Reality of P-680 chlorophyll protein – Identification of the site of primary photochemistry in oxygenic photosynthesis

Until recently nearly all available experimental evidence seemed to indicate that the largest subunit of about 50 kDa in the photosystem II core complex ( psb B gene product) is the site of primary photochemistry, and thus the name "P-680 apoprotcin" has been given to this subunit. The notion, however, has also been challenged on the basis of deduced amino acid sequence homology between the proteins in the photosystem II and those of the purple bacterial reaction center. The actual preparation of a functionally active photosystem II reaction center completely devoid of the psb B gene product, but consisting of D-1 and D-2 proteins and cytochrome b -559, has now been achieved.     

Thylakoid protein phosphorylation,state 1-state 2 transitions,and photosystem stoichiometry adjustment: redox control at multiple levels of gene expression

In photosynthesis in chloroplasts, control of thylakoid protein phosphorylation by redox state of inter-photosystem electron carriers makes distribution of absorbed excitation energy between the two photosystems self-regulating. During operation of this regulatory mechanism, reduction of plastoquinone activates a thylakoid protein kinase which phosphorylates the light-harvesting complex LHC II, causing a change in protein recognition that results in redistribution of energy to photosystem I at the expense of photosystem II, thus tending to oxidise the reduced plastoquinone pool. These events correspond to the transition from light-state 1 to light-state 2. The reverse transition (to light-state 1) is initiated by transient oxidation of plastoquinone, inactivation of the LHC II kinase, and return of dephosphorylated LHC II from photosystem I to photosystem II, supplying excitation energy to photosystem II and thereby reducing plastoquinone. State 1-state 2 transitions therefore operate by means of redox control of reversible, post-translational modification of pre-existing proteins. A balance in the rates of light utilization by photosystem I and photosystem II can also be achieved, on longer time-scales and between wider limits, by adjustment of the relative quantities, or stoichiometry, of photosystem I and photosystem II. Recent evidence suggests that adjustment of photosystem stoichiometry is also a response to perturbation of the redox state of inter-photosystem electron carriers, and involves specific redox control of de novo protein synthesis, assembly, and breakdown. It is therefore suggested that the same redox sensor initiates these different adaptations by control of gene expression at different levels, according to the time-scale and amplitude of the response. This integrated feedback control may serve to maintain redox homeostasis, and, as a result, quantum yield. Evidence for the components required by such systems is discussed.     

Inhibitors of photosystem II and the topology of the herbicide and QB binding polypeptide in the thylakoid membrane

The folding through the thylakoid membrane of the D-1 herbicide binding polypeptide and of the homologous D-2 subunit of photosystem II is predicted from comparison of amino acid sequences and hydropathy index plots with the folding of the subunits L and M of a bacterial photosystem. As the functional amino acids involved in Q and Fe binding in the bacterial photosystem of R. viridis, as indicated by the X-ray structure, are conserved in the homologous D-1 and D-2 subunits of photosystem II, a detailed topology of the binding niche of Q_B and of herbicides on photosystem II is proposed. The model is supported by the observed amino acid changes in herbicide tolerant plants and algae. These changes are all in the binding domain on the matrix side of the D-1 polypeptide, and turn out to be of functional significance in the Q_B binding.New inhibitors of Q_B function are described. Their chemical structure, i.e. pyridones, quinolones, chromones and benzodiones, contains the features of the phenolic type herbicides. Their essential elements, -charges at particular atoms, QSAR and steric requirements for optimal inhibitory potency are discussed and compared with the classical herbicides of the urea/triazine type.     

