Structure model of core proteins in photosystem I inferred from the comparison with those in photosystem II and bacteria; an application of principal component analysis to detect the similar regions between distantly related families of proteins.

A principal component analysis based on the physico-chemical properties of amino acid residues is developed to assign similar regions between distantly related families of proteins, taking account of the species diversities in respective families. The most important advantage of this analysis should be that it reflects different physico-chemical properties and thus can predict more detailed structural properties, including the transmembrane helices, than the hydropathy analysis. Its first application reconfirms the similarity between the core proteins of photosynthetic reaction center in purple bacteria and those of photosystem II, indicating that the low percentage of identical amino acid residues estimated previously between them is due to much allowance for amino acid substitutions in purple bacteria. The application of this analysis to the core proteins of photosystem I reveals that any of these proteins includes two domains, each showing high similarity to the amino acid sequences of core proteins in photosystem II and purple bacteria. A core structure model of A1 and A2 proteins folded into four layers of sheets of transmembrane helices is proposed to provide a molecular basis for the electron pathway suggested by spectroscopic experiments as well as for the interaction sites with plastocyanin, 9 kDa protein and LHC proteins.     

The evolutionary pathway from anoxygenic to oxygenic photosynthesis examined by comparison of the properties of photosystem II and bacterial reaction centers

In photosynthetic organisms, such as purple bacteria, cyanobacteria, and plants, light is captured and converted into energy to create energy-rich compounds. The primary process of energy conversion involves the transfer of electrons from an excited donor molecule to a series of electron acceptors in pigment–protein complexes. Two of these complexes, the bacterial reaction center and photosystem II, are evolutionarily related and structurally similar. However, only photosystem II is capable of performing the unique reaction of water oxidation. An understanding of the evolutionary process that lead to the development of oxygenic photosynthesis can be found by comparison of these two complexes. In this review, we summarize how insight is being gained by examination of the differences in critical functional properties of these complexes and by experimental efforts to alter pigment–protein interactions of the bacterial reaction center in order to enable it to perform reactions, such as amino acid and metal oxidation, observable in photosystem II.     

Nobel lecture. The photosynthetic reaction centre from the purple bacterium Rhodopseudomonas viridis.

In our lectures we first describe the history and methods of membrane protein crystallization, before we show how the structure of the photosynthetic reaction centre from the purple bacterium Rhodopseudomonas viridis was solved. Then the structure of this membrane protein complex is correlated with its function as a light-driven electron pump across the photosynthetic membrane. Finally we draw conclusions on the structure of the photosystem II reaction centre from plants and discuss the aspects of membrane protein structure. Sections 1 (crystallization), 4 (conclusions on the structure of photosystem II reaction centre and evolutionary aspects) and 5 (aspects of membrane protein structure) were presented and written by H.M., Sections 2 (determination of the structure) and 3 (structure and function) by J.D. We have arranged the paper in this way in order to facilitate continuous reading.     

Protein phosphorylation and control of excitation energy transfer in photosynthetic purple bacteria and cyanobacteria

The function of phosphorylation of light-harvesting polypeptides is well characterised in chloroplasts of green plants, but the prokaryotic cyanobacteria and purple photosynthetic bacteria have quite different light-harvesting polypeptides whose structure and function cannot be controlled in precisely the same way. Nevertheless, cyanobacteria show light-dependent phosphorylation of membrane polypeptides associated with photosystem II and with the light-harvesting phycobilisome, and purple bacteria show light-dependent phosphorylation of low molecular-weight chromatophore membrane polypeptides. In both cases membrane protein phosphorylation is associated with functional changes observed by chlorophyll fluorescence spectroscopy or chlorophyll fluorescence induction kinetics. Here we report on our recent protein sequence and other data concerning the identities of these phosphoproteins. We also discuss the significance of these findings for regulation by protein phosphorylation of photosynthesis in prokaryotes.     

