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How close is the analogy between the reaction centre of Photosystem II and that of purple bacteria?
... The occupation of the three remaining coordination sites by additional endogenous protein residues and/or exogenous ligands (e.g., substrate molecules) tunes to a great extent the properties of the metal center (Hegg and Que, 1997;Que, 2000). The iron of the BRC has perhaps the most inert coordination with 4 His plus a fixed glutamate bidentate ligand (Deisenhofer et al., 1985;Allen et al., 1988) and a high E m (Beijer and Rutherford, 1987;Diner and Petrouleas, 1987b). Based on extensive spectroscopic and sequence homologies (Rutherford, 1987;Michel and Deisenhofer, 1988;reviewed in Diner et al., 1991), the iron of PS II is also assumed to be coordinated by four histidines, two from each of the two protein subunits D1 and D2. ...
... The iron of the BRC has perhaps the most inert coordination with 4 His plus a fixed glutamate bidentate ligand (Deisenhofer et al., 1985;Allen et al., 1988) and a high E m (Beijer and Rutherford, 1987;Diner and Petrouleas, 1987b). Based on extensive spectroscopic and sequence homologies (Rutherford, 1987;Michel and Deisenhofer, 1988;reviewed in Diner et al., 1991), the iron of PS II is also assumed to be coordinated by four histidines, two from each of the two protein subunits D1 and D2. Compared to the BRC, there are important differences in the fifth and sixth coordination positions. ...
The flux of reducing equivalents out of Photosystem II (PS II) occurs through the two-electron gate function catalyzed by
the iron-quinone complex on the acceptor side. The mechanism of the two-electron gate has been studied more completely in
bacterial reaction centers, where an understanding of function has benefited from a structural context. However, the two-electron
gate was discovered in green plants, and a large body of work had suggested that the mechanism and main structural features
are similar in the two systems, and this is now confirmed by structures. In PS II a number of additional properties are found,
which result from the redox activity of the non-heme iron of the acceptor complex, and from the lability of its ligands. Pending
structures for PS II at a higher resolution, much of the discussion on the molecular architecture had borrowed the structural
context from the bacterial homologue. One theme in this chapter is the justification for this borrowing that comes from the
application of spectroscopic approaches to the PS II acceptor complex. This has been especially successful in studies of the
QA-site semiquinone, the magnetic interaction between the semiquinone formed at the site and the iron, and the interaction of
external ligands with the iron. A second theme, reflecting the poor stability of the semiquinone of the QB-site in isolated PS II preparations, is the use of indirect approaches, including kinetic studies and structural modeling,
to understand the structure-function interface. The crystallographic structures now available provide a gratifying validation
of these alternative approaches.
... Such an orientation would not be expected if P680 were a dimer of chlorophyll comparable with the primary donor of the purple bacterial reaction centre. This indicates, as suggested by Rutherford [36], that the triplet signal detected may not be located on P680 but originates from an accessory chlorophyll monomer. Our results indicate that illumination at low temperatures can indeed generate oxidised monomeric chlorophyll. ...
We have characterised the electron-transfer properties of the D1/D2/cytochrome b-559 complex using EPR spectrometry. The complex can transfer electrons to silicomolybdate and ferricyanide at cryogenic temperatures. In the presence of silicomolybdate or ferricyanide, two chlorophyll cation radicals were observed from P680+ (0.8 mT) and monomeric Chl (1.0 mT). Reduction of silicomolybdate was detected as a 2.7 mT signal at g = 1.942. A radical attributed to a tyrosine cation radical (D+/Z+) was also observed in a small percentage of centres.
... Photosystem II (PS II) is a membrane-bound pigmentprotein complex which catalyses the light-dependent oxidation of H 2 O to molecular oxygen and reduction of plastoquinone to plastoquinol (Nugent 1996). The study of transmembrane electron transfer in PS II has been helped significantly by structural (Michel and Deisenhofer 1988;Rhee et al. 1997) and functional (Rutherford 1986) similarities to the purple bacterial reaction center complex (RC) for which structures are available at high resolution (Deisenhofer et al. 1985;Allen et al. 1987;Deisenhofer et al. 1995). ...
Primary charge separation within Photosystem II (PS II) is much slower (time constant 21 ps) than the equivalent step in the related reaction center (RC) found in purple bacteria ( 3 ps). In the case of the bacterial RC, replacement of a specific tyrosine residue within the M subunit (at position 210 in Rhodobacter sphaeroides), by a leucine residue slows down charge separation to 20 ps. Significantly the analogous residue in PS II, within the D2 polypeptide, is a leucine not a tyrosine (at position D2-205, Chlamydomonas reinhardtii numbering). Consequently, it has been postulated [Hastings et al. (1992) Biochemistry 31: 7638–7647] that the rate of electron transfer could be increased in PS II by replacing this leucine residue with tyrosine. We have tested this hypothesis by constructing the D2-Leu205Tyr mutant in the green alga, Chlamydomonas reinhardtii, through transformation of the chloroplast genome. Primary charge separation was examined in isolated PS II RCs by time-resolved optical spectroscopy and was found to occur with a time constant of 40 ps. We conclude that mutation of D2-Leu205 to Tyr does not increase the rate of charge separation in PS II. The slower kinetics of primary charge separation in wild type PS II are probably not due to a specific difference in primary structure compared with the bacterial RC but rather a consequence of the P680 singlet excited state being a shallower trap for excitation energy within the reaction center.
