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The cyanobacterium Synechocystis sp. PCC 6803 has a bidirectional [NiFe]-hydrogenase (Hox hydrogenase) which reversibly reduces protons to H2. This enzyme is composed of a hydrogenase domain and a diaphorase moiety, which is distinctly homologous to the NADH input module of mitochondrial respiratory Complex I. Hox hydrogenase physiological function is still unclear, since it is not required for Synechocystis fitness under standard growth conditions. We analyzed the phenotype under prolonged darkness of three Synechocystis knock-out strains, lacking either Hox hydrogenase (ΔHoxE-H) or one of the proteins responsible for the assembly of its NiFe active site (ΔHypA1 and ΔHypB1). We found that Hox hydrogenase is required for Synechocystis growth under this condition, regardless of the functional status of its catalytic site, suggesting an additional role beside hydrogen metabolism. Moreover, quantitative proteomic analyses revealed that the expression levels of several subunits of the respiratory NADPH/plastoquinone oxidoreductase (NDH-1) are reduced when Synechocystis is grown in the dark. Our findings suggest that the Hox hydrogenase could contribute to electron transport regulation when both photosynthetic and respiratory pathways are down-regulated, and provide a possible explanation for the close evolutionary relationship between mitochondrial respiratory Complex I and cyanobacterial [NiFe]-hydrogenases.

Nannochloropsis is rapidly emerging as a model organism for the study of biofuel production in microalgae. Here, we report a high-quality genomic assembly of Nannochloropsis gaditana, consisting of large contigs, up to 500 kbp long, and scaffolds that in most cases span the entire length of the chromosomes. We identified 10646 complete genes and characterized possible alternative transcripts. The annotation of the predicted genes and the analysis of cellular processes revealed traits relevant for the genetic improvement of this organism such as genes involved in DNA recombination, RNA silencing, and cell wall synthesis. We also analyzed the modification of the transcriptional profile in nitrogen deficiency-a condition known to stimulate lipid accumulation. While the content of lipids increased, we did not detect major changes in expression of the genes involved in their biosynthesis. At the same time, we observed a very significant down-regulation of mitochondrial gene expression, suggesting that part of the Acetyl-CoA and NAD(P)H, normally oxidized through the mitochondrial respiration, would be made available for fatty acids synthesis, increasing the flux through the lipid biosynthetic pathway. Finally, we released an information resource of the genomic data of N. gaditana, available online at www.nannochloropsis.org.

Despite the important achievement of the high-resolution structures of several prokaryotic channels, current understanding of their physiological roles in bacteria themselves is still far from complete. We have identified a putative two transmembrane domain-containing channel, SynCaK, in the genome of the freshwater cyanobacterium Synechocystis sp. PCC 6803, a model photosynthetic organism. SynCaK displays significant sequence homology to MthK, a calcium-dependent potassium channel isolated from Methanobacterium thermoautotrophicum. Expression of SynCaK in fusion with enhanced GFP in mammalian Chinese hamster ovary cells' plasma membrane gave rise to a calcium-activated, potassium-selective activity in patch clamp experiments. In cyanobacteria, Western blotting of isolated membrane fractions located SynCaK mainly to the plasma membrane. To understand its physiological function, a SynCaK-deficient mutant of Synechocystis sp. PCC 6803, ΔSynCaK, has been obtained. Although the potassium content in the mutant organisms was comparable to that observed in the wild type, ΔSynCaK was characterized by a depolarized resting membrane potential, as determined by a potential-sensitive fluorescent probe. Growth of the mutant under various conditions revealed that lack of SynCaK does not impair growth under osmotic or salt stress and that SynCaK is not involved in the regulation of photosynthesis. Instead, its lack conferred an increased resistance to the heavy metal zinc, an environmental pollutant. A similar result was obtained using barium, a general potassium channel inhibitor that also caused depolarization. Our findings thus indicate that SynCaK is a functional channel and identify the physiological consequences of its deletion in cyanobacteria.

