Mutations of arginine 64 within the putative Ca(2+)-binding lumenal interhelical a-b loop of the photosystem II D1 protein disrupt binding of the manganese stabilizing protein and cytochrome c(550) in Synechocystis sp. PCC6803

Mutations D1-R64E, D1-R64Q, and D1-R64V in the putative calcium-binding lumenal interhelical a-b loop of the photosystem II (PSII) D1 protein were characterized in terms of impact on growth, extrinsic protein binding, photoactivation, and properties of the H(2)O-oxidation complex. The D1-R64E charge reversal mutation greatly weakened the binding of the extrinsic manganese-stabilizing protein (MSP) and, to a considerably lesser extent, weakened the binding of cytochrome c(550) (c550). Both D1-R64Q and D1-R64E exhibited an increased requirement for Ca(2+) in the cell growth medium. Bare platinum electrode measurements of O(2)-evolving membranes showed a retarded appearance of O(2) following single turn-over flashes, especially in the case of the D1-R64E mutant. The D1-R64E mutant also had a pronounced tendency to lose O(2) evolution activity in the dark and exhibited an increased relative quantum yield of photoactivation, which are characteristics shared by mutants that lack extrinsic proteins. S(2) and S(3) decay measurements in the isolated membranes indicate that D1-R64E and D1-R64Q have faster decays of these higher S-states as compared to the wild-type. However, fluorescence decay in the presence of DCMU, which monitors primarily Q(A)(-) charge recombination with PSII donors, showed somewhat slower decays. Taken together, the fluorescence and S-state decay indicate that the midpoint of either Q(B)(-) has been modified to be more negative in the mutants or that a recombination path presumably involving either Q(B)(-) or Y(D) has become kinetically more accessible.     

Probing the CD lumenal loop region of the D2 protein of photosystem II in Synechocystis sp. strain PCC 6803 by combinatorial mutagenesis

The CD lumenal loop region of the photosystem II reaction center protein D2 contains residues involved in oxygen evolution. Since detailed structural information about this region is unavailable, an M13-based combinatorial mutagenesis approach was used to investigate structure-function relationships in this vital region of D2 in Synechocystis sp. strain PCC 6803. The CD loop coding region contains close to 100 nucleotides, and for effective mutagenesis, it was subdivided into four regions of seven to eight codons. A gain-of-function selection protocol was employed such that all mutants that were selected contained a functional D2 protein. In this way, conservation patterns of residues along with numbers and types of amino acid substitutions accommodated at each position for each set of mutants would indicate which residues in the CD loop may play important structural and functional roles. Results of this study have substantiated the importance of residues previously studied by site-directed mutagenesis such as Arg180 and His189 and have identified other previously unremarkable residues in the CD loop (such as Ser166, Phe169, and Ala170) that cannot be replaced by many other residues. In addition, the pliability of the CD loop was further tested using deletion and D1-D2 substitution constructs in M13. This showed that the length of the loop was important to its function, and in two cases, D2 could accommodate homologous sequences from D1, which forms a heterodimer with D2 in photosystem II, but not the other way around. This study of the CD loop in D2 provides valuable clues regarding the structural and functional requirements of the region.     

Random chemical mutagenesis of a specific psbDI region coding for a lumenal loop of the D2 protein of photosystem II in Synechocystis sp. PCC 6803

To identify amino acid residues of the D2 protein that are critical for functional photosystem II (PS II), sodium bisulfite was utilized for in vitro random mutagenesis of the psbDI gene from Synechocystis sp. PCC 6803. Sodium bisulfite reacts specifically with cytosine in single-stranded regions of DNA and does not attack double-stranded DNA. Using a hybrid plasmid that was single-stranded in the region to be mutagenized and that was double-stranded elsewhere, mutations were targeted to a specific psbDI region coding for the lumenal A-B loop of the D2 protein. Several mutants were isolated with a total of 15 different amino acid changes in the loop. The majority of these mutations did not result in a loss of photoautotrophic growth or in significantly altered PS II function. However, mutation of Glu-69 to Lys, Ser-79 to Phe, and Ser-88 to Phe were found to influence photosystem II activity; the importance of the latter two residues for proper PS II function was unexpected. Cells carrying the double mutation S79F/S88F in D2 did not grow photoautotrophically and had no functionally active PS II centers. The single mutant S79F was also incapable of photoautrophic growth, but displayed reasonably stable oxygen evolution, while PS II function in the single mutant S88F appeared to be close to normal. Because of the more pronounced phenotype of the S79F/S88F strain as compared to the single mutants, both Ser residues appear to affect stable assembly and function of the PS II complex. The mechanism by which the S79F mutant loses photoautotrophic growth remains to be established. However, these results show the potential of targeted random mutagenesis to identify functionally important residues in selected regions of proteins.     

