Reconstitution of Photosystem Ⅱ Reaction Center with Cu-ChlorophyⅡ a

An Isolated photosystem （PS） II reaction center （RC） with altered pigment content was obtained by chemical exchange of native chlorophyll a （Chl） with externally added Cu-Chl a （Cu-Chl）. Pigment composition and spectroscopic properties of the RC exchanged with Cu-Chl were compared with native RC and RC treated with Chl In the same way. High-performance liquid chromatography analysis showed approximately 0.5 Cu-Chl per two pheophytln in the Cu-Chl-reconstltuted RC preparation. Insertion of Cu-Chl resulted in a decrease In absorption at 670 nm and an Increase at 660 nm, suggesting that the peripheral Chl may have been displaced. Fluorescence emission spectra of the Cu-Chl-reconstituted RC displayed a marked decrease In fluorescence yield and a blue shift of the band maximum, accompanied by the appearance of a broad peak at a shorter wavelength, Indicating that energy transfer In the modified RC was disturbed by Cu-Chl, a quencher of the excited state. However, there were few differences in the circular dichrolsm （CD） spectra, suggesting that the arrangement of pigments and proteins responsible for the CD signal was not significantly affected. In addition, no obvious change In peptlde components was found after the exchange procedure.     

Extraction and Reconstitution of Lyophilized Photosystem I Subchloroplast Fragments from Spinach Chloroplasts

Lyophilized photosystem I subchloroplast fragments prepared from spinach chloroplasts were extracted and reconstituted. Hexane extraction eliminated 26 % of the photosystem I activity without removing chlorophylls, and the reconstitution with β-carotene, an unkown lipid or chlorophyll a restored almost all the activity. Extraction with hexane–acetone (2:1, v/v) eliminated 81% of the activity with removal of 78% of the chlorophylls. In this case, reconstitution with chlorophyll a showed complete restoration of the activity, but β-carotene, the unknown lipid, and plastoquinone A caused a rather inhibitory effect. A possible explanation is that photosystem I reaction centers are closely surrounded by chlorophyll a; and β-carotene, the unknown lipid, and plastoquinone A function only through chlorophyll a.     

Photosystem II.

Electron crystallography of photosystem II has revealed the location of important subunits and photoactive pigment molecules within this large membrane protein complex. It has also demonstrated a close evolutionary link among all types of photosynthetic reaction centres.     

Energy transfer between Photosystem II and Photosystem I in chloroplasts

A model for the photochemical apparatus of photosynthesis is presented which accounts for the fluorescence properties of Photosystem II and Photosystem I as well as energy transfer between the two photosystems. The model was tested by measuring at ?196 °C fluorescence induction curves at 690 and 730 nm in the absence and presence of 5 mM MgCl₂ which presumably changes the distribution of excitation energy between the two photosystems. The equations describing the fluorescence properties involve terms for the distribution of absorbed quanta, α, being the fraction distributed to Photosystem I, and β, the fraction to Photosystem II, and a term for the rate constant for energy transfer from Photosystem II to Photosystem I,k_T(II→I). The data, analyzed within the context of the model, permit a direct comparison of α andk_T(II→I) in the absence (?) and presence (+) of Mg²⁺:α/^?α⁺= 1.2andk/^?_T(II→I)k⁺_T(II→I)= 1.9. If the criterion thatα + β = 1 is applied absolute values can be calculated: in the presence of Mg²⁺,a⁺ = 0.27 and the yield of energy transfer,φ⁺_T(II→I) varied from 0.065 when the Photosystem II reaction centers were all open to 0.23 when they were closed. In the absence of Mg²⁺,α^? = 0.32 andφ_T(II→I) varied from 0.12 to 0.28.The data were also analyzed assuming that two types of energy transfer could be distinguished; a transfer from the light-harvseting chlorophyll of Photosystem II to Photosystem I,k_T(II→I), and a transfer from the reaction centers of Photosystem II to Photosystem I,k_t(II→I). In that caseα/^?α⁺= 1.3,k/^?_T(II→I)k⁺_T(II→I)= 1.3 andk/^?_t(II→I)k⁺_(tII→I)= 3.0. It was concluded, however, that both of these types of energy transfer are different manifestations of a single energy transfer process.     