Although light is the source of energy for photosynthetic organisms, it causes oxidative stress when in excess. Plants and algae prevent reactive oxygen species (ROS) formation by activation of nonphotochemical quenching (NPQ), which dissipates excess excitation energy as heat. Although NPQ is found in both algae and plants, these organisms rely on two different proteins for its activation, Light harvesting complex stress-related (LHCSR) and Photosystem II subunit S (PSBS). In the moss Physcomitrella patens, both proteins are present and active. Several P. patens lines depleted in or over-expressing PSBS and/or LHCSR at various levels were generated by exploiting the ability of Physcomitrella to undergo homologous recombination. The analysis of the transgenic lines showed that either protein is sufficient, alone, for NPQ activation independently of the other, supporting the idea that they rely on different activation mechanisms. Modulation of PSBS and/or LHCSR contents was found to be correlated with NPQ amplitude, indicating that plants and algae can directly modulate their ability to dissipate energy simply by altering the accumulation level of one or both of these proteins. The availability of a large range of P. patens genotypes differing in PSBS and LHCSR content allowed comparison of their activation mechanisms and discussion of implications for the evolution of photoprotection during land colonization., (© 2012 The Authors. New Phytologist © 2012 New Phytologist Trust.)

[FeFe]-hydrogenases are iron-sulfur proteins characterized by a complex active site, the H-cluster, whose assembly requires three conserved maturases. HydE and HydG are radical S-adenosylmethionine enzymes that chemically modify a H-cluster precursor on HydF, a GTPase with a dual role of scaffold on which this precursor is synthesized, and carrier to transfer it to the hydrogenase. Coordinate structural and functional relationships between HydF and the two other maturases are crucial for the H-cluster assembly. However, to date only qualitative analysis of this protein network have been provided. In this work we showed that the interactions of HydE and HydG with HydF are distinct events, likely occurring in a precise functional order driven by different kinetic properties, independently of the HydF GTPase activity, which is instead involved in the dissociation of the maturases from the scaffold. We also found that HydF is able to interact with the hydrogenase only when co-expressed with the two other maturases, indicating that under these conditions it harbors per se all the structural elements needed to transfer the H-cluster precursor, thus completing the maturation process. These results open new working perspectives aimed at improving the knowledge of how these complex metalloenzymes are biosynthesized.

A potassium channel (SynK) of the cyanobacterium Synechocystis sp. PCC 6803, a photoheterotrophic model organism for the study of photosynthesis, has been recently identified and demonstrated to function as a potassium selective channel when expressed in a heterologous system and to be located predominantly to the thylakoid membrane in cyanobacteria. To study its physiological role, a SynK-less knockout mutant was generated and characterized. Fluorimetric experiments indicated that SynK-less cyanobacteria cannot build up a proton gradient as efficiently as WT organisms, suggesting that SynK might be involved in the regulation of the electric component of the proton motive force. Accordingly, measurements of flash-induced cytochrome b(6)f turnover and respiration pointed to a reduced generation of ΔpH and to an altered linear electron transport in mutant cells. The lack of the channel did not cause an altered membrane organization, but decreased growth and modified the photosystem II/photosystem I ratio at high light intensities because of enhanced photosensitivity. These data shed light on the function of a prokaryotic potassium channel and reports evidence, by means of a genetic approach, on the requirement of a thylakoid ion channel for optimal photosynthesis.

Biofuels from algae are highly interesting as renewable energy sources to replace, at least partially, fossil fuels, but great research efforts are still needed to optimize growth parameters to develop competitive large-scale cultivation systems. One factor with a seminal influence on productivity is light availability. Light energy fully supports algal growth, but it leads to oxidative stress if illumination is in excess. In this work, the influence of light intensity on the growth and lipid productivity of Nannochloropsis salina was investigated in a flat-bed photobioreactor designed to minimize cells self-shading. The influence of various light intensities was studied with both continuous illumination and alternation of light and dark cycles at various frequencies, which mimic illumination variations in a photobioreactor due to mixing. Results show that Nannochloropsis can efficiently exploit even very intense light, provided that dark cycles occur to allow for re-oxidation of the electron transporters of the photosynthetic apparatus. If alternation of light and dark is not optimal, algae undergo radiation damage and photosynthetic productivity is greatly reduced. Our results demonstrate that, in a photobioreactor for the cultivation of algae, optimizing mixing is essential in order to ensure that the algae exploit light energy efficiently.

[FeFe]-hydrogenases catalyze the reversible production of H2 in some bacteria and unicellular eukaryotes. These enzymes require ancillary proteins to assemble the unique active site H-cluster, a complex structure composed of a 2Fe center bridged to a [4Fe-4S] cubane. The first crystal structure of a key factor in the maturation process, HydF, has been determined at 3 Å resolution. The protein monomer present in the asymmetric unit of the crystal comprises three domains: a GTP-binding domain, a dimerization domain, and a metal cluster-binding domain, all characterized by similar folding motifs. Two monomers dimerize, giving rise to a stable dimer, held together mainly by the formation of a continuous β-sheet comprising eight β-strands from two monomers. Moreover, in the structure presented, two dimers aggregate to form a supramolecular organization that represents an inactivated form of the HydF maturase. The crystal structure of the latter furnishes several clues about the events necessary for cluster generation/transfer and provides an excellent model to begin elucidating the structure/function of HydF in [FeFe]-hydrogenase maturation.