Deletion mutations in a long hydrophilic loop in the photosystem II chlorophyll-binding protein CP43 in the cyanobacterium Synechocystis sp. PCC 6803

In order to investigate the role and function of the hydrophilic region between transmembrane regions V and CI in the photosystem II core antenna protein CP43, we introduced eight different deletions in psbC of Synechocystis sp; PCC 6803 resulting in a loss of 7–11 codons in evolutionary conserved domains in this region. All deletions resulted in an obligate photoheterotrophic phenotype (requirement of glucose for cell growth) and the absence of any detectable oxygen evolution activity. The various deletion mutations showed a different impact on the amount of CP43 in the thylakoid, ranging from wild-type levels of (a now slightly smaller) CP43 to no detectable CP43 at all. All deletions led to a decrease in the amount of the D1 and D2 proteins in the thylakoids with a larger effect on D2 than on D1. CP47, the other major chlorophyll-binding protein, was present in reduced but significant amounts in the thylakoid. Herbicide binding (diuron) was lost in all but one mutant indicating the PSII components are not assembled into functionally intact complexes. Fluorescence-emission spectra confirmed this notion. This indicates that the large hydrophilic loop of CP43 plays an important role in photosystem II, and even though a shortened CP43 is present in thylakoids of most mutants, functional characteristics resemble that of a mutant with interrupted psbC.Abbreviations CP chlorophyll-binding protein - DCPIP 2,6-dichlorophenolindophenol - DPC diphenylcarbazide - ferricyanide K₃Fe(CN)₆ - HEPES N-(2-hydroxyelthyl)piperazine-N-(2-hydroxypropane sulfonic acid) - MES 2-(N-morpholino)-ethanesulfonic acid - PCC Pasteur Culture Collection - PCR polymerase chain reaction - PS photosystem - Q_A first quinone acceptor in PSII - Q_B second quinone acceptor in PSII - Z redox-active tyrosine (Y161) in D1 serving as electron carrier between the Mn cluster and P680     

Site-specific mutations localized in the D-E loop of the D1 protein of photosystem II affect phototolerance in Synechocystis sp. PCC 6803 containing psbAII gene

Photosynthetic characteristics along with phototolerance and photoinhibition of photosystem II (PS II) were monitored in Synechocystis sp. PCC 6803 wild type (KC) and its psbAII mutants viz., I6 (N322I, I326F, and F328S), G6 (N267Y), and H7 (Y254C and I314V) that have up to three point mutations, localized in the D-E loop of the D1 polypeptide of PSII reaction centre. These strains exhibited entirely different growth trends upon shifting from 30 micormol m(-2)s(-1) to high irradiance (500 micromol m(-2)s(-1) , 30 degrees C). The I6 and H7 cells grew well, whereas KC and G6 cells showed inability for cell multiplication. The photosynthetic efficiency demonstrated about 50% loss in chlorophyll fluorescence of variable yield (Fv/Fm) within 20-30 min in all mutants, whereas the wild type (KC) cells could reach the same level of loss in 2 hr. I6 and H7 cells showed continuous cell growth and maintenance under long-term exposure of high light compared to G6 mutant and wild type cells. The wild type cells showed slow decrease in their photochemical activity and Fv/Fm values, compared to mutant cells. The recovery seemed to be almost identical, and also stimulated by growth light, inspite of differential photoinhibitory behaviours. Darkness and translational inhibitor lincomycin both were found to be unassociated with the restoration of photoinhibited process of PS II.     

Oxygen-evolving photosystem II preparation from wild type and photosystem II mutants of Synechocystis sp. PCC 6803.