This chapter discusses the composition, organization, and dynamics of the thylakoid membrane in relation to its function. The chapter reviews electron flow in photosynthesis, especially as it relates to membrane structure and function. The lipids of the thylakoid membrane can be divided into those that are saponifiable and those that are not. The saponifiable lipids are the diacylglycerolipids, which make up the matrix of the membrane, while the nonsaponifiable lipids are the various forms of pigments—chlorophylls and carotenoids—and quinones. The pigments are bound within protein complexes, and the quinines—mainly plastoquinone-9—are located in the hydrophobic lipid matrix. Despite the fact that polyunsaturated mono-galactosyldiglyceride (MGDG) is the dominant polar lipid of the thylakoids, there is no evidence that this membrane, under normal conditions, contains nonbilayer structures. Therefore, as for other biological membranes, it can be assumed that the thylakoid polar lipids exist predominantly as a bilayer in vivo.
Bicarbonate removal from the nonheme iron at the acceptor side of photosystem II (PSII) was shown recently to shift the midpoint potential of the primary quinone acceptor QA to a more positive potential and lowers the yield of singlet oxygen (1O2) production. The presence of QA- results in weaker binding of bicarbonate, suggesting a redox-based regulatory and protective mechanism where loss of bicarbonate or exchange of bicarbonate by other small carboxylic acids may protect PSII against 1O2 in vivo under photorespiratory conditions. Here, we compared the properties of QA in the Arabidopsis (Arabidopsis thaliana) photorespiration mutant deficient in peroxisomal HYDROXYPYRUVATE REDUCTASE1 (hpr1-1), which accumulates glycolate in leaves, with the wild type. Photosynthetic electron transport was affected in the mutant, and chlorophyll fluorescence showed slower electron transport between QA and QB in the mutant. Glycolate induced an increase in the temperature maximum of thermoluminescence emission, indicating a shift of the midpoint potential of QA to a more positive value. The yield of 1O2 production was lowered in thylakoid membranes isolated from hpr1-1 compared with the wild type, consistent with a higher potential of QA/QA- In addition, electron donation to photosystem I was affected in hpr1-1 at higher light intensities, consistent with diminished electron transfer out of PSII. This study indicates that replacement of bicarbonate at the nonheme iron by a small carboxylate anion occurs in plants in vivo. These findings suggested that replacement of the bicarbonate on the nonheme iron by glycolate may represent a regulatory mechanism that protects PSII against photooxidative stress under low-CO2 conditions.
Photosystem II (PS II) is responsible for the light-driven redox reactions transfering electrons from water to the plastoquinone pool resulting in the evolution of oxygen (Vermaas and Ikeuchi, 1991; Andersson and Styring, 1991). PS II consists of four major and several smaller (≤ 10,000 Mr) integral membrane proteins and also several peripheral polypeptides. The integral proteins include CP43, CP47, D1, D2, PSII-I, and cytochrome b559. CP43 and CP47 are chlorophyll-binding proteins and act as light-harvesting antenna. D1 and D2 are the two reaction center proteins that together create the binding environment for the reaction center components, for the quinone-type electron acceptors, QA and QB, and presumably for Mn involved in water splitting. The functions of cytochrome b559 and of PS II-I, the product of the psbI gene, are still unknown. The most important peripheral protein is the 33 kDa manganese-stabilizing protein (MSP), also known as PS II-O or OEE-1 with OEE standing for oxygen-evolving enhancer. This 33 kDa protein is located on the lumenal side of the membrane and is thought to be involved in stabilizing the manganese that is necessary for the water-splitting process. However, recent in vivo experiments have shown that in the cyanobacterium Synechocystis sp. PCC 6803 the MSP is not absolutely essential for the stable assembly of reaction centers and for oxygen evolution (Burnap et al., 1991; Philbrick et al., 1991; S. Mayes and J. Barber, personal communication). However, in the alga Chlamydomonas reinhardtii (Mayfield et al., 1987), the 33kDa protein appears to be required for oxygenic photosynthesis and PS II stability.
On the electron donor side of Photosystem II (PSII) there are two components, Z and D, which give rise to free radical EPR signals at g = 2.0046 (for a review, see ref. 1). These components appear to be spectroscopically identical but they have different kinetic properties. Firstly, Z gives rise to an EPR signal when it is oxidized by the photooxidized reaction center chlorophyll, P680+. The EPR signal from Z+ is designated Signal II very fast since Z+ is rapidly reduced by electrons from water via the manganese cluster of the oxygen evolving enzyme. When the manganese cluster is destroyed or inhibited the decay rate of Z+ becomes slower; under these circumstances the EPR signal from Z+ is designated Signal II fast (1). The component Z, then, is usually thought of as a transient electron carrier between P680+ and the Mn of the oxygen evolving enzyme. In contrast, the second of the components, D+, is very stable in the dark and present in virtually all intact PSII-containing material even after many hours of dark adaptation. The EPR signal arising from D+ is designated Signal II slow. Despite its stability it is usually assumed that D/D+ is a rather oxidizing redox couple with an estimated Em of 760 mV (2). Its stability probably arises from its location in a protein site inaccessible to reduction.