Structural Analysis of Photosystem II: Comparative Study of Cyanobacterial and Higher Plant Photosystem II Complexes

Oxygen evolving photosystem II (PSII-OEC) complexes and PSII core complexes were isolated from spinach and the thermophilic cyanobacteriumSynechococcussp. OD24 and characterized by gel electrophoresis, immunoblotting, and absorbance spectroscopy. The mass of the core complexes was determined by scanning transmission electron microscopy (STEM) and found to be 281 ± 65 kDa for spinach and 313 ± 52 kDa forSynechococcussp. OD24. The mass of the spinach PSII-OEC complex was 327 ± 64 kDa. Digital images of negatively stained PSII-OEC and PSII core complexes were recorded by STEM and analyzed by single particle averaging. All monomeric complexes showed similar morphologies and were of comparable length (14 nm) and width (10 nm). The averages revealed a pseudo-twofold symmetry axis, which is a prominent structural element of the monomeric form. Difference maps between the averaged projections of the oxygen evolving complexes and the core complexes from both species indicated where the 33-kDa extrinsic manganese stabilizing protein is bound. A symmetric organization of the PSII complex, with the PsbA and the PsbD proteins in the center and symmetrically arranged PsbB and PsbC proteins at the periphery of the monomeric complex, is proposed.     

Inhibition of Photosystem II activity by saturating single turnover flashes in calcium-depleted and active Photosystem II

Inhibition of Photosystem II (PS II) activity induced by continuous light or by saturating single turnover flashes was investigated in Ca²⁺-depleted, Mn-depleted and active PS II enriched membrane fragments. While Ca²⁺- and Mn-depleted PS II were more damaged under continuous illumination, active PS II was more susceptible to flash-induced photoinhibition. The extent of photoinactivation as a function of the duration of the dark interval between the saturating single turnover flashes was investigated. The active centres showed the most photodamage when the time interval between the flashes was long enough (32 s) to allow for charge recombination between the S₂ or S₃ and Q_B ⁻ to occur. Illumination with groups of consecutive flashes (spacing between the flashes 0.1 s followed by 32 s dark interval) resulted in a binary oscillation of the loss of PS II-activity in active samples as has been shown previously (Keren N, Gong H, Ohad I (1995), J Biol Chem 270: 806–814). Ca²⁺- and Mn-depleted PS II did not show this effect. The data are explained by assuming that charge recombination in active PS II results in a back reaction that generates P₆₈₀ triplet and thence singlet oxygen, while in Ca²⁺- and Mn-depleted PS II charge recombination occurs through a different pathway, that does not involve triplet generation. This correlates with an up-shift of the midpoint potential of Q_A in samples lacking Ca²⁺ or Mn that, in term, is predicted to result in the triplet generating pathway becoming thermodynamically less favourable (G.N. Johnson, A.W. Rutherford, A. Krieger, 1995, Biochim. Biophys. Acta 1229, 201–207). The diminished susceptibility to flash-induced photoinhibition in Ca²⁺- and Mn-depleted PS II is attributed at least in part to this mechanism. This revised version was published online in June 2006 with corrections to the Cover Date.     

Reconstitution of iron-sulfur center B of Photosystem I damaged by mercuric chloride

Incubation of thylakoid membranes from spinach with low concentrations of mercuric chloride induces the loss of one of the iron-sulfur centers, F_B, in Photosystem I (PS I) and inhibits the electron transfer from PS I to the soluble electron carrier, ferredoxin. Reconstitution of this damaged iron-sulfur center has been carried out by incubating treated thylakoid membranes with exogenous FeCl₃ and Na₂S in the presence of-mercaptoethanol under anaerobic conditions. Low temperature EPR measurements indicate that center F_B is largely restored. Kinetic experiments show that the restored F_B can be photoreduced from P700. However, these reconstituted thylakoid membranes are still incompetent in the photoreduction of ferredoxin and NADP⁺, even though ferredoxin binding to the modified membranes was not impaired, indicating additional changes in the structure of the PS I complex must have occurred.     