We present here a simple and rapid method which allows relatively large quantities of oxygen-evolving photosystem II- (PS-II-) enriched particles to be obtained from wild-type and mutants of the cyanobacterium Synechocystis 6803. This method is based on that of Burnap et al. [Burnap, R., Koike, H., Sotiropoulou, G., Sherman, L. A., & Inoue, Y. (1989) Photosynth. Res. 22, 123-130] but is modified so that the whole preparation, from cells to PS-II particles, is achieved in 10 h and involves only one purification step. The purified preparation exhibits a 5-6-fold increase of O2-evolution activity on a chlorophyll basis over the thylakoids. The ratio of PS-I to PS-II is about 0.14:1 in the preparation. The secondary quinone electron acceptor, QB, is present in this preparation as demonstrated by thermoluminescence studies. These PS-II particles are well-suited to spectroscopic studies as demonstrated by the range of EPR signals arising from components of PS-II that are easily detectable. Among the EPR signals presented are those from a formal S3-state, attributed to an oxidized amino acid interacting magnetically with the Mn complex in Ca(2+)-deficient PS-II particles, and from S2 modified by the replacement of Ca2+ by Sr2+. Neither of these signals has been previously reported in cyanobacteria. Their detection under these conditions indicates a similar lesion caused by Ca2+ depletion in both plants and cyanobacteria. The protocol has also been applied to mutants which have site-specific changes in PS-II. Data are presented on mutants having changes on the electron donor (Y160F) and electron acceptor (G215W) side of the D2 polypeptide.     

Kinetic characterization of His-tagged CP47 photosystem II in Synechocystis sp. PCC6803

Recently, construction of strains of Synechocystis sp. PCC6803 having a His(6) extension (His-tag) of the carboxyl terminus of the CP47 protein has been reported (T.M. Bricker et al, Biochim. Biophys. Acta 1409 (1998) 50; M.J. Reifler et al., in: Garab, Pusztai (Eds.) Proc. XIth International Congress on Photosynthesis, 1998). While these initial reports suggest a minimal impact of the His-tag upon Photosystem (PS) II function, a more thorough analysis of the kinetic properties of the modified complex is essential. This communication reports on a more detailed kinetic analysis to assess possible perturbations of PS II due to the genetic addition of the His-tag on the CP47 protein. It was found that: (1) Patterns of flash O(2) yield exhibited normal period four oscillations and the associated fits of the Kok-Joliot S-state cycling parameters were virtually identical to the wild type; (2) O(2) release kinetics during the S(3)-S(0) transition were experimentally indistinguishable from the wild type; (3) S-state decay measurements indicate slightly faster decays of the S(2) and S(3) states compared to the wild type; (4) fluorescence measurements indicate that the kinetics of the forward reaction of electron transfer from Q(A)(-) to Q(B) and back-reactions of Q(A)(-) with PS II electron donors are similar in the His-tag and wild-type strains. It is therefore concluded that the addition of the His-tag results in a minimal perturbation of PS II function.     

Functional analysis of combinatorial mutants with changes in the C-terminus of the CD loop of the D2 protein in photosystem II of Synechocystis sp. PCC 6803

Photosystem II properties were investigated in a set of combinatorial mutants containing changes in the C-terminal end of the CD lumenal loop (Gly187-Asn194) in the D2 protein of Synechocystis sp. PCC 6803. Initial screening of variable fluorescence (F(v)) induction and decay in the presence of DCMU showed that all but one of the combinatorial strains tested had an increased rate of Q(A)(-) reoxidation. Two strains showed an increase in the amplitude of constant fluorescence (F(o)). Examination of the primary sequence of the combinatorial strains combined with results obtained from analysis of site-directed mutants suggested that alterations in residue 191 of D2 increased the rate of charge recombination. Indeed, reintroduction of Trp191, the residue present in wild type, slowed the Q(A)(-) reoxidation rate in the presence of DCMU by 2-3-fold. However, the nature of other residues, in particular at codon 192, was also important in determining charge recombination rates. The increase in F(o) yield was due to an increased fluorescence lifetime of open reaction centers in intact cells and may reflect a decreased excitation trapping rate in the reaction center. This change was reversed by reintroduction of Trp191 even though a mutant lacking just Trp191 was normal in this respect. Trapping efficiency therefore was decreased only when multiple changes were present at the same time. We interpret Trp191 and neighboring residues to influence the midpoint redox potential of P680/P680(+) and in certain sequence contexts to affect the energy trapping efficiency by P680. The stability or environment of Y(D)(ox) was essentially unaffected in the mutants. Interestingly, many combinatorial mutants displayed an increased requirement for chloride for photoautotrophic growth, and two mutants, C8-10 and C8-23, also required more calcium. This indicates that this CD loop region of D2 not only affects properties of P680 but also affects properties of the oxygen-evolving complex.     