Plants are able to deal with variable environmental conditions; when exposed to strong illumination, they safely dissipate excess energy as heat and increase their capacity for scavenging reacting oxygen species. Both these protection mechanisms involve activation of the xanthophyll cycle, in which the carotenoid violaxanthin is converted to zeaxanthin by violaxanthin de-epoxidase, using ascorbate as the source of reducing power. In this work, following determination of the three-dimensional structure of the violaxanthin de-epoxidase catalytic domain, we identified the putative binding sites for violaxanthin and ascorbate by in silico docking. Amino acid residues lying in close contact with the two substrates were analyzed for their involvement in the catalytic mechanism. Experimental results supported the proposed substrate-binding sites and point to two residues, Asp-177 and Tyr-198, which are suggested to participate in the catalytic mechanism, based on complete loss of activity in mutant proteins. The role of other residues and the mechanistic similarity to aspartic proteases and epoxide hydrolases are discussed.

Light is the source of energy for photosynthetic organisms; when in excess, however, it also drives the formation of reactive oxygen species and, consequently, photoinhibition. Plants and algae have evolved mechanisms to regulate light harvesting efficiency in response to variable light intensity so as to avoid oxidative damage. Nonphotochemical quenching (NPQ) consists of the rapid dissipation of excess excitation energy as heat. Although widespread among oxygenic photosynthetic organisms, NPQ shows important differences in its machinery. In land plants, such as Arabidopsis thaliana, NPQ depends on the presence of PSBS, whereas in the green alga Chlamydomonas reinhardtii it requires a different protein called LHCSR. In this work, we show that both proteins are present in the moss Physcomitrella patens. By generating KO mutants lacking PSBS and/or LHCSR, we also demonstrate that both gene products are active in NPQ. Plants lacking both proteins are more susceptible to high light stress than WT, implying that they are active in photoprotection. These results suggest that NPQ is a fundamental mechanism for survival in excess light and that upon land colonization, photosynthetic organisms evolved a unique mechanism for excess energy dissipation before losing the ancestral one found in algae.

Elucidation of the structure-function relationship of a small number of prokaryotic ion channels characterized so far greatly contributed to our knowledge on basic mechanisms of ion conduction. We identified a new potassium channel (SynK) in the genome of the cyanobacterium Synechocystis sp. PCC6803, a photosynthetic model organism. SynK, when expressed in a K(+)-uptake-system deficient E. coli strain, was able to recover growth of these organisms. The protein functions as a potassium selective ion channel when expressed in Chinese hamster ovary cells. The location of SynK in cyanobacteria in both thylakoid and plasmamembranes was revealed by immunogold electron microscopy and Western blotting of isolated membrane fractions. SynK seems to be conserved during evolution, giving rise to a TPK (two-pore K(+) channel) family member which is shown here to be located in the thylakoid membrane of Arabidopsis. Our work characterizes a novel cyanobacterial potassium channel and indicates the molecular nature of the first higher plant thylakoid cation channel, opening the way to functional studies.

Background: Photosystems are composed of two moieties, a reaction center and a peripheral antenna system. In photosynthetic eukaryotes the latter system is composed of proteins belonging to Lhc family. An increasing set of evidences demonstrated how these polypeptides play a relevant physiological function in both light harvesting and photoprotection. Despite the sequence similarity between antenna proteins associated with the two Photosystems, present knowledge on their physiological role is mostly limited to complexes associated to Photosystem II., Results: In this work we analyzed the physiological role of Photosystem I antenna system in Arabidopsis thaliana both in vivo and in vitro. Plants depleted in individual antenna polypeptides showed a reduced capacity for photoprotection and an increased production of reactive oxygen species upon high light exposure. In vitro experiments on isolated complexes confirmed that depletion of antenna proteins reduced the resistance of isolated Photosystem I particles to high light and that the antenna is effective in photoprotection only upon the interaction with the core complex., Conclusion: We show that antenna proteins play a dual role in Arabidopsis thaliana Photosystem I photoprotection: first, a Photosystem I with an intact antenna system is more resistant to high light because of a reduced production of reactive oxygen species and, second, antenna chlorophyll-proteins are the first target of high light damages. When photoprotection mechanisms become insufficient, the antenna chlorophyll proteins act as fuses: LHCI chlorophylls are degraded while the reaction center photochemical activity is maintained. Differences with respect to photoprotection strategy in Photosystem II, where the reaction center is the first target of photoinhibition, are discussed.