Interaction of 1,4-benzoquinones with Photosystem II in thylakoids and Photosystem II membrane fragments from spinach

The interaction of exogenous quinones with the Photosystem II (PS II) acceptor side has been analyzed by measurements of flash-induced 320 nm absorption changes, transient flash-induced variable fluorescence changes, thermoluminescence emission and oxygen yield in dark-adapted thylakoids and PS II membrane fragments. Two classes of 1,4-benzoquinones were shown to give rise to remarkably different reaction patterns. (A) Phenyl-p-benzoquinone (Ph-p-BQ) -type compounds give rise to a marked binary oscillation of the initial amplitudes of 320 nm absorption changes induced by a flash train in dark-adapted PS II membrane fragments and a retardation of the decay kinetics of the flash-induced variable fluorescence. The electron transfer reactions to these type of quinones are severely inhibited by 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU). (B) In the presence of tribromotoluquinone (TBTQ) a different oscillation pattern of the 320 nm absorption changes is observed characterized by a marked relaxation after the first flash in the 5 ms domain. This relaxation is insensitive to 10 μM DCMU. Likewise the decay of the flash-induced variable fluorescence in TBTQ-treated samples is much less sensitive to DCMU than in control. The thermoluminescence emission exhibits an oscillation in samples incubated for 5 min with TBTQ before addition of 30 μM DCMU. Under the same conditions a significant flash-induced oxygen evolution is observed only after the third and fourth flash, respectively, whereas in the presence of TBTQ alone a normal oscillation pattern is observed. The different functional patterns of PS II caused by the two types of classes of exogenous quinones are interpreted by their binding properties: a noncovalent association with the Q_B-site of Ph-p-BQ-type quinones versus a tight (covalent?) binding in the vicinity of Q_A (possibly also at the Q_B-site) in the case of halogenated 1,4-benzoquinones. The mechanistic implications of these findings are discussed.     

Current perceptions of Photosystem II

In the last few years our knowledge of the structure and function of Photosystem II in oxygen-evolving organisms has increased significantly. The biochemical isolation and characterization of essential protein components and the comparative analysis from purple photosynthetic bacteria (Deisenhofer, Epp, Miki, Huber and Michel (1984) J Mol Biol 180: 385–398) have led to a more concise picture of Photosystem II organization. Thus, it is now generally accepted that the so-called D1 and D2 intrinsic proteins bind the primary reactants and the reducing-side components. Simultaneously, the nature and reaction kinetics of the major electron transfer components have been further clarified. For example, the radicals giving rise to the different forms of EPR Signal II have recently been assigned to oxidized tyrosine residues on the D1 and D2 proteins, while the so-called Q₄₀₀ component has been assigned to the ferric form of the acceptor-side iron. The primary charge-separation has been meaured to take place in about 3 ps. However, despite all recent major efforts, the location of the manganese ions and the water-oxidation mechanism still remain largely unknown. Other topics which lately have received much attention include the organization of Photosystem II in the thylakoid membrane and the role of lipids and ionic cofactors like bicarbonate, calcium and chloride. This article attempts to give an overall update in this rapidly expanding field.     

Cyanobacterial Acclimation to Photosystem I or Photosystem II Light

The organization and function of the photochemical apparatus of Synechococcus 6301 was investigated in cells grown under yellow and red light regimes. Broadband yellow illumination is absorbed preferentially by the phycobilisome (PBS) whereas red light is absorbed primarily by the chlorophyll (Chl) pigment beds. Since PBSs are associated exclusively with photosystem II (PSII) and most of the Chl with photosystem I (PSI), it follows that yellow and red light regimes will create an imbalance of light absorption by the two photosystems. The cause and effect relationship between light quality and photosystem stoichiometry in Synechococcus was investigated. Cells grown under red light compensated for the excitation imbalance by synthesis/assembly of more PBS-PSII complexes resulting in high PSII/PSI = 0.71 and high bilin/Chl = 1.30. The adjustment of the photosystem stoichiometry in red light-grown cells was necessary and sufficient to establish an overall balanced absorption of red light by PSII and PSI. Cells grown under yellow light compensated for this excitation imbalance by assembly of more PSI complexes, resulting in low PSII/PSI = 0.27 and low bilin/Chl = 0.42. This adjustment of the photosystem stoichiometry in yellow light-grown cells was necessary but not quite sufficient to balance the absorption of yellow light by the PBS and the Chl pigment beds. A novel excitation quenching process was identified in yellow light-grown cells which dissipated approximately 40% of the PBS excitation, thus preventing over-excitation of PSII under yellow light conditions. It is hypothesized that State transitions in O₂ evolving photosynthetic organisms may serve as the signal for change in the stoichiometry of photochemical complexes in response to light quality conditions.     