Modification of excitation energy distribution to photosystem I by protein phosphorylation and cation depletion during thylakoid biogenesis in wheat

The effects of protein phosphorylation and cation depletion on the electron transport rate and fluorescence emission characteristics of photosystem I at two stages of chloroplast development in light-grown wheat leaves are examined. The light-harvesting chlorophyll a/b protein complex associated with photosystem I (LHC I) was absent from the thylakoids at the early stage of development, but that associated with photosystem II (LHC II) was present. Protein phosphorylation produced an increase in the light-limited rate of photosystem I electron transport at the early stage of development when chlorophyll b was preferentially excited, indicating that LHC I is not required for transfer of excitation energy from phosphorylated LHC II to the core complex of photosystem I. However, no enhancement of photosystem I fluorescence at 77 K was observed at this stage of development, demonstrating that a strict relationship between excitation energy density in photosystem I pigment matrices and the long-wavelength fluorescence emission from photosystem I at 77 K does not exist. Depletion of Mg²⁺ from the thylakoids produced a stimulation of photosystem I electron transport at both stages of development, but a large enhancement of the photosystem I fluorescence emission was observed only in the thylakoids containing LHC I. It is suggested that the enhancement of PS I electron transport by Mg²⁺-depletion and phosphorylation of LHC II is associated with an enhancement of fluorescence at 77 K from LHC I and not from the core complex of PS I.     

Protein composition of the photosystem II core complex in genetically engineered mutants of the cyanobacterium Synechocystis sp. PCC 6803

The presence of four photosystem II proteins, CP47, CP43, D1 and D2, was monitored in mutants of Synechocystis sp. PCC 6803 that have modified or inactivated genes for CP47, CP43, or D2. It was observed that: (1) thylakoids from mutants without a functional gene encoding CP47 are also depleted in D1 and D2; (2) inactivation of the gene for CP43 leads to decreased but significant levels of CP47, D1 and D2; (3) deletion of part of both genes encoding D2, together with deletion of part of the CP43-encoding gene causes a complete loss of CP47 and D1; (4) thylakoids from a site-directed mutant in which the His-214 residue of D2 has been replaced by asparagine do not contain detectable photosystem II core proteins. However, in another site-directed mutant, in which His-197 has been replaced by tyrosine, some CP47 as well as breakdown products of CP43, but no D1 and D2, can be detected. These data could indicate a central function of CP47 and D2 in stable assembly of the photosystem II complex. CP43, however, is somewhat less critical for formation of the core complex, although CP43 is required for a physiologically functional photosystem II unit. A possible model for the assembly of the photosystem II core complex is proposed.     

Photoinhibition of photosynthesis and growth responses at different light levels in psbA gene mutants of the cyanobacterium Synechococcus

Photoinhibition of photosynthesis and growth responses at diffrent light levels (10, 120 and 250 μmol m⁻² s⁻¹) were studied in psbA gene mutants R2S2C3 ( psbAI gene present) and R2K1 ( psbAIIIpsbAIII genes present) of the cyanobacterium Synechococcus sp . PCC 7942 ( Anacystis nidulans R2). Mutant R2K1 (possessing form II of the D1 protein of photosystem II) was much more resistant to photoinhibition than the mutant R2S2C3 (possessing form I of the D1 protein). At moderate inhibitory light levels (100 to 300 μmol m⁻² s⁻¹) this was largely ascribed to an increased rsistance of the photosystem II reaction cetres possessing form II of the D1 protein. However, at higher light levels the higher resistance mutant R2K1 was assigned to a higher rate of photosystem II repair, i.e. turnover of the D1 protein. Moreover, our results support the hypothesis that photoinhibition of photosystem II and photoinhibitory induced quenching are due to separate processes. Results from growth experiments show that the R2K1 mutant has a slower growth rate than the R2S2C3 mutant but shows an increased survival under high light stress conditions. It is hypothesized that high resistance to photoinhibition, though allowing a better survival under high light, is not advantageous for optimal growth.     

Characterisation of the PsbX protein from Photosystem II and light regulation of its gene expression in higher plants

The location and expression of the previously uncharacterised photosystem II subunit PsbX have been analysed in higher plants. We show that this protein is a component of photosystem II (PSII) core particles but absent from light-harvesting complexes or PSII reaction centres. PsbX is, however, localised to the near vicinity of the reaction centre because it can be cross-linked to cytochrome b559, which is known to be associated with the D1/D2 dimer. We also show that the expression of this protein is tightly regulated by light, since neither protein nor mRNA is found in dark-grown plants.     