Among the many similarities noted between the isolated bacterial and photosystem II (PSII) reaction center (RC) complexes are analogous electron transport components [1], protein sequence homologies [2], and charge separation rates [3]. On the other hand, there are some striking differences. For example, the PSII RC contains Mn-binding sites not reported for the bacterial RC which is consistent with the O2-evolution role of PSII [4], the triplet state of chlorophyll (Chl) in the PSII RC is monomeric and oriented perpendicular to the analogous bacteriochlorophyll (BChl) triplet observed in the bacterial RC [5], and the excited singlet state lifetime of P680 (the primary donor in PSII) is much longer than that reported for P870 (the primary donor in the RC of purple bacteria) [3]. Furthermore, whereas P870 is red-shifted up to 100 nm compared to the other porphyrins in the bacterial RC, P680 is red-shifted about 10 nm, and there is significant spectral overlap between all of the PSII pigments in red region. Consequently, the purpose of this paper is to further examine the spectral properties of stabilized PSII RC complex with emphasis on the nature of the primary donor.
Although there have been numerous studies to understand the role of cytochrome b-559 in photosynthesis, no clear picture has yet emerged. The study of this cytochrome is complicated by the fact that there seems to be two distinctly different pools. One pool consists of a low potential form (E~ −30 mV) and is located in the unappressed stromal regions of higher plant chloroplasts (1). This low potential form may be in some way linked to electron and proton translocation through the cytochrome b6-f complex (2). The other pool is localized in the appressed regions which constitute the grana (3,4) and normally seems to exist in a high potential form (E~ 380 mV). In the earlier literature there was some confusion about the redox properties of the high potential form because its midpoint potential can be significantly modified after certain treatments which affect membrane structure especially by the action of detergents (5,6). In this case the Em changes from 380 mV to about 80 mV. Apparently this shift is due to a perturbation of the environment around the cytochrome and can be reversed by adding back polar lipids derived from the thylakoid, particularly digalactosyldiacylglycerol (7,8). Reconstitution of a certain extrinsic polypeptide (23 kDa) associated with water oxidation, has also been reported to revert the cytochrome to its high potential form (9).
In 1987 Nanba and Satoh (1) reported the isolation of a pigment protein complex which was capable of photochemical charge separation and contained the D1 and D2 proteins but not the other chlorophyll binding polypeptides of photosystem two (PS2). This gave the first firm experimental support to the suggestion that the D1 and D2 polypeptides are the two essential elements of the PS2 reaction centre and confirmed that D1 and D2 are analogous to the L and M subunits of the reaction centre of purple bacteria (2–5). It was then possible to obtain a good idea of the structure of the PS2 reaction centre by reference to the detailed knowledge of the bacterial complex derived by X-ray crystallographic studies (6,7). The isolated D1–D2 complex has provided the experimental opportunities not only to establish reaction centre structure and study photochemical charge separation but also to determine how the reaction centre interacts with other thylakoid components to give the full water-plastoquinone oxido-reductase activity of the complete PS2.
The experimental data available suggested the monomeric nature of the cation radicals and triplets of P680 and P700, the primary electron donors in plant photosynthesis1. Interactions of monomeric Chlorophyll a /Chl.a/ electrostatics /for a review, see2 /, are proposed as a factors, responsible for the observed in vivo behavior of these complexes. Chl.a-water interactions have been used for modelling of P680 and P700 /for a review, see3 /. Recent works4,5, have demonstrated that polar solvents can be used for modelling of protein environment and four types of specific solvation, depending on solvents’ empirical parameters /electrophilicity, nucleophilicity and steric factors/, have been distingished. Thus, bisligation and hydrogen-bonding of Chl.a in the case of methanol and only bisligation in the case of pyridine have been shown4.
Photosystem II reaction centers (RC) consist of D1 and D2 polypeptides and a bound cytochrome b 559. Isolated RC, named D1/D2 particles, contain 4–6 Chl a, 1 β-carotene and 1 or 2 cytochrome b 559 per two pheophytin a molecules (1). Polypeptides D1 and D2 exhibit marked homologies with the L and M subunits of the RC of purple bacteria, respectively (2). PS II shares a number of functional similarities with bacterial RCs. In these particles, absorption of a photon results in a rapid charge separation between the primary electron donor, P680, and a Pheo molecule; because of the lack of a secondary electron acceptor, relaxation of the D1/D2 reaction centers occurs through the formation of a 900 µs-lived triplet state involving Chl a molecule(s), named PR (3).
