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This study describes an analysis of different treatments that influence the relative content and the midpoint potential of HP Cyt b559 in PS II membrane fragments from higher plants. Two basically different types of irreversible modification effects are distinguished: the HP form of Cyt b559 is either predominantly affected when the heme group is oxidized ("O-type" effects) or when it is reduced ("R-type" effects). Transformation of HP Cyt b559 to lower potential redox forms (IP and LP forms) by the "O-type" mechanism is induced by high pH and detergent treatments. In this case the effects consist of a gradual decrease in the relative content of HP Cyt b559 while its midpoint potential remains unaffected. Transformation of HP Cyt b559 via an "R-type" mechanism is caused by a number of exogenous compounds denoted L: herbicides, ADRY reagents and tetraphenylboron. These compounds are postulated to bind to the PS II complex at a quinone binding site designated as Q(C) which interacts with Cyt b559 and is clearly not the Q(B) site. Binding of compounds L to the Q(C) site when HP Cyt b559 is oxidized gives rise to a gradual decrease in the E(m) of HP Cyt b559 with increasing concentration of L (up to 10 K(ox)(L) values) while the relative content of HP Cyt b559 is unaffected. Higher concentrations of compounds L required for their binding to Q(C) site when HP Cyt b559 is reduced (described by K(red)(L)) induce a conversion of HP Cyt b559 to lower potential redox forms ("R-type" transformation). Two reaction pathways for transitions of Cyt b559 between the different protein conformations that are responsible for the HP and IP/LP redox forms are proposed and new insights into the functional regulation of Cyt b559 via the Q(C) site are discussed.

Photosystem II core complex (PSII CC) absorbs light energy and triggers a series of electron transfer reactions by oxidizing water while producing molecular oxygen. Synthetic lipids with different alkyl chains and spacer lengths bearing functionalized headgroups were specifically designed to bind the Q(B) site and to anchor this large photosynthetic complex (240 kDa) in order to attempt two-dimensional crystallization. Among the series of different compounds that have been tested, oxygen evolution measurements have shown that dichlorophenyl urea (DCPU) binds very efficiently to the Q(B) site of PSII CC, and therefore, that moiety has been linked covalently to the headgroup of synthetic lipids. The analysis of the monolayer behavior of these DCPU-lipids has allowed us to select ones bearing long spacers for the anchoring of PSII CC. Oxygen evolution measurements demonstrated that these long-spacer DCPU-lipids specifically bind to PSII CC and inhibit electron transfer. With the use of atomic force microscopy (AFM) and scanning near-field optical microscopy (SNOM), it was possible to visualize domains of PSII CC bound to DCPU-lipid monolayers. SNOM imaging has enabled us to confirm that domains observed by AFM were composed of PSII CC. Indeed, the SNOM topography images presented similar domains as those observed by AFM, but in addition, it allowed us to determine that these domains are fluorescent. Electron microscopy of these domains, however, has shown that the bound PSII CC was not crystalline.

Here we show that herbicide binding influences the redox potential (Em) of the plastoquinone QA/QA- redox couple in Photosystem II (PSII). Phenolic herbicides lower the Em by approximately 45 mV, while DCMU raises the Em by 50 mV. These shifts are reflected in changes in the peak temperature of thermoluminescence bands arising from the recombination of charge pairs involving QA-. The herbicide-induced changes in the Em of QA/QA- correlate with earlier work showing that phenolic herbicides increase the sensitivity of PSII to light, while DCMU protects against photodamage. This correlation is explained in terms of the following hypothesis which is based on reactions occurring in the bacterial reaction center. The back-reaction pathway for P680+QA- is assumed to be modulated by the free-energy gap between the P680+QA- and the P680+Ph- radical pairs. When this gap is small (i.e., when the Em of QA/QA- is lowered), a true back-reaction is favored in which P680+Ph- is formed, a state which decays forming a significant yield of P680 triplet. This triplet state of chlorophyll reacts with oxygen, forming singlet oxygen, a species likely to be responsible for photodamage. When the free-energy gap is increased (i.e., when the Em of QA/QA- is raised), the yield of the P680+Ph- is diminished and a greater proportion of the P680+QA- radical pair decays by an alternative, less damaging, route. We propose that at least some of the phytotoxic properties of phenolic herbicides may be explained by the fact that they render PSII ultrasensitive to light due to this mechanism.