Electric-field-induced luminescence emission spectra of Photosystem I and Photosystem II from chloroplasts

The electroluminescence induced by external electric fields in blebs prepared from chloroplasts consists of two kinetically different phases, rapid (R) and slow (S), which were shown to be linked to Photosystem I (PS I) and Photosystem II (PS II) activities, respectively (Symons, M., Korenstein, R. and Malkin, S. (1985) Biochim. Biophys. Acta 806, 305–310). In this report we describe conditions involving heat treatment of broken chloroplasts, which make it possible to observe R phase electroluminescence essentially devoid of any contribution by the S phase. This allowed the precise measurement of the emission spectrum of PS I electroluminescence. The emission spectrum of PS II electroluminescence was obtained using regular broken chloroplasts, which show only S-type emission. The latter emission spectrum is identical to the one obtained for ordinary prompt fluorescence, peaking at 685 nm with a bandwidth of about 25 nm. The PS I emission spectrum is symmetric around 705 nm and is much broader, about 60 nm.     

Temperature-dependent changes in Photosystem II heterogeneity support a cycle of Photosystem II during photoinhibition

High light treatments were given to attached leaves of pumpkin (Cucurbita pepo L.) at room temperature and at 1°C where the diffusion- and enzyme-dependent repair processes of Photosystem II are at a minimum. After treatments, electron transfer activities and fluorescence induction were measured from thylakoids isolated from the treated leaves. When the photoinhibition treatment was given at 1°C, the Photosystem II electron transfer assays that were designed to require electron transfer to the plastoquinone pool showed greater inhibition than electron transfer from H₂O to paraphenyl-benzoquinone, which measures all PS II centers. When the light treatment was given at room temperature, electron transfer from H₂O to paraphenyl-benzoquinone was inhibited more than whole-chain electron transfer. Variable fluorescence measured in the presence of ferricyanide decreased only during room-temperature treatments. These results suggest that reaction centers of one pool of Photosystem II, non-Q_B-PS II, replace photoinhibited reaction centers at room temperature, while no replacement occurs at 1°C. A simulation of photoinhibition at 1°C supports this conclusion.Abbreviations BSA bovine serum albumin - Chl chlorophyll - DCMU 3-(3,4,-dichlorophenyl)-1,1,-dimethylurea - DCPIP dichlorophenol-indophenol (2,6-dichloro-4((4-hydroxyphenyl)imino)-2,5-cyclohexadien-1-one) - DPC diphenyl carbazide (2,2-diphenylcarbonic dihydrazide) - FeCN ferricyanide (hexacyanoferrate(III)) - _app apparent quantum yield of photosynthetic oxygen evolution - MV methyl viologen (1,1-dimethyl-4,4-bipyridinium dichloride) - PPBQ phenyl-p-benzoquinone - PPFD photosynthetic photon flux density - PQ pool plastoquinone - Q_B secondary quinone acceptor of PS II - RT room temperature - WC whole chain electron transfer     

Photosystem II photoinhibition-repair cycle protects Photosystem I from irreversible damage