Photosystem II (PS II) is a pigment-protein complex in thylakoid membranes from all oxygenic photosynthetic organisms (cyanobacteria and photosynthetic eukaryotes). It catalyzes the light-induced reduction of plastoquinone by water through a number of redox reactions. The electron transport chain in PS II is composed of various protein-bound components, which are held in close proximity and suitable orientation with respect to each other by the protein environment, so that a rapid and efficient electron transport is feasible. The PS II complex consists of at least five integral membrane proteins in the thylakoid (together forming the PS II “core complex”), in addition to several peripheral proteins. Many of these polypeptides interact with one or more components of the electron transport chain or with light-harvesting pigments, such that these protein ligands can fulfill a specific function in PS 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.
A photosystem II core from spinach containing the chlorophyll-binding proteins 47 kDa, 43 kDa, the reaction center proteins D1, D2 and cytochromeb
559
and three low molecular weight polypeptides (MW < 10 kDa) was isolated, its three-dimensional crystals were prepared, and both core and crystals were studied by spectroscopic techniques and electron microscopy. The absorption spectra of the crystallized form of the core indicate a specific orientation of the various pigments within the crystal.
A recent report (Nanba O, Satoh K: Proc. Natl. Acad. Sci. USA 84: 109-112, 1987) described the isolation from spinach of a putative photosystem 2 reaction centre which contained cytochrome b-559 and three other electrophoretically resolvable polypeptide bands, two of which have molecular weights comparable to the D1 and D2 polypeptides. We have used in vivo labelling with radioactive methionine and probed with D1 and D2 monospecific antibodies (raised against synthetically expressed sequences of the psbA and psbD genes) for specific detection of these proteins in a similarly prepared photosystem 2 reaction centre preparation. These techniques identified a 32 000 dalton D1 band, a 30 000 dalton D2 band and a 55 000 dalton D1/D2 aggregate, the latter apparently arising from the detergent treatments employed. Digestions with a lysine-specific protease further confirmed the identification of the lysine-free D1 polypeptide and also confirmed that the D1 molecules in the 55 000 dalton band were in aggregation with other bands and not in self-aggregates. The D1 and D2 polypeptides (including the aggregate) are considerably enriched in the photosystem two reaction centre preparation compared to the other resolved fractions.
A hypothetical model for the structure and function of photosystem II is proposed that attempts to incorporate different phenomena related to the variable chlorophyll fluorescence inherent in this photosystem. The involvement of pheophytin redox chemistry on both the acceptor and donor side of photosystem II is postulated to achieve redox potentials high enough to oxidize water The presence of this symmetry would be the cause of inefficient photochemistry in photosystem II when, under unbalanced carbon metabolism. a surplus charge remains on the reaction centre. In addition, such a scheme would enable an efficient dissipation of surplus energy in the reaction centre itself, and would be the origin of the energy-dependent quenching of chlorophyll fluorescence. q(E).
. The structure of chloroplast membrane proteins and their organization into photosynthetically-active multimeric complexes is described. Extensive use has been made of information derived from gene sequencing and other biochemical studies to predict likely protein conformations. These predictions have been assimilated into structural models of the various thylakoid complexes. The enzymatic activities of the complexes have also been described and where possible related to individual polypeptides.
In order to determine the origin of the photoexcited pheophytin triplet state (3Phe) in plant Photosystem II preparations ( complexes) an absorption and fluorescence detected magnetic resonance (ADMR, FDMR) study was conducted in zero magnetic field at low temperatures. The ADMR signal intensity dependence on excitation light flux was linear showing that 3Phe is formed by a 1-photon process. Upon successive exposure of the sample to strong white light it was shown that 3Phe is not directly correlated with the primary donor triplet state 3P680. This is consistent with the absence of a double resonance signal connecting 3Phe with 3P680 down to an instrumental sensitivity of ) below 10−6. An intermediate state of photoinhibition between intact and fully degraded reaction centres is responsible for the formation of 3Phe. It may be a conformationally changed state of the reaction centre protein, most likely having a larger distance between P680 and the pheophytin whose triplet state is observed. Factor analysis of the absorption spectra of a series of gradually degraded samples yielded two spectral components which were interpreted as the spectra of fully intact and completely degraded reaction centres.
Transient absorption changes in photosystem II reaction centres have been measured using silicomolybdate as electron acceptor. Comparison of changes at 740 nm and 820 nm, in the presence and absence of oxygen, clearly distinguish two decay components due to 3P680 and a long-lived charge separated state. The lifetime of the latter was found to be dependent on silicomolybdate concentration. Absorption difference spectra of the charge-stabilised state indicate a maximum absorption decrease at 680 nm with a shoulder at 670 nm. There is no clearly detectable difference in the decay kinetics of the long-lived component at 670 nm and 680 nm although this point is under further investigation. We cannot conclusively determine whether this shoulder is an integral part of the ground state absorption spectrum of P680 or is due to a second chlorophyll species, but evidence for these two theories is discussed.
In oxygen-evolving organisms, two sequential light-driven reactions occur in two distinct membrane-bound chlorophyll-protein complexes, namely PS2 and PS1. In purple bacteria (Rhodospirillales), use of the energy of light requires only one photoreaction, which occurs in the membrane-bound bacterial reaction center (RC). The bacterial RC shares a number of common functional properties with PS2 (1). Moreover, it has been shown that a highly significant degree of sequence homology exists between the two membrane-embedded subunits of the bacterial RCs (L and M) and the two subunits of the PS2 which are the location of the primary electron transfers, namely D1 and D2 (2). Unlike PS2, PS1 has not benefited from the analogy to the bacterial reaction center and many questions concerning its structure remain unresolved. Nevertheless, the sequence of the polypeptides, psa A and psa B, thought to carry the first electron carriers in PS1 was obtained for maize in 1985 (3).