Seven genes seem to encode for putative ClC chloride channels (AtClC-a to AtClC-g) in Arabidopsis thaliana. Their function and localization is still largely unknown. AtClC-f shares considerable sequence similarity with putative ClC channel proteins from Synechocystis, considered to represent the precursor of chloroplasts. We show by biochemical and mass spectrometry analysis that ClC-f is located in the outer envelope membrane of spinach chloroplasts. Consistent with the plastidial localization of ClC-f, p-chlorophenoxy-acetic acid (CPA) reduces photosynthetic activity and the protein is expressed in etioplasts and chloroplasts but not in root tissue. These findings may represent a step toward the molecular identification of ion channel activities in chloroplast membranes.

Efficient photosynthesis is of fundamental importance for plant survival and fitness. However, in oxygenic photosynthesis, the complex apparatus responsible for the conversion of light into chemical energy is susceptible to photodamage. Oxygenic photosynthetic organisms have therefore evolved several protective mechanisms to deal with light energy. Rapidly inducible non-photochemical quenching (NPQ) is a short-term response by which plants and eukaryotic algae dissipate excitation energy as heat. This review focuses on recent advances in the elucidation of the molecular mechanisms underlying this protective quenching pathway in higher plants.

In higher plants, the PsbS subunit of photosystem II (PSII) plays a crucial role in pH- and xanthophyll-dependent nonphotochemical quenching of excess absorbed light energy, thus contributing to the defense mechanism against photoinhibition. We determined the amino acid sequence of Zea mays PsbS and produced an antibody that recognizes with high specificity a region of the protein located in the stroma-exposed loop between the second and third putative helices. By means of this antiserum, the thylakoid membranes of various higher plant species revealed the presence of a 42-kDa protein band, indicating the formation of a dimer of the 21-kDa PsbS protein. Crosslinking experiments and immunoblotting with other antisera seem to exclude the formation of a heterodimer with other PSII protein components. The PsbS monomer/dimer ratio in isolated thylakoid membranes was found to vary with luminal pH in a reversible manner, the monomer being the prevalent form at acidic and the dimer at alkaline pH. In intact chloroplasts and whole plants, dimer-to-monomer conversion is reversibly induced by light, known to cause luminal acidification. Sucrose-gradient centrifugation revealed a prevalent association of the PsbS monomer and dimer with light-harvesting complex and PSII core complexes, respectively. The finding of the existence of a light-induced change in the quaternary structure of the PsbS subunit may contribute to understanding the mechanism of PsbS action during nonphotochemical quenching.

Photosystem I-less Synechocystis 6803 mutants carrying modified PsbH proteins, derived from different combinations of wild-type cyanobacterial and maize genes, were constructed. The mutants were analyzed in order to determine the relative importance of the intra- and extramembrane domains of the PsbH subunit in the functioning of photosystem (PS) II, by a combination of biochemical, biophysical, and physiological approaches. The results confirmed and extended previously published data showing that, besides D1, the whole PsbH protein is necessary to determine the correct structure of a QB/herbicide-binding site. The different turnover of the D1 protein and chlorophyll photobleaching displayed by mutant cells in response to photoinhibitory treatment revealed for the first time the actual role of the PsbH subunit in photoprotection. A functional PsbH protein is necessary for (i) rapid degradation of photodamaged D1 molecules, which is essential to avoid further oxidative damage to the PSII core, and (ii) insertion of newly synthesized D1 molecules into the thylakoid membrane. PsbH is thus required for both initiation and completion of the repair cycle of the PSII complex in cyanobacteria.

The smallest extrinsic polypeptide of the water-oxidizing complex (PsbQ) was extracted and purified from spinach (Spinacia oleracea) photosystem II (PSII) membranes. It was then crystallized in the presence of Zn(2+) and its structure was determined by X-ray diffraction at 1.95-A resolution using the multi-wavelength anomalous diffraction method, with the zinc as the anomalous scatterer. The crystal structure shows that the core of the protein is a four-helix bundle, whereas the amino-terminal portion, which possibly interacts with the photosystem core, is not visible in the crystal. The distribution of positive and negative charges on the protein surface might explain the ability of PsbQ to increase the binding of Cl(-) and Ca(2+) and make them available to PSII.