Spectral hole burning: examples from photosynthesis

The optical spectra of photosynthetic pigment–protein complexes usually show broad absorption bands, often consisting of a number of overlapping, ‘hidden’ bands belonging to different species. Spectral hole burning is an ideal technique to unravel the optical and dynamic properties of such hidden species. Here, the principles of spectral hole burning (HB) and the experimental set-up used in its continuous wave (CW) and time-resolved versions are described. Examples from photosynthesis studied with hole burning, obtained in our laboratory, are then presented. These examples have been classified into three groups according to the parameters that were measured: (1) hole widths as a function of temperature, (2) hole widths as a function of delay time and (3) hole depths as a function of wavelength. Two examples from light-harvesting (LH) 2 complexes of purple bacteria are given within the first group: (a) the determination of energy-transfer times from the chromophores in the B800 ring to the B850 ring, and (b) optical dephasing in the B850 absorption band. One example from photosystem II (PSII) sub-core complexes of higher plants is given within the second group: it shows that the size of the complex determines the amount of spectral diffusion measured. Within the third group, two examples from (green) plants and purple bacteria have been chosen for: (a) the identification of ‘traps’ for energy transfer in PSII sub-core complexes of green plants, and (b) the uncovering of the lowest k = 0 exciton-state distribution within the B850 band of LH2 complexes of purple bacteria. The results prove the potential of spectral hole burning measurements for getting quantitative insight into dynamic processes in photosynthetic systems at low temperature, in particular, when individual bands are hidden within broad absorption bands. Because of its high-resolution wavelength selectivity, HB is a technique that is complementary to ultrafast pump–probe methods. In this review, we have provided an extensive bibliography for the benefit of scientists who plan to make use of this valuable technique in their future research.     

Photosynthetic and phylogenetic primers for detection of anoxygenic phototrophs in natural environments.

Primer sets were designed to target specific 16S ribosomal DNA (rDNA) sequences of photosynthetic bacteria, including the green sulfur bacteria, the green nonsulfur bacteria, and the members of the Heliobacteriaceae (a gram-positive phylum). Due to the phylogenetic diversity of purple sulfur and purple nonsulfur phototrophs, the 16S rDNA gene was not an appropriate target for phylogenetic rDNA primers. Thus, a primer set was designed that targets the pufM gene, encoding the M subunit of the photosynthetic reaction center, which is universally distributed among purple phototrophic bacteria. The pufM primer set amplified DNAs not only from purple sulfur and purple nonsulfur phototrophs but also from Chloroflexus species, which also produce a reaction center like that of the purple bacteria. Although the purple bacterial reaction center structurally resembles green plant photosystem II, the pufM primers did not amplify cyanobacterial DNA, further indicating their specificity for purple anoxyphototrophs. This combination of phylogenetic- and photosynthesis-specific primers covers all groups of known anoxygenic phototrophs and as such shows promise as a molecular tool for the rapid assessment of natural samples in ecological studies of these organisms.     

A general model for regulation of photosynthetic unit function by protein phosphorylation

We propose that regulatory effects of membrane protein phosphorylation in photosynthetic systems result in all cases from simultaneous phosphorylation by a single kinase of the polypeptides of two intrinsic pigment-protein complexes, with phosphorylation leading to their mutual electrostatic repulsion in a direction parallel to the membrane plane and therefore to decreased excitation energy transfer between them. One complex is a peripheral light-harvesting complex and the other is bound to the reaction centre and functions as a link in excitation energy transfer. Immediate effects of phosphorylation are therefore decreased absorption cross-section together with decreased cooperativity of photosynthetic units. This general model applies equally to photosystem II of green plants, algae and cyanobacteria, as well as to the single photosystem of purple bacteria. Special cases of this general model permit increased excitation energy transfer to one type of reaction centre at the expense of another, and this may occur even in laterally homogeneous membranes that are uniformly unappressed.     

Structure of donor side components in photosystem II predicted by computer modelling.

Thirty-one and eleven sequences for the photosystem II reaction centre proteins D1 and D2 respectively, were compared to identify conserved single amino acid residues and regions in the sequences. Both proteins are highly conserved. One important difference is that the lumenal parts of the D1 protein are more conserved than the corresponding parts in the D2 protein. The three-dimensional structures around the electron donors tyrosineZ and tyrosineD on the oxidizing side of photosystem II have been predicted by computer modelling using the photosynthetic reaction centre from purple bacteria as a framework. In the model the tyrosines occupy two cavities close to the lumenal surface of the membrane. They are symmetrically arranged around the primary donor P680 and the distances between the centre of the tyrosines and the closest Mg ion in P680 are around 14 A. Both tyrosineZ and tyrosineD are suggested to form a hydrogen bond with histidine 190 from the loop connecting helices C and D in the D1 and D2 proteins, respectively. The Mn cluster in the oxygen evolving complex has been localized by using known and estimated distances from the tyrosine radicals. It is suggested that a binding region for the Mn cluster is constituted by the lumenal ends of helices A and B and the loop connecting them in the D1 protein. This part of the D1 protein contains a large number of strictly conserved carboxylic acid residues and histidines which could participate in the Mn binding. There is little probability that the Mn cluster binds on the lumenal surface of the D2 protein.     