D1 protein of photosystem II: The light sensor in chloroplasts

Light, controls the “blueprint” for chloroplast development, but at high intensities is toxic to the chloroplast. Excessive light intensities inhibit primarily photosystem II electron transport. This results in generation of toxic singlet oxygen due to impairment of electron transport on the acceptor side between pheophytin and Q_B -the secondary electron acceptor. High light stress also impairs electron transport on the donor side of photosystem II generating highly oxidizing species Z⁺ and P680⁺. A conformationsl change in the photosystem II reaction centre protein Dl affecting its Q_B-binding site is involved in turning the damaged protein into a substrate for proteolysis. The evidence indicates that the degradation of D1 is an enzymatic process and the protease that degrades D1 protein has been shown to be a serine protease Although there is evidence to indicate that the chlorophyll a-protein complex CP43 acts as a serine-type protease degrading Dl, the observed degradation of Dl protein in photosystem II reaction centre particlesin vitro argues against the involvement of CP43 in Dl degradation. Besides the degradation during high light stress of Dl, and to a lesser extent D2-the other reaction centre protein, CP43 and CP29 have also been shown to undergo degradation. In an oxygenic environment, Dl is cleaved from its N-and C-termini and the disassembly of the photosystem II complex involves simultaneous release of manganese and three extrinsic proteins involved in oxygen evolution. It is known that protein with PEST sequences are subject to degradation; D1 protein contains a PEST sequence adjacent to the site of cleavage on the outer side of thylakoid membrane between helices IV and V. The molecular processes of “triggering” of Dl for proteolytic degradation are not clearly understood. The changes in structural organization of photosystem II due to generation of oxy-radicals and other highly oxidizing species have also not been resolved. Whether CP43 or a component of the photosystem II reaction centre itself (Dl. D2 or cy1 b559 subunits), which may be responsible for degradation of Dl, is also subject to light modification to become an active protease, is also not known. The identity of proteases degrading Dl, LHCII and CP43 and C29 remains to be established     

The presequence of the precursor to the nucleus-encoded 30 kDa protein of photosystem II in Euglena gracilis Z includes two hydrophobic domains

A cDNA clone for the extrinsic 30 kDa protein (OEC30) of photosystem II in Euglena gracilis Z was isolated and characterized. The open reading frame of the cDNA encoded a polypeptide of 338 amino acids, which consisted of a long presequence of 93 amino acids and a mature polypeptide of 245 amino acids. Two hydrophobic domains were identified in the presequence, in contrast to the presence of a single hydrophobic domain in the presequence of the corresponding proteins from higher plants. At the N- and C-terminal regions, respectively, of the presequence, a signal-peptide-like sequence and a thylakoid-transfer domain were identified. The presence of a long and unique presequence in the precursor to OEC30 is probably related to the complexity of the intracellular processes required for the synthesis and/or transport of the protein in Euglena.Abbreviations ER endoplasmic reticulum - cDNA complementary DNA - SSU small subunit; Rubisco, ribulose 1,5-bisphosphate carboxylase/oxygenase - Rubico, ribulose 1,5 bisphosphate carboxylase/oxygenase - LHC II light-harvesting chlorophyll protein of photosystem II - PS II photosystem II - OEC30 the extrinsic 30 kDa protein of photosystem II in Euglena - PCR polymerase chain reaction - SDS sodium dodecyl sulfate - TE a solution containing 10 mM Tris-HCl and 1 mM EDTA pH 8.0 - SSPE a solution containing 0.15 M NaCl, 10 mM NaH₂PO₄ and 1 mM EDTA pH 7.4 - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis - PVDF poly(vinylidene difluoride)     

Influence of thylakoid protein phosphorylation on photosynthetic electron transport and photophosphorylation

Data are reported which show that thylakoid protein phosphorylation decreases photosystem II fluorescence yield and enhances the photosystem I dependent photophosphorylation catalyzed by phenazinemethosulphate in the presence of DCMU. The stimulation is larger at low light intensity, but is still observed at high intensity. These observations are interpreted to demonstrate that thylakoid protein phosphorylation causes a transfer of excitation energy from PS II to PS I, but may also have an independent stimulatory effect on PS I dependent photophosphorylation.     