Our previous work using the "diphenylcarbazide (DPC)-inhibition assay" has identified four amino acid (two carboxyls and two histidyls) ligands to four Mn2+ bound with high affinity on Tris-washed photosystem II (PSII) membrane fragments [Preston and Seibert (1991) Biochemistry 30, 9615-9624, 9625-9633]. One of the ligands binds a photooxidizable Mn, specifically, and the others bind either nonphotooxidizable Mn2+, Zn2+, or Co2+ [Ghirardi et al. (1996) Biochemistry 35, 1820-1828]. The current paper shows the following: (a) the high-affinity photooxidizable Mn, which donates to the oxidized primary PSII donor (YZ*), is bound to a carboxyl residue with a KM = 1.5 microM or Kd = 0.94 microM in the absence of DPC, and a Ki = 1.3 microM in the presence of DPC (both steady-state and flash approaches were used); (b) if this carboxyl is chemically modified using 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide hydrochloride (EDC), Mn2+ is photooxidized at a lower affinity (Kd = 25 microM) site that does not involve carboxyl ligands; (c) low-affinity Mn is photooxidized (possibly by YD*, the oxidized form of the alternative PSII donor) with a KM = 220 microM at a completely different site that also requires a carboxyl ligand; (d) photooxidation of high-affinity DPC by YZ* with a KM of 40-42 microM or Kd of 49-58 microM occurs at a site that does not require carboxyl residues; (e) photooxidation of low-affinity DPC with a KM = 1200 microM occurs at a site (possibly near YD) that is not affected by carboxyl modification with EDC. Due to the similarities between the binding of the high-affinity photooxidizable Mn to EDC-treated membranes and to PSII complexes from Asp170D1 mutants [Nixon and Diner (1992) Biochemistry 31, 942-948], we identify its carboxyl residue ligand as Asp170 on D1, one of the reaction-center proteins. The second carboxyl ligand identified using the DPC-inhibition assay binds Mn (but not a photooxidizable one), Zn, or Co ions. At least one of the two histidyl ligands (either His337 on D1 or another unidentified histidyl) that bind nonphotooxidizable, high-affinity Mn2+ also binds Zn2+ and Co2+.

Upon introduction of random mutations in a region of the psbDI gene that encodes the D2 protein in the cyanobacterium Synechocystis sp. PCC 6803, an obligate photoheterotrophic mutant was isolated that contained three mutations: V247M, A249T, and M329I. This mutant evolved oxygen in the absence of added electron acceptors, but oxygen evolution was inhibited by micromolar concentrations of several artificial quinones. Complementation analysis showed that the V247M and/or A249T mutations were responsible for this phenotype. Using fluorescence induction and decay measurements, the site of inhibition by the quinones was found to be at the level of the primary electron-accepting quinone in photosystem II, QA. Duroquinone inhibited by blocking reduction of QA, and in the presence of other quinones such as 2,5-dichloro-p-benzoquinone, 2, 5-dimethyl-p-benzoquinone, and p-benzoquinone, QA could be reduced but could not efficiently transfer an electron to QB. To distinguish the effects of the V247M and A249T mutations, single mutants were created. V247M was photoautotrophic and had an essentially normal phenotype. The A249T mutant, although photoautotrophic, was affected by artificial quinones, but less than the mutant carrying both the V247M and A249T changes. The results indicate a decreased plastoquinone affinity at the QA site in the strains carrying a A249T mutation, such that after dark-adaptation a significant percentage of the QA sites is empty or is occupied by an artificial quinone. In light, the percentage of photosystem II centers with plastoquinone bound at the QA site appears to increase, which may be due in part to an increased affinity of the semiquinone versus that of the quinone at the QA site.

A three-dimensional model of the photosystem II (PSII) reaction center from the cyanobacterium Synechocystis sp. PCC 6803 was generated based on homology with the anoxygenic purple bacterial photosynthetic reaction centers of Rhodobacter sphaeroides and Rhodopseudomonas viridis, for which the X-ray crystallographic structures are available. The model was constructed with an alignment of D1 and D2 sequences with the L and M subunits of the bacterial reaction center, respectively, and by using as a scaffold the structurally conserved regions (SCRs) from bacterial templates. The structurally variant regions were built using a novel sequence-specific approach of searching for the best-matched protein segments in the Protein Data Bank with the "basic local alignment search tool" (Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ, 1990, J Mol Biol 215:403-410), and imposing the matching conformational preference on the corresponding D1 and D2 regions. The structure thus obtained was refined by energy minimization. The modeled D1 and D2 proteins contain five transmembrane alpha-helices each, with cofactors (4 chlorophylls, 2 pheophytins, 2 plastoquinones, and a non-heme iron) essential for PSII primary photochemistry embedded in them. A beta-carotene, considered important for PSII photoprotection, was also included in the model. Four different possible conformations of the primary electron donor P680 chlorophylls were proposed, one based on the homology with the bacterial template and the other three on existing experimental suggestions in literature. The P680 conformation based on homology was preferred because it has the lowest energy. Redox active tyrosine residues important for P680+ reduction as well as residues important for PSII cofactor binding were analyzed. Residues involved in interprotein interactions in the model were also identified. Herbicide 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) was also modeled in the plastoquinone QB binding niche using the structural information available from a DCMU-binding bacterial reaction center. A bicarbonate anion, known to play a role in PSII, but not in anoxygenic photosynthetic bacteria, was modeled in the non-heme iron site, providing a bidentate ligand to the iron. By modifying the previous hypothesis of Blubaugh and Govindjee (1988, Photosyn Res 19:85-128), we modeled a second bicarbonate and a water molecule in the QB site and we proposed a hypothesis to explain the mechanism of QB protonation mediated by bicarbonate and water. The bicarbonate, stabilized by D1-R257, donates a proton to QB2- through the intermediate of D1-H252; and a water molecule donates another proton to QB2-. Based on the discovery of a "water transport channel" in the bacterial reaction center, an analogous channel for transporting water and bicarbonate is proposed in our PSII model. The putative channel appears to be primarily positively charged near QB and the non-heme iron, in contrast to the polarity distribution in the bacterial water transport channel. The constructed model has been found to be consistent with most existing data.