The effect of visible light on photosystem II reaction centre D1 protein in plants treated with ultraviolet-B light was studied. It was found that a 20 kDa C-terminal fragment of D1 protein generated during irradiation with ultraviolet-B light was stable when plants were incubated in the dark, but was degraded when plants were incubated in visible light. In this condition the recovery of photosynthetic activity was also observed. Even a low level of white light was sufficient to promote both further degradation of the fragment and recovery of activity. During this phase, the D1 protein is the main synthesized thylakoid polypeptide, indicating that other photosystem II proteins are recycled in the recovery process. Although both degradation of the 20 kDa fragment and resynthesis of D1 are light-dependent phenomena, they are not closely related, as degradation of the 20 kDa fragment may occur even in the absence of D1 synthesis. Comparing chemical and physical factors affecting the formation of the fragment in ultraviolet-B light and its degradation in white light, it was concluded that the formation of the fragment in ultraviolet-B light is a photochemical process, whereas the degradation of the fragment in white light is a protease-mediated process.

A photosystem II (PSII) core complex lacking the internal antenna CP43 protein was isolated from the photosystem II of Synechocystis PCC6803, which lacks photosystem I (PSI). CP47-RC and reaction centre (RCII) complexes were also obtained in a single procedure by direct solubilization of whole thylakoid membranes. The CP47-RC subcore complex was characterized by SDS/PAGE, immunoblotting, MALDI MS, visible and fluorescence spectroscopy, and absorption detected magnetic resonance. The purity and functionality of RCII was also assayed. These preparations may be useful for mutational analysis of PSII RC and CP47-RC in studying primary reactions of oxygenic photosynthesis.

Photosystem II of higher plants and cyanobacteria is composed of more than 20 polypeptide subunits. The pronounced hydrophobicity of these proteins hinders their purification and subsequent analysis by mass spectrometry. This paper reports the results obtained by application of matrix-assisted laser desorption/ionization mass spectrometry directly to isolated complexes and thylakoid membranes prepared from cyanobacteria and spinach. Changes in protein contents following physiopathological stimuli are also described. Good correlations between expected and measured molecular masses allowed the identification of the main, as well as most of the minor, low molecular weight components of photosystem II. These results open up new perspectives for clarifying the functional role of the various polypeptide components of photosystems and other supramolecular integral membrane complexes.

This study examines the effects of ecologically important levels of ultraviolet B radiation on protein D1 turnover and stability and lateral redistribution of photosystem II. It is shown that ultraviolet B light supported only limited synthesis of protein D1, one of the most important components of photosystem II, whereas it promoted significant degradation of proteins D1 and D2. Furthermore, dephosphorylation of photosystem II subunits was specifically elicited upon exposure to ultraviolet B light. Structural modifications of photosystem II and changes in its lateral distribution between granum membranes and stroma-exposed lamellae were found to be different from those observed after photoinhibition by strong visible light. In particular, more complete dismantling of photosystem II cores was observed. Altogether, the data reported here suggest that ultraviolet B radiation alone fails to activate the photosystem II repair cycle, as hypothesized for visible light. This failure may contribute to the toxic effect of ultraviolet B radiation, which is increasing as a consequence of depletion of stratospheric ozone.

A Synechocystis 6803 mutant carrying a chimaeric photosystem II (PSII), in which the Zea mays PsbH subunit (7.7 kDa calculated molecular mass) replaces the cyanobacterial copy (7.0 kDa), was constructed. With the exception of the N-terminal 12 amino acid extension, which has a phosphorylatable threonine, the eukaryotic polypeptide is 78% homologous to its bacterial counterpart. Biochemical characterization of this mutant shows that it expresses the engineered gene correctly and is competent for photoautotrophic growth. Fluorescence analysis and oxygen evolution measurements in the presence of exogenous acceptors indicate that the observed phenotype results from a chimaeric PSII rather than from the absence of function associated with PsbH, suggesting that the heterologous protein is assembled into a functional PSII. Inhibition of oxygen evolution by herbicides belonging to different classes shows that the sensitivity of the mutant PSII is changed only towards phenolic compounds. This result indicates slight conformational modification of the QB/herbicide binding pocket of the D1 polypeptide caused by the bulky PsbH protein in the mutant, and also suggests close structural interaction of the D1 and PsbH subunits in the topological arrangement of PSII.