Photosynthetic and Phylogenetic Primers for Detection of Anoxygenic Phototrophs in Natural Environments

Primer sets were designed to target specific 16S ribosomal DNA (rDNA) sequences of photosynthetic bacteria, including the green sulfur bacteria, the green nonsulfur bacteria, and the members of the Heliobacteriaceae (a gram-positive phylum). Due to the phylogenetic diversity of purple sulfur and purple nonsulfur phototrophs, the 16S rDNA gene was not an appropriate target for phylogenetic rDNA primers. Thus, a primer set was designed that targets the pufM gene, encoding the M subunit of the photosynthetic reaction center, which is universally distributed among purple phototrophic bacteria. The pufM primer set amplified DNAs not only from purple sulfur and purple nonsulfur phototrophs but also from Chloroflexus species, which also produce a reaction center like that of the purple bacteria. Although the purple bacterial reaction center structurally resembles green plant photosystem II, the pufM primers did not amplify cyanobacterial DNA, further indicating their specificity for purple anoxyphototrophs. This combination of phylogenetic- and photosynthesis-specific primers covers all groups of known anoxygenic phototrophs and as such shows promise as a molecular tool for the rapid assessment of natural samples in ecological studies of these organisms.     

Pheophytin-protein interactions in photosystem II studied by resonance Raman spectroscopy of modified reaction centers

Soret-excited resonance Raman spectra of two types of pheophytin-exchanged photosystem II RCs are reported. The cofactor composition of the reaction centers was modified by exchanging pheophytin a for 13(1)-deoxo-13(1)-hydroxypheophytin a, yielding one preparation with selective replacement of the photochemically inactive pheophytin (H(B)) and a second one exhibiting total replacement of H(B) and 40% replacement of H(A), the primary electron acceptor. Resonance Raman spectra indicate that the other bound cofactors present are not significantly perturbed by Pheo substitution. The resonance Raman contributions from H(A) and H(B) in the carbonyl stretching region are identified at 1679 and 1675 cm(-)(1), respectively, indicating that both pheophytin molecules in the photosystem II reaction center have hydrogen-bonded keto-carbonyl groups. This conclusion differs from what is observed in the functionally related RCs of purple non-sulfur bacteria, where the keto-carbonyl group of H(B) is not hydrogen bonded, but confirms predictions from models based on protein sequence alignments.     

Photosystem II of rye. Nucleotide sequence of the psbK gene and regions adjacent to it]

The structure of the rye chloroplast DNA which contains the psbK gene coding for a subunit of photosystem II is determined. The gene psbI encoding an other protein of photosystem II is located 407 bp downstream from the stop codon of this gene. The determination of structure of the intergenic region between the psbI and psbD genes is fully elucidated. The rye BamHI fragment, comprising the psbK gene, is structurally similar to the corresponding fragment of the barley genome.     

Photochemically induced dynamic nuclear polarization in the reaction center of the green sulphur bacterium Chlorobium tepidum observed by 13C MAS NMR

Photochemically induced dynamic nuclear polarization has been observed in reaction centres of the green sulphur bacterium Chlorobium tepidum by (13)C magic-angle spinning solid-state NMR under continuous illumination with white light. An almost complete set of chemical shifts of the aromatic ring carbons of a BChl a molecule has been obtained. All light-induced (13)C NMR signals appear to be emissive, which is similar to the pattern observed in the reaction centers of plant photosystem I and purple bacterial reaction centres of Rhodobacter sphaeroides wild type. The donor in RCs of green sulfur bacteria clearly differs from the substantially asymmetric special pair of purple bacteria and appears to be similar to the more symmetric donor of photosystem I.     

Too much light? How beta-carotene protects the photosystem II reaction centre.