Lifetimes of photosystem I and II proteins in the cyanobacterium Synechocystis sp. PCC 6803

The half-life times of photosystem I and II proteins were determined using (15)N-labeling and mass spectrometry. The half-life times (30-75h for photosystem I components and <1-11h for the large photosystem II proteins) were similar when proteins were isolated from monomeric vs. oligomeric complexes on Blue-Native gels, suggesting that the two forms of both photosystems can interchange on a timescale of <1h or that only one form of each photosystem exists in thylakoids in vivo. The half-life times of proteins associated with either photosystem generally were unaffected by the absence of Small Cab-like proteins.     

Ammonia triggers photodamage of photosystem II in the cyanobacterium Synechocystis sp. strain PCC 6803

Ammonia has long been known to be toxic for many photosynthetic organisms; however, the target for its toxicity remains elusive. Here, we show that in the cyanobacterium Synechocystis sp. strain PCC 6803, ammonia triggers a rapid photodamage of photosystem II (PSII). Whereas wild-type cells can cope with this damage by turning on the FtsH2-dependent PSII repair cycle, the FtsH2-deficient mutant is highly sensitive and loses PSII activity at millimolar concentration of ammonia. Ammonia-triggered PSII destruction is light dependent and occurs already at low photon fluence rates. Experiments with monochromatic light showed that ammonia-promoted PSII photoinhibition is executed by wavebands known to directly destroy the manganese cluster in the PSII oxygen-evolving complex, suggesting that the oxygen-evolving complex may be a direct target for ammonia toxicity.     

In situ effects of mutations of the extrinsic cytochrome c550 of photosystem II in Synechocystis sp. PCC6803

The H(2)O oxidizing domain of the cyanobacterial photosystem II (PSII) complex contains a low potential, c-type cytochrome termed c(550) that is essential for the in vivo stability of the PSII complex. A mutant lacking cytochrome c(550) (DeltapsbV) in Synechocystis sp. PCC6803 has been further analyzed together with a construct in which the distal axial heme iron ligand, histidine 92, has been substituted with a methionine (C550-H92M). Heme staining of SDS-PAGE showed that the C550-H92M mutation did not disturb the accumulation and heme-binding properties of the cytochrome. In DeltapsbV cells, the number of charge separating PSII centers was estimated to be 56% of the wild type, but of the existing centers, 33% lacked photooxidizable Mn ions. C550-H92M did not discernibly affect the intrinsic PSII electron-transfer kinetics compared to the wild type nor did it exhibit a significant fraction of centers lacking photooxidizable Mn; however, the number of charge separating PSII centers in mutant cells was 69% of the wild type. C550-H92M lost photoautotrophic growth ability in the absence of Ca(2+), but its growth was not affected by depletion of Cl(-), which differs from DeltapsbV. Taken together, the results suggest that in the absence of cytochrome c(550) electron transfer on the donor side is retarded perhaps at the level of Y(z) to P680(+) transfer, the heme ligand. His92 is not absolutely required for assembly of functional PSII centers; however, replacement by methionine prevents normal accumulation of PSII centers in the thylakoid membranes and alters the Ca(2+) requirement of PSII. The results are discussed in terms of current understanding of the Ca(2+) site of PSII.     