Light-induced fluorescence quenching in the light-harvesting chlorophyll a/b protein complex

Summary Irradiation of the principal photosystem II light-harvesting chlorophyll-protein antenna complex, LHC II, with high light intensities brings about a pronounced quenching of the chlorophyll fluorescence. Illumination of isolated thylakoids with high light intensities generates the formation of quenching centres within LHC II in vivo, as demonstrated by fluorescence excitation spectroscopy. In the isolated complex it is demonstrated that the light-induced fluorescence quenching: a) shows a partial, biphasic reversibility in the dark; b) is approximately proportional to the light intensity; c) is almost independent of temperature in the range 0–30°C; d) is substantially insensitive to protein modifying reagents and treatments; e) occurs in the absence of oxygen. A possible physiological importance of the phenomenon is discussed in terms of a mechanism capable of dissipating excess excitation energy within the photosystem II antenna.Abbreviations chla chlorophyll a - chlb chlorophyll b - F₀ fluorescence yield with reaction centers open - F_m fluorescence yield with reaction centres closed - F_i fluorescence at the plateau level of the fast induction phase - LHC II light-harvesting chlorophyll a/b protein complex II - PS II photosystem II - PSI photosystem I - Tricine N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine     

Spinach-thylakoid phosphorylation: Studies on the kinetics of changes in photosystem antenna size, spill-over and phosphorylation of light-harvesting chlorophyll a/b protein

The kinetics of LHCP phosphorylation and associated changes in photosystem cross-section and energy ‘spill-over’ from PS II to PS I have been examined in isolated spinach chloroplasts. During an initial phosphorylation period of 3–6 min, in the presence of saturating concentrations of Mg²⁺, the increase in PS I and decrease in PS II cross-section are largely completed, as judged by both measurements of the steady-state redox state of Q and fluorescence yield changes. This corresponds to a period of rapid ³²P incorporation into the low-molecular weight LHCP polypeptide. Subsequent to this initial 3–6-min period there is substantial further phosphorylation of both LHCP polypeptides, which is not accompanied by significant changes in photosystem cross-section, even after the chloroplasts had been unstacked with extensive mixing of PS I and PS II by Mg-removal. It is suggested that there exists a specific ‘mobile’ population of LHCP molecules which is rapidly phosphorylated and which may be enriched in the low-molecular-weight polypeptide. In addition, measurements of the kinetics of the ‘spill-over’ changes upon either Mg²⁺ addition or removal indicate that the continued phosphorylation of LHCP is able to increase the ‘spill-over’ process under favourable ionic conditions.     

The structure and function of the 33 kDa extrinsic protein of Photosystem II: A critical assessment

In this review the structure and function of the 33 kDa protein of Photosystem II is examined. Significant controversies exist concerning the solution secondary structure of the protein, the location of its binding site(s) within Photosystem II, the amino acid residues of the 33 kDa protein required for binding and its stoichiometry within the photosystem. The studies which examine these topics are considered from a critical perspective. A hypothetical model of the folding of the 33 kDa extrinsic protein which is supported by site-specific labeling studies and site-directed mutagenesis experiments is presented. Additionally, the function of the protein within the photosystem is unclear. We present a hypothesis that the 33 kDa protein is involved in maintaining the chloride associated with photosynthetic oxygen evolution in close proximity to the oxygen-evolving site.     

Relationship between excitation energy transfer, trapping, and antenna size in photosystem II

We present a systematic study of the effect of antenna size on energy transfer and trapping in photosystem II. Time-resolved fluorescence experiments have been used to probe a range of particles isolated from both higher plants and the cyanobacterium Synechocystis 6803. The isolated reaction center dynamics are represented by a quasi-phenomenological model that fits the extensive time-resolved data from photosystem II reaction centers and reaction center mutants. This representation of the photosystem II "trapping engine" is found to correctly predict the extent of, and time scale for, charge separation in a range of photosystem II particles of varying antenna size (8-250 chlorins). This work shows that the presence of the shallow trap and slow charge separation kinetics, observed in isolated D1/D2/cyt b559 reaction centers, are indeed retained in larger particles and that these properties are reflected in the trapping dynamics of all larger photosystem II preparations. A shallow equilibrium between the antennae and reaction center in photosystem II will certainly facilitate regulation via nonphotochemical quenching, and one possible interpretation of these findings is therefore that photosystem II is optimized for regulation rather than for efficiency.     