Fractionation of photosynthetic membranes from the cyanobacterium Synechocystis 6803 by polyacrylamide gel electrophoresis in the presence of Deriphat-160 allowed the isolation of a number of pigmented bands. Two of them, with molecular masses of 240+/-20 and 110+/-15 kDa respectively, showed peroxidase activity and, by means of polypeptide composition, immunoblotting and N-terminal sequencing, were identified as dimeric and monomeric cytochrome b6/f complexes, containing 1.3+/-0.35 chlorophyll molecules per cytochrome f. Further fractionation of monomeric complexes by mild gel electrophoresis in the presence of sodium dodecyl sulfate indicated that it is the cytochrome b6 polypeptide which provides the actual binding site for the chlorophyll molecule observed in the complex.

The effects of ultraviolet-B radiation (280-320 nm) on photosystem II of Synechocystis sp. PCC 6303 were investigated at the functional and structural levels. Loss of oxygen-evolving and electron-transport activity, measured by various techniques including Clark electrode polarography, fluorescence induction and fluorescence relaxation after a single turnover flash, are discussed in terms of two types of damage caused by ultraviolet-B radiation: (a) depletion of the plastoquinone pool; (b) perturbation and degradation of the D1 protein, with cleavage in the second transmembrane segment. These findings are in full agreement with those obtained, both in vivo and in vitro for higher plants for which a donor-side mechanism involving the water-splitting Mn cluster has been proposed for the main cleavage of the D1 protein. At the structural level, complete disruption of the photosystem II core is documented as a consequence of (or in parallel with) degradation of the D1 protein. From this point of view, ultraviolet-B-induced photoinhibition is unlike the visible-induced type and less susceptible to repair by synthesis and reinsertion of new D1 protein.

Photosystem I and II core complexes were resolved in a single step from the thylakoid membrane of Synechocystis sp. PCC 6803 by using a mild solubilization procedure in dodecyl beta-D-maltoside and Deriphat/PAGE. For each photosystem, two green bands were obtained containing oligomeric and monomeric forms of the core complexes of either photosystem. The oligomers are likely to be trimers in the case of photosystem I and dimers for photosystem II. The absorption spectra, polypeptide and pigment composition of green bands corresponding to either photosystem I or photosystem II were identical for monomeric and oligomeric forms. The cytochrome b-559 content of photosystem II was evaluated to be one cytochrome b-559/reaction centre both in the monomeric and dimeric forms. Two new 15-kDa and 22-kDa carotenoid-binding protein were isolated and their polypeptides purified to homogeneity.

The in vivo effects of ultraviolet-B radiation (280-320 nm) on photosystem-II activity and degradation of the D1 protein are investigated and compared with the in vitro results on isolated thylakoids and other detergent-extracted photosystem-II preparations. A cleavage site in the second transmembrane segment of the D1 protein, giving rise to a 20-kDa C-terminal and a 13-kDa N-terminal fragment pair, is detected after irradiation of entire leaves as well as in all photosystem-II preparations, irrespective of their actual ability to evolve oxygen but depending on the presence of Mn ions associated with the water-splitting system. Damage to the plastoquinone moiety, observed by other authors, is confirmed and is proposed to be responsible for the impairment of electron-transport activity, but not for the observed cleavage of the D1 protein.

Exposure of isolated reaction centres of photosystem II to UV-B radiation generates specific breakdown products of the D2 protein. When the quinone, 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone is present a 22 kDa fragment containing the N-terminus of the mature protein is generated. Concomitant with the appearance of the N-terminal fragment, two fragments containing the C-terminus of the D2 protein having apparent molecular masses around 10-12 kDa are observed. It is concluded that the primary cleavage occurs in the hydrophilic loop linking putative transmembrane segments IV and V. No such cleavage was observed when silicomolybdate was used as an electron acceptor, suggesting that this UV-B damage is dependent on binding of the added quinone to the QA site.

The nearest neighbour relationships within the D1/D2/cyt b559 complex (PSIIRC) and the CP47/D1/D2/cyt b559 complex (RC-CP47) were investigated by using different length bifunctional crosslinking agents. The crosslinking products were identified by immunoblotting with polyclonal antibodies and by two-dimensional gel electrophoresis. Seven products (CP47/D2, D1/D2/alpha, D1/D2, D2/alpha, D1/alpha, alpha/alpha, alpha/beta) have been revealed in both complexes. The crosslinking of both complexes does not increase their photostability. The photocrosslinking products (D1/alpha and D2/alpha) appeared under illumination of complexes with light of high intensity.