Requirement of carotene isomerization for the assembly of photosystem II in Synechocystis sp. PCC 6803

Carotene isomerase mutant (crtH mutant) cells of Synechocystis sp. PCC 6803 can accumulate beta-carotene under light conditions. However, the mutant cells grown under a light-activated heterotrophic growth condition contained detectable levels of neither beta-carotene nor D1 protein of the photosystem (PS) II reaction center, and no oxygen-evolving activity of PSII was detected. beta-Carotene and D1 protein appeared and a high level of PSII activity was detected after the cells were transferred to a continuous light condition. The PSI activities of thylakoid membranes from mutant cells were almost the same as those of thylakoid membranes from wild-type cells, both before and after transfer to the continuous light condition. These results suggest that beta-carotene is required for the assembly of PSII but not for that of PSI.     

Role of the PsbI protein in photosystem II assembly and repair in the cyanobacterium Synechocystis sp. PCC 6803

The involvement of the PsbI protein in the assembly and repair of the photosystem II (PSII) complex has been studied in the cyanobacterium Synechocystis sp. PCC 6803. Analysis of PSII complexes in the wild-type strain showed that the PsbI protein was present in dimeric and monomeric core complexes, core complexes lacking CP43, and in reaction center complexes containing D1, D2, and cytochrome b-559. In addition, immunoprecipitation experiments and the use of a histidine-tagged derivative of PsbI have revealed the presence in the thylakoid membrane of assembly complexes containing PsbI and either the precursor or mature forms of D1. Analysis of PSII assembly in the psbI deletion mutant and in strains lacking PsbI together with other PSII subunits showed that PsbI was not required for formation of PSII reaction center complexes or core complexes, although levels of unassembled D1 were reduced in its absence. However, loss of PsbI led to a dramatic destabilization of CP43 binding within monomeric and dimeric PSII core complexes. Despite the close structural relationship between D1 and PsbI in the PSII complex, PsbI turned over much slower than D1, whereas high light-induced turnover of D1 was accelerated in the absence of PsbI. Overall, our results suggest that PsbI is an early assembly partner for D1 and that it plays a functional role in stabilizing the binding of CP43 in the PSII holoenzyme.     

Mutations in the CD-loop region of the D2 protein in Synechocystis sp. PCC 6803 modify charge recombination pathways in photosystem II in vivo

The lumenal CD-loop region of the D2 protein of photosystem II contains residues that interact with the primary electron donor P680 and the redox active tyrosyl residue Y(D). Photosystem II properties were studied in a number of photoautotrophic mutants of Synechocystis sp. PCC 6803, most of which carried combinatorial mutations in residues 164-170, 179-186, or 187-194 of the D2 protein. To facilitate characterization of photosystem II properties in the mutants, the CD-loop mutations were introduced into a photosystem I-less background. According to variable fluorescence decay measurements in DCMU-treated cells, charge recombination of Q(A)(-) with the donor side was faster in the majority of mutants (t(1/2) = 45-140 ms) than in the control (t(1/2) = 180 ms). However, in one mutant (named C7-3), the decay of Q(A)(-) was 2 times slower than in the control (t(1/2) = 360 ms). The decay half-time of each mutant correlated with the yield of the Q-band of thermoluminescence (TL) emitted due to S(2)Q(A)(-) charge recombination. The C7-3 mutant had the highest TL intensity, whereas no Q-band was detected in the mutants with fast Q(A)(-) decay (t(1/2) = 45-50 ms). The correlated changes in the rate of recombination and in TL yield in these strains suggest the existence of a nonradiative pathway of charge recombination between Q(A)(-) and the donor side. This may involve direct electron transfer from Q(A)(-) to P680(+) in a way not leading to formation of excited chlorophyll. Many mutations in the CD-loop appear to increase the equilibrium P680(+) concentration during the lifetime of the S(2)Q(A)(-) state, for example, by making the midpoint potential of the P680(+)/P680 redox couple more negative. The nonradiative charge recombination pathway involves a low activation energy and is less temperature-dependent than the formation of excited P680 that leads to TL emission. Therefore, during the TL measurements in these mutants, the S(2)Q(A)(-) state can recombine nonradiatively before temperatures are reached at which radiative charge recombination becomes feasible. The results presented here highlight the presence of two charge recombination pathways and the importance of the CD-loop of the D2 protein in determination of the energy gap between the P680(+)S(1) and P680S(2) states.     