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Using affinity chromatography with the extrinsic 33 kDa protein as the immobilised ligand, it was demonstrated that the reaction centre complex of photosystem II, composed of the D1, D2 and cytochrome b-559 polypeptides, can directly interact with the 33 kDa protein. By this approach it was possible to purify the reaction centre from solubilised photosystem II core complexes since neither the 47 kDa nor the 43 kDa protein would bind to the ligand.
D1/D2-cyt b559 complexes from Pisum sativum were investigated by absorption detected magnetic resonance (ADMR) in zero field. Two different triplet states were detected and further characterized by temperature dependent and time resolved ADMR, microwave holeburning studies, and the magnetic field dependence of the ADMR signals. Their microwave induced absorption (MIA) spectra showed maxima at 683 and 680 nm. They were identified as the triplet states of the primary electron donor P-3(680) and of a pheophytin a molecule possibly connected with partially degraded D1/D2-complexes. From comparison with bacterial reaction centers (RC) it can be concluded that the triplet state of the primary donor in plant photosystem II (P-3(680)) is located on a monomeric chlorophyll a molecule.
An excitonic pentamer model (an adaptation of the multimer model; Durrant et al. Proc. Natl. Acad. Sci. U.S.A. 1995, 92, 4798) is proposed for the core Qy states of the photosystem II reaction center (PSII RC). The core chlorins consist of four chlorophyll a molecules (P1, P2, Chl1, Chl2) and two pheophytin a molecules (Pheo1, Pheo2). In the pentamer model Pheo2 on the inactive D2 branch is, for all intents and purposes, decoupled from the other five chlorins. This model is the result of theoretical simulations of several types of spectra obtained at liquid helium temperatures in the Qy region and the Pheo Qx region of the absorption spectrum. They include bleaching spectra obtained by reduction of Pheo2 with dithionite (in the dark) and reduction of the active Pheo1 with dithionite and white light illumination, triplet bottleneck hole spectra, and femtosecond pump−probe spectra (from S. R. Greenfield et al. J. Phys. Chem. B 1999, 103, 8364). The model structure of Svensson et al. of PSII RC (Biochemistry 1996, 35, 14486) and the recent X-ray RC structure of Zouni et al. (Nature 2001, 409, 739) were used to construct hexamer excitonic Hamiltonians. Both Hamiltonians, with uncorrelated site excitation energy disorder taken into account, yield similar results and acceptable fits to the spectra but only if Pheo2 is decoupled. Such decoupling would require a significant weakening of the Pheo2-Chl2 interaction predicted by the RC structures. Possible reasons for weakening are given. Our findings include the following: (1) The localized Qx/Qy transitions of Pheo2 are at 541.2 and 668.3 nm with absorption bandwidths of 200 cm-1. (2) The Qx transition of Pheo1 is at 544.4 nm with an absorption bandwidth of 200 cm-1. (3) Within the pentamer model four of the five Qy states are delocalized over both the D1 and D2 branches. The delocalization results in significant narrowing (40%) of inhomogeneous spectral broadening that stems from the width of the site (chlorin) excitation energy distribution functions. (4) The contributions of P1 and P2 to the lowest energy (primary donor, P680*) state are, on average, the largest although the contributions from the other three chlorins are significant. (5) The triplet state associated with the bottleneck spectrum appears to be localized on Chl1 (or P2). (6) The combined absorption dipole strength of Pheo1 associated with the two lowest energy and strongly absorbing states (separated by only 80 cm-1) is equivalent to that of 1.8 monomer Pheo molecules. This finding provides a plausible explanation for the results of Greenfield et al. The paper ends with discussion of the nature of P680* and the triplet state(s) formed by charge recombination of the primary radical ion pair.
High-level quantum chemical methods (hybrid density-functional type) are applied to the light-driven charge-separation process in the photosynthetic reaction centers of both bacteria and photosystem II in green plants. Structural information on the bacterial system provides the basis for choosing the models used in the calculations. The energetics of the electron transfer from the (bacterio)chlorophyll to the quinone are calculated as well as those of the intermediate step involving the (bacterio)pheophytin. The surrounding protein is treated as a dielectric medium, and the cavities around the solute molecules are determined by isodensity surfaces. The dielectric effects on the charge-separation processes are calculated to be as large as 50 kcal/mol. It is shown that hydrogen bonding between the chromophores and certain peptide residues as well as the axial histidine ligand on the (bacterio)chlorophylls contributes substantially to the energetics. Good agreement with experimentally estimated driving forces of the different steps is obtained within 2 kcal/mol for the bacteriosystem and within 8 kcal/mol for photosystem II. The results for photosystem II have a lower accuracy, as expected, due to the lack of detailed structural information on this system. Recent low-resolution data indicating a weaker coupling between the chlorophylls in P680 as compared to that of the special pair in the bacterial systems are taken into account in the calculations. In the bacterial system, charge separation to the accessory bacteriochlorophyll is essentially thermoneutral and the P865+BPheo- state is stable by 7.5 kcal/mol. In photosystem II, charge separation to the P680+Pheo- state is much less strongly driven, and the absence of an axial histidine ligand to the accessory chlorophyll appears necessary to allow its formation. The creation of the tyrosine radical (YZ) by proton-coupled electron transfer to the photoionized reaction center chlorophyll in photosystem II is also studied. In this case as well, hydrogen bonding to other peptide residues plays an important role in the overall energetic balance of the reaction.