When isolated photosystem II reaction centers from spinach are exposed to photoinhibitory light in the presence of an electron acceptor, breakdown products of the D2 protein at 28, 25, 23, 18, 9, 5 and 4.5 kDa are detected by immunoblotting with a monospecific anti-D2 polyclonal antibody. In a time-course experiment the 23 and 4.5 kDa fragments show a transient appearance, whilst the others are photoaccumulated. The regions of the D2 protein containing the cleavage sites for the 28 and 18 kDa photoinduced fragments have been identified. Significant degradation of D2 takes place only in the presence of an electron acceptor, and breakdown of the protein is partially prevented by serine-type protease inhibitors.

The structural and topological stability of thylakoid components under photoinhibitory conditions (4,500 microE.m-2.s-1 white light) was studied on Mn depleted thylakoids isolated from spinach leaves. After various exposures to photoinhibitory light, the chlorophyll-protein complexes of both photosystems I and II were separated by sucrose gradient centrifugation and analysed by Western blotting, using a set of polyclonals raised against various apoproteins of the photosynthetic apparatus. A series of events occurring during donor side photoinhibition are described for photosystem II, including: (a) lowering of the oligomerization state of the photosystem II core; (b) cleavage of 32-kD protein D1 at specific sites; (c) dissociation of chlorophyll-protein CP43 from the photosystem II core; and (d) migration of damaged photosystem II components from the grana to the stroma lamellae. A tentative scheme for the succession of these events is illustrated. Some effects of photoinhibition on photosystem I are also reported involving dissociation of antenna chlorophyll-proteins LHCI from the photosystem I reaction center.

When isolated reaction centres of photosystem II are subjected to photoinhibitory illumination, a 41 kDa SDS-PAGE band is observed under all experimental conditions. The same band is also found, together with lower molecular weight fragments of the D1 protein, in whole thylakoids and in all PSII sub-particles investigated up to now. In the case of isolated reaction centres the 41 kDa band is represented by a heterodimer of the D1 polypeptide and the alpha-subunit of cytochrome b559. The cross-linkage between D1 and alpha-cyt b559 involves a region on D1 between the N-terminal residue and Arg-225, and is an early event in photo-induced damage to the D1 protein.

Photoinduced degradation of the photosystem II (PSII) reaction center D1 protein was studied in isolated thylakoids and different PSII subparticles. A 16 kDa fragment corresponding to the C-terminus of the protein is detected in thylakoids when they are inactivated at the donor side before illumination. The same D1 fragment is found in different types of PSII preparations at different integration levels characterized by different polypeptide compositions so long as they have an inactivated donor side and an active electron acceptor for the reduced pheophytin. However, when the PSII particle is equal to or smaller than the 43-less PSII core complex, other fragments are observed which are not found in more integrated systems.

When isolated reaction centres of photosystem 2 from pea or wheat are exposed to photoinhibitory illumination in the presence of an electron acceptor, breakdown products of the D1-protein are observed having molecular masses ranging from about 24 to 10 kDa. By using antibodies raised to the C-terminal or N-terminal portions of D1 it was shown that the major breakdown fragment of 24 kDa was derived from the C-terminus. This conclusion was supported by phosphorylation studies and from the digestion pattern obtained by lysine specific endoprotease-induced proteolysis. The complementary N-terminal breakdown fragment was found to have an apparent molecular mass of 10 kDa. The implications of these data are discussed in terms of the possible relationship between the 24 kDa C-terminal fragment and the 23.5 kDa breakdown fragment detected in vivo by Greenberg et al. [1987, EMBO J. 6, 2865-2869] and it is suggested, based on limited proteolysis using papain, that the latter may not be derived from the N-terminus as previously thought but also originates from the C-terminus.

The magnetic susceptibilities of solutions of deoxyhemoglobin Zürich and deoxyhemoglobin A have been found quite close together, in contrast to previous findings. Therefore, any magnetic inequivalence between alpha- and beta-hemes must be confined to finer details.