Nitric oxide represses photosystem II and NDH-1 in the cyanobacterium Synechocystis sp. PCC 6803

Photosynthetic electron transfer comprises a series of light-induced redox reactions catalysed by multiprotein machinery in the thylakoid. These protein complexes possess cofactors susceptible to redox modifications by reactive small molecules. The gaseous radical nitric oxide (NO), a key signalling molecule in green algae and plants, has earlier been shown to bind to Photosystem (PS) II and obstruct electron transfer in plants. The effects of NO on cyanobacterial bioenergetics however, have long remained obscure. In this study, we exposed the model cyanobacterium Synechocystis sp. PCC 6803 to NO under anoxic conditions and followed changes in whole-cell fluorescence and oxidoreduction of P700 in vivo. Our results demonstrate that NO blocks photosynthetic electron transfer in cells by repressing PSII, PSI, and likely the NDH dehydrogenase-like complex 1 (NDH-1). We propose that iron?sulfur clusters of NDH-1 complex may be affected by NO to such an extent that ferredoxin-derived electron injection to the plastoquinone pool, and thus cyclic electron transfer, may be inhibited. These findings reveal the profound effects of NO on Synechocystis cells and demonstrate the importance of controlled NO homeostasis in cyanobacteria.     

Slr2013 is a novel protein regulating functional assembly of photosystem II in Synechocystis sp. strain PCC 6803

The Synechocystis sp. strain PCC 6803, which has a T192H mutation in the D2 protein of photosystem II, is an obligate photoheterotroph due to the lack of assembled photosystem II complexes. A secondary mutant, Rg2, has been selected that retains the T192H mutation but is able to grow photoautotrophically. Restoration of photoautotrophic growth in this mutant was caused by early termination at position 294 in the Slr2013 protein. The T192H mutant with truncated Slr2013 forms fully functional photosystem II reaction centers that differ from wild-type reaction centers only by a 30% higher rate of charge recombination between the primary electron acceptor, QA-, and the donor side and by a reduced stability of the oxidized form of the redox-active Tyr residue, YD, in the D2 protein. This suggests that the T192H mutation itself did not directly affect electron transfer components, but rather affected protein folding and/or stable assembly of photosystem II, and that Slr2013 is involved in the folding of the D2 protein and the assembly of photosystem II. Besides participation in photosystem II assembly, Slr2013 plays a critical role in the cell, because the corresponding gene cannot be deleted completely under conditions in which photosystem II is dispensable. Truncation of Slr2013 by itself does not affect photosynthetic activity of Synechocystis sp. strain PCC 6803. Slr2013 is annotated in CyanoBase as a hypothetical protein and shares a DUF58 family signature with other hypothetical proteins of unknown function. Genes for close homologues of Slr2013 are found in other cyanobacteria (Nostoc punctiforme, Anabaena sp. strain PCC 7120, and Thermosynechococcus elongatus BP-1), and apparent orthologs of this protein are found in Eubacteria and Archaea, but not in eukaryotes. We suggest that Slr2013 regulates functional assembly of photosystem II and has at least one other important function in the cell.     

PratA, a periplasmic tetratricopeptide repeat protein involved in biogenesis of photosystem II in Synechocystis sp. PCC 6803

The light reactions of oxygenic photosynthesis are mediated by multisubunit pigment-protein complexes situated within the specialized thylakoid membrane system. The biogenesis of these complexes is regulated by transacting factors that affect the expression of the respective subunit genes and/or the assembly of their products. Here we report on the analysis of the PratA gene from the cyanobacterium Synechocystis sp. PCC 6803 that encodes a periplasmic tetratricopeptide repeat protein of formerly unknown function. Targeted inactivation of PratA resulted in drastically reduced photosystem II (PSII) content. Protein pulse labeling experiments of PSII subunits indicated that the C-terminal processing of the precursor of the reaction center protein D1 is compromised in the pratA mutant. Moreover, a direct interaction of PratA and precursor D1 was demonstrated by applying yeast two-hybrid analyses. This suggests that PratA represents a factor facilitating D1 maturation via the endoprotease CtpA. The periplasmic localization of PratA supports a model that predicts the initial steps of PSII biogenesis to occur at the plasma membrane of cyanobacterial cells.