The iron quinone-complex of the reaction centers of photosystem II and the purple non-sulphur photosynthetic bacteria contains two quinones, QA and QB connected in series with respect to electron transfer, and separated by a non-heme iron coordinated by amino acid residues. It is the site of inhibition of many of the common photosynthetic herbicides, which act by displacing QB from the center. The complex is responsible for reducing QB to QBH2 in two successive one-electron photo acts. OBH2 dissociates from the center, to be replaced by a new QB molecule and reduces the following membrane-bound electron-transfer complex (cytochrome b6/for b/c1). The energetic, kinetics and mechanism of complex function are reviewed here. Recent crystallographic, spectroscopic and molecular biological evidence has produced a considerable quantity of structural information on this complex. These data have given a less formal and more molecular view of how the complex functions. They have also revealed fundamental differences between the photo system II and bacterial complexes, particularly with respect to the coordination of the iron and its chemistry. The comparative anatomy of the complexes is reviewed and its implications for function discussed.
Absorption changes at 830 nm, ΔA830, induced by repetitive (1 Hz) strong actinic 35 ps laser pulses (λ = 532 nm; E = 4 · 1015 photons / cm2 per pulse) were detected in oxygen-evolving Photosystem (PS) II membrane fragments with a time resolution of about 500 ps. The following results were obtained: (a) in the presence of 2 mM K3(Fe(CN)6) the relaxation of ΔA830 is dominated by the multiphasic P-680+-reduction kinetics (ns and μs range) and the fast I⨪ reoxidation, (b) if the reaction centers are kept largely in the reduced state P-680IQ⨪A, the initial amplitude of ΔA830 remains almost unaffected, but the decay mainly occurs via a kinetics of at most 500 ps, (c) if the reaction centers are transformed into the state P-680I⨪Q⨪A by strong illumination in the presence of Na2S2O4, then the initial amplitude of ΔA830 becomes drastically diminished, (d) after a few cycles of strong continuous actinic light and dark recovery (4 min) ΔA830 is dominated by a 3 ns relaxation kinetics. Comparative measurements of the fluorescence decay after excitation with weak 620 nm pulses (E = 2 · 1010 photons / cm2 per pulse) exhibited the following dependencies of the kinetics of the redox state of the reaction center: up to 200 ps kinetics dominate for the states P-680IQA and for P-680I⨪Q⨪A, whereas for the state P-680IQ⨪A the fluorescence relaxes predominantly via 1.3 ns kinetics. The analysis of these results led to the following conclusions. (i) The PS II reaction center provides a rather shallow trap for excitons. (ii) The fluorescence emission in the state P-680IQ⨪a is probably not a recombination fast delayed light emission but represents a prompt fluorescence of the antenna. (iii) If the yield of flash-induced radical pair formation in reaction centers of the state P-680IQ⨪A is significant (at least 25%), then the recombination reaction P-680+I•Q•A → P-680IQ•A takes place with .
Similarities in the piginent and subunit composition and sequence homologies suggest a common evolutionary origin for the reaction centers (RCs) of photosynthetic purple bacteria and photosystem II (PS II) of green plants [1]. Besides these similarities, significant functional differences exist that make comparative studies between both types of RCs interesting. We report here (i) on the electric detection of two kinetically distinct phases of the primary charge separation in PS II with 100 and 500 ps reflecting trapping and charge stabilization, and (ii) the determination of the dielectrically weighted transmembrane distances between the primary donor (P), the pheophytin intermediary acceptor (I), and the first quinoic acceptor (QA). These data are compared with those for purple bacteria. In PS II the reduction of QA is 2–3-times slower, although the dielectric distance between I and QA appears to be significantly shorter than in purple bacteria.
Experimental evidence for a new model of the structure of the photosystem two reaction centre of oxygenic organisms is rapidly accumulating and is being extended to incorporate the organizational relationships between it and the components involved in the splitting of water to molecular oxygen.
In the last few years various advances have contributed to an increased understanding of Photosystem II (PS II). Most notably, the X-ray diffraction analysis of crystallized bacterial reaction centers, along with the recognition that there is functional and structural homology between the bacterial reaction center and PS II, has led to detailed information regarding the potential function of individual proteins and residues in the PS II complex. In-depth studies of PS II structure and function, however, require the availability of specific mutants in which certain proteins have been altered. Recombinant DNA technology has provided the methodology by which generation of such mutants has become feasible. This minireview focuses on methods for mutagenesis of PS II components and on the impact of mutant analysis on the understanding of PS II structure and function.