Photodamage of Photosystem II (PSII) has been considered as an unavoidable and harmful reaction that decreases plant productivity. PSII, however, has an efficient and dynamically regulated repair machinery, and the PSII activity becomes inhibited only when the rate of damage exceeds the rate of repair. The speed of repair is strictly regulated according to the energetic state in the chloroplast. In contrast to PSII, Photosystem I (PSI) is very rarely damaged, but when occurring, the damage is practically irreversible. While PSII damage is linearly dependent on light intensity, PSI gets damaged only when electron flow from PSII exceeds the capacity of PSI electron acceptors to cope with the electrons. When electron flow to PSI is limited, for example in the presence of DCMU, PSI is extremely tolerant against light stress. Proton gradient (ΔpH)-dependent slow-down of electron transfer from PSII to PSI, involving the PGR5 protein and the Cyt b6f complex, protects PSI from excess electrons upon sudden increase in light intensity. Here we provide evidence that in addition to the ΔpH-dependent control of electron transfer, the controlled photoinhibition of PSII is also able to protect PSI from permanent photodamage. We propose that regulation of PSII photoinhibition is the ultimate regulator of the photosynthetic electron transfer chain and provides a photoprotection mechanism against formation of reactive oxygen species and photodamage in PSI.     

Regulation des photosynthetischen Elektronentransports zwischen Photosystem II und Photosystem I

Synchronkulturen einzelliger Grünalgen stellen ein ausgezeichnetes Untersuchungsmaterial zum Studium von Änderungen im Photosyntheseapparat ohne Anwendung externer Einflüsse dar. Vorausgegangene Untersuchungen legen es nahe, den begrenzenden Faktor für die Photosynthesekapazität im Elektronentransport zwischen PS II und PS I zu suchen. Die Regulation des Elektronentransportes zwischen PS'II und PS I während der Entwicklungszyklen von Scenedesmus und Chlamydomonas ist Gegenstand der vorliegenden Untersuchungen. Messungen der Poolgrößen des Plastochinons und der Cytochrome ergaben während der Entwicklungszyklen größere Differenzen für Chlamydomonas als für Scenedesmus. Jedoch waren die Differenzen nicht groß genug, um die Schwankungen in der Photosynthesekapazität zu erklären. Aus den Messungen konnte indirekt geschlossen werden, daß die Poolgröße des Quenchers Q während der Entwicklungszyklen konstant bleibt. Experimente mit den Photosynthesehemmstoffen DCMU und DBMIB an ganzen Zellen und photosynthetisch aktiven Partikeln führten zu dem Schluß, daß die Reoxidationskapazität von Plastochinon den geschwindigkeitsbestimmenden Schritt und somit die Regulation des nichtzyklischen Elektronentransports darstellt. Die Möglichkeit, daß während der Abnahme des nichtzyklischen Elektronentransports die Kapazität von PS I für zusätzliche zyklische Photophosphorylierung genutzt wird, wird diskutiert. Wir danken Herrn Prof. Dr. A. Trebst für die freundliche Überlassung von DBMIB und der Deutschen Forschungsgemeinschaft für apparative Unterstützung.     

Photosystem II: evolutionary perspectives

Based on the current model of its structure and function, photosystem II (PSII) seems to have evolved from an ancestor that was homodimeric in terms of its protein core and contained a special pair of chlorophylls as the photo-oxidizable cofactor. It is proposed that the key event in the evolution of PSII was a mutation that resulted in the separation of the two pigments that made up the special chlorophyll pair, making them into two chlorophylls that were neither special nor paired. These ordinary chlorophylls, along with the two adjacent monomeric chlorophylls, were very oxidizing: a property proposed to be intrinsic to monomeric chlorophylls in the environment provided by reaction centre (RC) proteins. It seems likely that other (mainly electrostatic) changes in the environments of the pigments probably tuned their redox potentials further but these changes would have been minor compared with the redox jump imposed by splitting of the special pair. This sudden increase in redox potential allowed the development of oxygen evolution. The highly oxidizing homodimeric RC would probably have been not only inefficient in terms of photochemistry and charge storage but also wasteful in terms of protein or pigments undergoing damage due to the oxidative chemistry. These problems would have constituted selective pressures in favour of the lop-sided, heterodimeric system that exists as PSII today, in which the highly oxidized species are limited to only one side of the heterodimer: the sacrificial, rapidly turned-over D1 protein. It is also suggested that one reason for maintaining an oxidizable tyrosine, TyrD, on the D2 side of the RC, is that the proton associated with its tyrosyl radical, has an electrostatic role in confining P(+) to the expendable D1 side.