigated in terms of the number of the conjugated double bonds for a series of carotenoids ranging from n = 5-19, giving an estimated first excited singlet state energy from 20,700 cm-1 to 10,120 cm-1. At pH 7.8 the addition of exogenous carotenoids with >=10 conjugated double bonds (including zeaxanthin) stimulated fluorescence quenching relative to the control with no added carotenoid, whereas those with n 相似文献