Abstract—Spruce needles collected from several trees of the Black Forest were investigaled by EPR spectroscopy. These needles show in the g = 2.00 region a signal IIS(Tyr D +) and a light-induced signal I(P700+) and a Mn2+ hyperfine structure which superimposes the other absorptions. Difference spectra, light minus dark, partly eliminate the manganese hyperfine structure, and P700+ can be observed. By comparison of these EPR signals with those of spinach chloroplast or thylakoid membranes described in the literature, significant deviations were observed, whereas several trees grown in the vicinity of Tubingen exhibit the well known D+ and P700+ EPR spectra. After treatment of branches of these ‘normal’ trees with herbicides like Amitrol and Roundup or chemicals like toluene or trichlormethane the EPR signals obtained are comparable with those observed with needles of the Black Forest.
The photosystem II (PSII) complex catalyzes the crucial and highly labile photosynthetic water-splitting reaction that uses
solar energy to reduce plastoquinone using electrons derived from water. This chapter presents a brief summary of the structure,
function, and damage repair cycle of the PSII complex. A bioinformatic analysis of the PSII proteins, utilizing the amino
acid sequence conservation patterns projected onto the three dimensional structure is presented. The analysis illustrates
structural domains of the PSII complex where amino acid substitutions appear to have been subjected to selective constraint
and areas that have had freedom to mutate. In addition to the expected conservation at known functional sites within the proteins,
the analysis shows conserved surface features that likely form the interfaces with other proteins, such as antenna complexes,
that are not present in the x-ray crystal structure. This chapter also discusses how patterns of structural divergence can
be attributed to functional variations that have been selected on the basis of different environmental regimes, such as high
versus low light intensity conditions. Finally, a model of extrinsic protein evolution involving the duplication and divergence
of assembly factor proteins is proposed to account for the phyletic variations in the protein composition surrounding the
manganese cluster of the H2O-oxidation domain of PSII. In this model, PsbP is proposed to spatially replace PsbV, whereas PsbQ is proposed to bind to
the outward-facing surface of the e-loop of CP43.
Many herbicides inhibit the photosynthetic electron transfer in photosystem II by binding to the polypeptide D1. A point mutation in the chloroplast gene psbA, which leads to a change of the amino acid residue 264 of D1 from serine to glycine, is responsible for atrazine resistance in higher plants. We have changed serine 264 to glycine in Synechococcus PCC7942 and compared its phenotype to a mutant with a serine to alanine shift in the same position. The results show that glycine at position 264 in D1 gives rise to a similar phenotype in cyanobacteria and in higher plants, indicating a similar structure of the binding site for herbicides and for the quinone QB in the two systems. A possible mode of binding of phenyl-urea herbicides to D1 is predicted from the difference in herbicidal cross-resistance between glycine and alanine substitutions of serine 264.
Inhibition of electron transport and damage to the protein subunits by visible light has been studied in isolated reaction
centers of the non-sulfur purple bacterium Rhodobacter sphaeroides. Illumination by 1100 μEm−2 s−1 light induced only a slight effect in wild type, carotenoid containing 2.4.1. reaction centers. In contrast, illumination
of reaction centers isolated from the carotenoidless R26 strain resulted in the inhibition of charge separation as detected
by the loss of the initial amplitude of absorbance change at 430 nm arising from the P+QB
− → PQB recombination. In addition to this effect, the L, M and H protein subunits of the R26 reaction center were damaged as shown
by their loss on Coomassie stained gels, which was however not accompanied by specific degradation products. Both the loss
of photochemical activity and of protein subunits were suppressed in the absence of oxygen. By applying EPR spin trapping
with 2,2,6,6-tetramethylpiperidine we could detect light-induced generation of singlet oxygen in the R26, but not in the 2.4.1.
reaction centers. Moreover, artificial generation of singlet oxygen, also led to the loss of the L, M and H subunits. Our
results provide evidence for the common hypothesis that strong illumination by visible light damages the carotenoidless reaction
center via formation of singlet oxygen. This mechanism most likely proceeds through the interaction of the triplet state of
reaction center chlorophyll with the ground state triplet oxygen in a similar way as occurs in Photosystem II.
Transient absorption spectroscopy has been used to study the isolated D1/D2/cytochrome b-559 reaction centre complex at 4°C. The D1/D2 reaction centre is observed to have an increased susceptibility to photodamage under aerobic conditions. This is attributed to oxygen quenching of a P680 triplet state, which results in the formation of highly oxidising singlet oxygen. This P680 triplet state is observed to have a lifetime of (1.0±0.1) ms under anaerobic conditions, shortening to (33±3) μs in the presence of oxygen. This state, which has a quantum yield of approx. 30%, is identified as residing upon the primary electron donor P680 by the transient bleaching of its reddest absorption band, which peaks at (680.5±0.5) nm. The shape of the P680 triplet-minus-singlet absorption difference spectrum, and particularly the (12±1) nm bandwidth of the red absorption band bleach, indicate that P680 is probably a pair of excitonically coupled chlorophyll molecules, with the P680 triplet state being localised upon one of these chlorophyll molecules. The red absorption band bleached by P680 triplet formation has a peak extinction coefficient of 133000 M−1 · cm−1 and an oscillator strength 1.1-times larger than that of the Qy-band of a chlorophyll a monomer in ether. It is shown that this P680 triplet state is formed primarily by charge recombination from the primary radical pair state at 4°C. A 3% quantum yield of a carotenoid triple state characterised by an absorption peak at 526 nm is also observed. The observed P680 triplet does not appear to be quenched by this carotenoid.