The photosystem II reaction centre of all oxygenic organisms is subject to photodamage by high light i.e. photoinhibition. In this review I discuss the reasons for the inevitable and unpreventable oxidative damage that occurs in photosystem II and the way in which beta-carotene bound to the reaction centre significantly mitigates this damage. Recent X-ray structures of the photosystem II core complex (reaction centre plus the inner antenna complexes) have revealed the binding sites of some of the carotenoids known to be bound to the complex. In the light of these X-ray structures and their known biophysical properties it is thus possible to identify the two beta-carotenes present in the photosystem II reaction centre. The two carotenes are both bound to the D2 protein and this positioning is discussed in relation to their ability to act as quenchers of singlet oxygen, generated via the triplet state of the primary electron donor. It is proposed that their location on the D2 polypeptide means there is more oxidative damage to the D1 protein and that this underlies the fact that this latter protein is continuously re-synthesised, at a far greater rate than any other protein involved in photosynthesis. The relevance of a cycle of electrons around photosystem II, via cytochrome b(559), in order to re-reduce the beta-carotenes when they are oxidised and hence restore their ability to quench singlet oxygen, is also discussed.     

Study of the structure of the photosynthetic reaction center of the green thermophilic bacteria Chloroflexus aurantiacus

Analysis of the Chloroflexus aurantiacus reaction centre (RC) using both protein and recombinant DNA techniques resulted in determination of its polypeptide composition and the primary structures of its two subunits. A model of the polypeptide chains' folding in the membrane is suggested based on: i) homology between L- and M-subunits of Chloroflexus aurantiacus RC and their counterparts in purple bacteria; ii) comparison of their hydropathy plots, and iii) data on the tertiary structures of purple bacteria RCs. The role of a number of functionally important amino acid residues in the RC electron transport activity is discussed. Limited proteolysis of the RC under non-denaturing conditions was used to determine the contribution of the N-terminal regions to its thermal stability.     

Light-induced quinone reduction in photosystem II

The photosystem II core complex is the water:plastoquinone oxidoreductase of oxygenic photosynthesis situated in the thylakoid membrane of cyanobacteria, algae and plants. It catalyzes the light-induced transfer of electrons from water to plastoquinone accompanied by the net transport of protons from the cytoplasm (stroma) to the lumen, the production of molecular oxygen and the release of plastoquinol into the membrane phase. In this review, we outline our present knowledge about the "acceptor side" of the photosystem II core complex covering the reaction center with focus on the primary (Q(A)) and secondary (Q(B)) quinones situated around the non-heme iron with bound (bi)carbonate and a comparison with the reaction center of purple bacteria. Related topics addressed are quinone diffusion channels for plastoquinone/plastoquinol exchange, the newly discovered third quinone Q(C), the relevance of lipids, the interactions of quinones with the still enigmatic cytochrome b559 and the role of Q(A) in photoinhibition and photoprotection mechanisms. This article is part of a Special Issue entitled: Photosystem II.     

Dynamic Interaction between the D1 protein,CP43 and OEC33 at the lumenal side of photosystem II in spinach chloroplasts: evidence from light-induced cross-Linking of the proteins in the donor-side photoinhibition

During the donor-side photoinhibition of spinach photosystem II, the reaction center D1 protein cross-linked with the antenna chlorophyll binding protein CP43 of photosystem II lacking the oxygen-evolving complex (OEC) subunit proteins. The cross-linking did not occur upon illumination of photosystem II samples that retained the OEC33, nor when OEC33-depleted photosystem II samples were reconstituted with the OEC33 prior to illumination. These results suggest that the D1 protein, CP43 and the OEC33 are located in close proximity at the lumenal side of photosystem II, and that the OEC33 suppresses the unnecessary contact between the D1 protein and CP43. Previously we presented data showing the D1 protein located adjacent to CP43 on the stromal side of photosystem II [Ishikawa et al. (1999) BIOCHIM: Biophys. Acta 1413: 147]. The present data suggest that the spatial arrangement of the D1 protein and CP43 at the lumenal side of photosystem II in spinach chloroplasts is similar to that at the stromal side of photosystem II and is consistent with the assignment of these proteins recently proposed on the crystal structures of the photosystem II complexes from cyanobacteria [Zouni et al. (2001) Nature 409: 739, Kamiya and Shen 2003 PROC: Natl. Acad. Sci. USA, 100: 98]. Moreover, the data suggest that the binding condition and positioning of the OEC33 in the photosystem II complex from higher plants may be different from those in cyanobacteria.     