The present paper reports a spectroscopic and kinetic study of the reaction with oxygen and carbon monoxide of (a) the abnormal hemoglobin Zürich (beta 63, E7 His leads to Arg), (b) its isolated abnormal chains (beta ZH), and (c) a reconstituted hybrid containing cobalt instead of iron on the normal alpha chains (Co alpha)2-(Fe beta ZH)2. The abnormal beta ZH chains, isolated from hemoglobin Zürich (HbZH) tetramer, display very peculiar spectral properties in the Soret region which were determined, for the oxy and deoxy derivatives, by kinetic difference spectra. The spectral properties of abnormal chains are maintained in the native and reconstituted hybrid tetramer. The rate constants for CO and O2 binding to isolated beta ZH chains were compared with those of the normal alpha and beta chains, It is confirmed that the CO combination rate constant to beta ZH is much higher than that of normal chains, whereas that for O2 is similar for the normal and abnormal chains. The CO binding rate constant for Fe beta ZH chains in the hybrid tetramer is the same as in the native HbZH molecule, i.e. much higher than that of normal chain. The new data are fully consistent with the sequential mechanism for CO binding previously proposed by us; on the other hand, on the basis of the spectral and kinetic results, and contrary to what has been suggested by other authors, a sequential binding of the ligand is excluded in the case of O2.

The pH dependence in the range 3--7 of the optical absorption and electron paramagnetic resonance of the nitric oxide adduct of ferrous Aplysia myoglobin is reported. Optical spectra in the Soret region show a transition between two conformers with an apparent pK in the range 3.5--5 depending on the presence of carboxylic anions as third component. In the same pH range, the EPR spectrum undergoes a change from a 9-line to a 3-line hyperfine pattern in the g. region, similar to that reported for synthetic heme derivatives and for other hemoproteins. The structural interpretation of the pH-induced transition experienced by Aplysia myoglobin nitric oxide is that of a proton-linked cleavage of the proximal bond as suggested by several lines of evidence. Temperature-jump measurements allowed an estimation of the relaxation time for the process, which is of the order of 0.3 ms at 25 degrees C.

Resonance Raman spectra of ferric Aplysia myoglobin in the ligand-free and the azide-bound forms have been studied over a wide pH range to determine the coordination states of the heme iron atom. In the hydroxide form at high pH (approximately 9) the iron is six-coordinate and is in a high/low spin equilibrium. As the pH is lowered below the acid/alkaline transition (pKa = 7.5), the heme becomes five-coordinate. When the pH is lowered even further no other changes in the resonance Raman spectrum are detected; thus, the heme remains five-coordinate down to pH 4, the lowest value studied. For ferric azide-bound Aplysia myoglobin, the iron is six-coordinate in a high/low spin equilibrium at all pH values (4.8-9). These data indicate (i) that the unusual reactivity toward azide previously observed at neutral pH is indeed related to the absence of a coordinated water molecule, and (ii) that causes other than the heme coordination are responsible for the spectral differences and the ligand-binding kinetics differences observed below pH 6.

The rates of reaction of myoglobin with carbon monoxide at low pH are reported. The pH versus rate profile of these kinetics resembles that found for heme model compounds, revealing an increase in combination rate at low pH. These facts suggest that CO binding by myoglobin changes from a mechanism of "direct ligant association" at pH 5 to a mechanism, similar to that proposed for heme model compounds, which assumes a tetracoordinated intermediate as a result of the protonation of the proximal imidazole.

The chlorophyll-protein complexes of purified maize photosystem II membranes were separated by a new mild gel electrophoresis system under conditions which maintained all of the major chlorophyll a/b-protein complex (LHCII) in the oligomeric form. This enabled the resolution of three chlorophyll a/b-proteins in the 26-31-kDa region which are normally obscured by monomeric LHCII. All chlorophyll a/b-proteins had unique polypeptide compositions and characteristic spectral properties. One of them (CP26) has not previously been described, and another (CP24) appeared to be identical to the connecting antenna of photosystem I (LHCI-680). Both CP24 and CP29 from maize had at least one epitope in common with the light-harvesting antennae of photosystem I, as shown by cross-reactivity with a monoclonal antibody raised against LHCI from barley thylakoids. A complex designated Chla.P2, which was capable of electron transport from diphenylcarbazide to 2,6-dichlorophenolindophenol, was isolated by nondenaturing gel electrophoresis. It lacked CP43, which therefore can be excluded as an essential component of the photosystem II reaction center core. Fractionation of octyl glucoside-solubilized photosystem II membranes in the presence and absence of Mg2+ enabled the isolation of the Chla . P2 complex and revealed the existence of a light-harvesting complex consisting of CP29, CP26, and CP24. This complex and the major light-harvesting system (LHCII) are postulated to transfer excitation energy independently to the photosystem II reaction center via CP43.