A photosystem II reaction centre has been isolated from peas and found to consist of D1, D2 polypeptides and the apoproteins of cytochrome b-559, being similar to that reported for spinach by Nanba and Satoh [(1987) Proc. Natl. Acad. Sci. USA 84, 109–112]. The complex binds chlorophyll a, pheophytin and the haem of cytochrome b-559 in an approximate ratio of 4:2:1 and also contains about one molecule of β-carotene. It binds no plastoquinone-9 or manganese but does contain at least one non-haem iron. In addition to a light-induced signal due to Pheo− seen under reducing conditions, a light-induced P680+ signal is seen when the reaction centre is incubated with silicomolybdate. In the presence of diphenylcarbazide, the P680+ signal is partially inhibited and net electron flow to silicomolybdate occurs. This net electron flow is insensitive to o-phenanthroline, 3-(3,4-dichlorophenyl)-1,1-dimethyl urea and 2-(3-chloro-4-trifluoromethyl)anilino-3,5-dinitrothiophene but is inhibited by proteolysis with trypsin and by other treatments. Fluorescence, from the complex, peaks at 682 nm at room temperature and at 685 nm at 77 K. This emission is significantly quenched when either the P680+Pheo or P680Pheo− states are established indicating that the fluorescence emanates from the back reaction between P680+ and Pheo−.
Light-induced Fourier transform infrared (FTIR) difference spectroscopy has been applied for the first time to primary reactions in green plant photosynthesis. Photooxidation of the primary electron donor (P700) in photosystem I-enriched particles as well as in thylakoids, and photoreduction of the pheophytin (Pheo) intermediary electron acceptor in photosystem II-enriched particles, have led to reproducible difference spectra. In the spectral range investigated (between 1800 and 1000 cm−1) several bands are tentatively attributed to changes in intensity and position of the keto and ester carbonyl vibrations of the chlorophyll or Pheo molecule(s) involved. For some of these groups, possible interpretations in terms of changes of their environment or type of bonding to the protein are given. The intensity of the differential features in the amide I and amide II spectral region allows the exclusion of the eventuality of large protein conformational changes occurring upon primary charge separation.
Absorbance difference spectroscopy has been used to study electron transfer reactions at low temperature in isolated Photosystem II complexes from Synechococcus, when the first quinone acceptor is in the oxidized form. (1) The flash-induced absorbance difference spectrum attributed to the formation of P680+QA− has been measured at 77 K between 300 nm and 900 nm. The The difference spectrum in the QY region exhibits a marked temperature dependence. At 77 K the spectrum includes the main bleaching band at 675 nm, an absorbance increase at 681 nm, a smaller bleaching at 686 nm and a positive band at around 667 nm. The width of the main bleaching band is only ≈ 6 nm compared to about 15 nm of the 680 nm band at room temperature. It is proposed that a strong electrochromic red shift of an absorption band dominates the shape of the spectrum giving rise to an absorbance decrease at 675 nm and an absorbance increase at 681 nm. (2) While multiphasic decay kinetics of the secondary radical pair, P680+QA−, are found at room temperature, the decay becomes close to mono-exponential with an apparent half-life of 3 ms below 200 K. However, with a signal to noise ratio ≥ 50, two components with half-lives of ≈ 1.8 and ≈ 5 ms could clearly be resolved at 77 K. This biphasicity is attributed to frozen conformational states with a slightly different distance between P680 and QA. (3) In agreement with earlier work in the literature performed with PS II preparations of higher plants, we identified in PS II of Synechococcus three secondary donors oxidized with low quantum yield at 77 K: Cyt b-559, a carotenoid and a chlorophyll a characterized by a bleaching at 667 nm. Between 650 and 700 nm the light-induced absorbance difference spectra due to the oxidition of the secondary donor(s) and the reduction of QA exhibit also strong electrochromic band shifts. Predominant is a red-shift of an absorption band similar to that proposed to be present in the difference spectrum. (4) On the basis of kinetic data, regarding all three secondary donors, we conclude that Car and Cyt b-559 donate electrons to P680+ in parallel pathways. As the rise of Car + formation is faster than the decay of P680+QA−, it is proposed that Car+ is rapidly rereduced by Chl down to an equilibrium level.
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- W O L F G A N G Weiss G E R N O T Renger
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40x
Functional and structural aspects of photosynthetic water oxidation
G E R N O T RENGER and W O L F G A N G WEISS
Mu.\.-Vr)lt?icir-lti.stititt,fur Bioph?wikuli.~~,~ii~ und
Pli~~.vikrrli.sc~h~~ Cliiwiic, Strape des I7 Juni 135,