Dielectric and photoelectric properties of photosynthetic reaction centers

A brief review of studies of dielectric and photoelectric properties of photosynthetic reaction centers of purple bacteria as well as photosystem I and photosystem II of cyanobacteria and higher plants is given. A simple kinetic model of the primary processes of electron transfer in photosynthesis is used to discuss possible mechanisms of correlation between rate constant of charge transfer reaction, free energy of electron transition, and effective dielectric constant in the locus of corresponding carriers.Translated from Biokhimiya, Vol. 70, No. 2, 2005, pp. 315–322.Original Russian Text Copyright © 2005 by Chamorovsky, Chamorovsky, Semenov.This revised version was published online in April 2005 with corrections to the post codes.     

Quantitation of the rapid electron donors to P700, the functional plastoquinone pool, and the ratio of the photosystems in spinach chloroplasts

Recent studies of chloroplast architecture have emphasized the segregation of photosystem I and photosystem II in different regions of the lamellar membrane. The apparent localization of photosystem II reaction centers in regions of membrane appression and of photosystem I reaction centers in regions exposed to the chloroplast stroma has focused attention on the intervening electron carriers, carriers which must be present to catalyze electron transfer between such spatially separated reaction sites. Information regarding the stoichiometries of these intermediate carriers is essential to an understanding of the processes that work together to establish the mechanism and to determine the rate of the overall process. We have reinvestigated the numbers of photosystem I and photosystem II reaction centers, the numbers of intervening cytochrome b6/f complexes, and the numbers of molecules of the relatively mobile electron carriers plastoquinone and plastocyanin that are actively involved in electron transfer. Our investigations were based on a new experimental technique made possible by the use of a modified indophenol dye, methyl purple, the reduction of which provides a particularly sensitive and accurate measure of electron transfer. Using this dye, which accepts electrons exclusively from photosystem I, it was possible to drain electrons from each of the carriers. Thus, by manipulation of the redox condition of the various carriers and through the use of specific inhibitors we could measure the electron storage capacity of each carrier in turn. We conclude that the ratio of photosystem I reaction centers to cytochrome b6/f complexes to photosystem II reaction centers is very nearly 1:1:1. The pool of rapid donors of electrons to P700 includes not only the 2 reducing equivalents stored in the cytochrome b6/f complex but also those stored in slightly more than 2 molecules of plastocyanin per P700. More slowly available are the electrons from about 6 plastoquinol molecules per P700.     

Structural characteristics of channels and pathways in photosystem II including the identification of an oxygen channel

Here we use crystal structures to investigate and review channels and pathways for the transfer of substrates (water, plastoquinone (PQ)) and products (electrons, protons, oxygen, reduced PQ (PQH(2))) to, and from, the redox active catalytic sites of photosystem II (PSII). A putative oxygen channel has been identified which is about 21A in length, leading from the water splitting site to the lumen. This channel follows a path along the lumenal surface of CP43, passing across the interface of the large extrinsic loop which joins the fifth and sixth transmembrane helices of this chlorophyll binding protein. In so doing it seems to minimise interactions with the excited states of chlorophylls bound within the PSII complex, especially those that constitute the primary electron donor, P680. Two additional channels leading from the water splitting site, and also exiting at the lumen, were also identified. Their hydrophilic nature suggests that they probably facilitate the delivery of water to, and protons from, the catalytic site. Also discussed are unique features in the electron transfer pathway of PSII, as compared with those of purple photosynthetic bacteria, and structural implications of the PSII Q(B)-site in terms of PQ protonation and PQ/PQH(2) diffusion.     

The effect of o-phenanthroline on the midpoint potential of the primary electron acceptor of photosystem II.

The primary electron acceptor of Photosystem II has a midpoint oxidation-reduction potential of +95 mV at pH 7.0 in Photosystem II chloroplast fragments prepared by digitonin treatment. The midpoint potential of the acceptor has a pH dependence of -60 mV/pH unit. At concentrations that inhibit oxygen evolution, o-phenanthroline shifts the midpoint potential of the primary acceptor by +70 mV. The shifted potential retains the same dependence on pH. The effect of o-phenanthroline suggests that it interacts directly with the primary electron acceptor of photosystem II in a manner similar to that reported previously for the primary electron acceptor in purple photosynthetic bacteria.