Photochemical Apparatus Organization in the Chloroplasts of Two Beta vulgaris Genotypes

The composition and structural organization of thylakoid membranes of a low chlorophyll mutant of Beta vulgaris was investigated using spectroscopic, kinetic and electrophoretic techniques. The data obtained were compared with those of a standard F1 hybrid of the same species. The mutant was depleted in chlorophyll b relative to the hybrid and it had a higher photosystem II/photosystem I reaction center (Q/P₇₀₀) ratio and a smaller functional chlorophyll antenna size. Analysis of thylakoid membranes by sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that the mutant lacked a portion of the chlorophyll a/b light-harvesting complex but was enriched in the photosystem II reaction center chlorophyll protein complex. Comparison of functional antenna sizes and of photosystem stoichiometries determined electrophoretically were in good agreement with those determined spectroscopically. Both approaches indicated that about 30% of the total chlorophyll was associated with photosystem I and about 70% with photosystem II. A greater proportion of photosystem II_β was detected in the mutant. The results suggest that a higher photosystem II to photosystem I ratio in the sugar beet mutant has apparently compensated for the smaller photosystem II chlorophyll light-harvesting antenna in its chloroplasts. Moreover, a lack of chlorophyll a/b light-harvesting complex correlates with the abundance of photosystem II_β. It is proposed that a developmental relationship exists between the two types of photosystem II where photosystem II_β is a precursor form of photosystem II_α occurring prior to the addition of the chlorophyll a/b light-harvesting complex and grana formation.     

Photosynthetic acclimation: structural reorganisation of light harvesting antenna--role of redox-dependent phosphorylation of major and minor chlorophyll a/b binding proteins

In order to carry out photosynthesis, plants and algae rely on the co-operative interaction of two photosystems: photosystem I and photosystem II. For maximum efficiency, each photosystem should absorb the same amount of light. To achieve this, plants and green algae have a mobile pool of chlorophyll a/b-binding proteins that can switch between being light-harvesting antenna for photosystem I or photosystem II, in order to maintain an optimal excitation balance. This switch, termed state transitions, involves the reversible phosphorylation of the mobile chlorophyll a/b-binding proteins, which is regulated by the redox state of the plastoquinone-mediating electron transfer between photosystem I and photosystem II. In this review, we will present the data supporting the function of redox-dependent phosphorylation of the major and minor chlorophyll a/b-binding proteins by the specific thylakoid-bound kinases (Stt7, STN7, TAKs) providing a molecular switch for the structural remodelling of the light-harvesting complexes during state transitions. We will also overview the latest X-ray crystallographic and electron microscopy-derived models for structural re-arrangement of the light-harvesting antenna during State 1-to-State 2 transition, in which the minor chlorophyll a/b-binding protein, CP29, and the mobile light-harvesting complex II trimer detach from the light-harvesting complex II-photosystem II supercomplex and associate with the photosystem I core in the vicinity of the PsaH/L/O/P domain.     

Effects of desiccation on the excitation energy distribution from phycoerythrin to the two photosystems in the red alga Porphyra perforata

Low temperature (77°K) fluorescence emission and excitation spectra were recorded for wet and desiccated thalli of Porphyra perforata . The photosystem I (F730) and photosystem II (F695) fluorescence emission kinetics during photosystem II trap closure were also recorded at 77°K. Desiccation induced a lowering of the fluorescence yield over the whole emission spectrum but the decrease was most pronounced for the photosystem II fluorescence bands, F688 and F695. It was shown that the desiccation-induced changes of the phycoerythrin sensitized emission spectrum were due to 1) a decrease in the fluorescence yield of the photosystem I antenna, 2) an even stronger decrease in the fluorescence of photosystem II, which was mediated by an increased spillover (k_T(II→I)) of excitation to photosystem I and an increase in the absorption cross section, α, for photosystem I. We hypothesize that the increase of both k_T(II→I) and α are part of a mechanism by which the desiccation-tolerant, high light exposed, Porphyra can avoid photodynamic damage to photosystem II, when photosynthesis becomes inhibited as a result of desiccation during periods of low tide.     

Effects of winter stress on photosynthetic electron transport and energy distribution between the two photosystems of pine as assayed by chlorophyll fluorescence kinetics

The fluorescence kinetics of both intact needles and isolated chloroplasts of summer active and winter stressed Pinus sylvestris were measured at both room temperature and 77 K. It was confirmed that winter stress inhibited the photochemical capacity of photosystem II but also that winter stress caused the strongest inhibition of the electron transport at the site where the plastoquinone pool is reduced. Parallel analyses of the fluorescence characteristics of photosystem II (F₆₉₃) and photosystem I (F₇₂₉) during photosystem II trap closure furthermore revealed that the yield of spillover of excitation energy from photosystem II to photosystem I decreased upon winter stress. We suggest that this is because of an increased radiationless decay of excitation energy both at the reaction center and antennae levels of photosystem II. There is, however, also a possibility that the decreased yield of spill-over is accentuated by a partial detachment of the light harvesting chlorophyll a/b complex from photosystem II upon winter stress.Paper presented at the FESPP meeting in Strasbourg (1984).     

The identity of the water oxidizing enzyme in photosystem II is still controversial

In recent years great advances in the understanding of photosystem II have been achieved. The process of photochemical charge separation seems to be fairly well understood, while the identity of the water oxidizing enzyme in photosystem II has remained uncertain. In the first part of the paper a brief review on structural and functional aspects of photosystem II is given, and in the second part the nature of the elusive water oxidizing enzyme is considered. Two models are discussed. The first model, favoured by the majority of groups working in this area, suggests that the reaction center polypeptide D1 (in association with other known photosystem II polypeptides) is the site of water oxidation. The second model, mainly based on our results with cyanobacteria, predicts that the water oxidizing enzyme is a separate polypeptide in the 30 kDa region, distinct from D1 and D2, in addition to the seven polypeptides so far recognized in minimal O₂ evolving photosystem II complexes     

Photosynthetic activity of far-red light in green plants

We have found that long-wavelength quanta up to 780 nm support oxygen evolution from the leaves of sunflower and bean. The far-red light excitations are supporting the photochemical activity of photosystem II, as is indicated by the increased chlorophyll fluorescence in response to the reduction of the photosystem II primary electron acceptor, Q_A. The results also demonstrate that the far-red photosystem II excitations are susceptible to non-photochemical quenching, although less than the red excitations. Uphill activation energies of 9.8 ± 0.5 kJ mol⁻¹ and 12.5 ± 0.7 kJ mol⁻¹ have been revealed in sunflower leaves for the 716 and 740 nm illumination, respectively, from the temperature dependencies of quantum yields, comparable to the corresponding energy gaps of 8.8 and 14.3 kJ mol⁻¹ between the 716 and 680 nm, and the 740 and 680 nm light quanta. Similarly, the non-photochemical quenching of far-red excitations is facilitated by temperature confirming thermal activation of the far-red quanta to the photosystem II core. The observations are discussed in terms of as yet undisclosed far-red forms of chlorophyll in the photosystem II antenna, reversed (uphill) spill-over of excitation from photosystem I antenna to the photosystem II antenna, as well as absorption from thermally populated vibrational sub-levels of photosystem II chlorophylls in the ground electronic state. From these three interpretations, our analysis favours the first one, i.e., the presence in intact plant leaves of a small number of far-red chlorophylls of photosystem II. Based on analogy with the well-known far-red spectral forms in photosystem I, it is likely that some kind of strongly coupled chlorophyll dimers/aggregates are involved. The similarity of the result for sunflower and bean proves that both the extreme long-wavelength oxygen evolution and the local quantum yield maximum are general properties of the plants.