Functional identity and significant similarities in cofactors and sequence exist between the L and M reaction center proteins of the photosynthetic bacteria and the D1 and D2 photosystem-II reaction center proteins of cyanobacteria, algae, and plants. A model of the quinone (QB) binding site of the D1 protein is presented based upon the resolved structure of the QB binding pocket of the L subunit, and introducing novel quantitative notions of complementarity and contact surface between atoms. This model, built without using traditional methods of molecular mechanics and restricted to residues in direct contact with QB, accounts for the experimentally derived functional state of mutants of the D1 protein in the region of QB. It predicts the binding of both the classical and phenol-type PSII herbicides and rationalizes the relative levels of tolerance of mutant phenotypes.

Several site-directed photosystem II mutants with substitutions at Asp-170 of the D1 polypeptide were characterized by noninvasive methods in vivo. In several mutants, including some that evolve oxygen, a significant fraction of photosystem II reaction centers are shown to lack photooxidizable Mn ions. In this fraction of reaction centers, either the high-affinity site from which Mn ions rapidly reduce the oxidized secondary electron donor, YZ+, is devoid of Mn ions or the Mn ion(s) bound at this site are unable to reduce YZ+. It is concluded that the Mn clusters in these mutants are unstable or are assembled inefficiently in vivo. Mutants were constructed in the unicellular cyanobacterium Synechocystis sp. PCC 6803. The in vivo characterization procedures employed in this study involved measuring changes in the yield of variable chlorophyll a fluorescence following a saturating flash or brief illumination given in the presence of the electron transfer inhibitor 3-(3,4-dichlorophenyl)-1,1-dimethylurea, or following each of a series of saturating flashes given in the absence of this inhibitor. These procedures are easily applied to mutants that evolve little or no oxygen, facilitate the characterization of mutants with labile oxygen-evolving complexes, permit photosystem II isolation efforts to be concentrated on mutants having the stablest Mn clusters, and guide systematic spectroscopic studies of isolated photosystem II particles to mutants of particular interest.

The filamentous nitrogen-fixing cyanobacterium Anabaena variabilis ATCC 29413 is capable of heterotrophic growth in complete darkness. After 6 months of continuous dark growth, both the autotrophic and heterotrophic cultures were found to have the same doubling time of 14 h. On a cellular basis, the chlorophyll content remained the same and the phycobilin content showed an increase in the dark-grown cultures. Fluorescence emission spectra at 77 K of dark-grown cells indicated that the phycobilisomes are functionally associated with photosystem II (PSII). Moreover, upon transfer to light, the dark-grown cells readily evolved oxygen. Although photosystem I (PSI) and whole chain-mediated electron transfer rates were comparable in both types of cultures, the rate of PSII-mediated electron transfer was found to be 20% higher in dark-grown cells. The PSI to PSII ratio changed from 6:1 in autotrophic cultures to 4:1 in the dark-grown cells. These changes in the rate of PSII electron transfer and in the stoichiometry between the two photosystems under dark, heterotrophic growth conditions were brought about by a preferential increase in the number of PSII units while the number of PSI units remained unchanged. The advantages of using this organism in the selection of PSI-deficient mutants are discussed.

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 resembled that of a mutant with interrupted psbC.

The two reaction-centre proteins of the photosystem I (PSI) complex are encoded by two adjacent genes named psaA and psaB. We have performed targeted mutagenesis to insertionally inactivate each of these genes in the filamentous cyanobacterium Anabaena variabilis ATCC 29413. The resulting mutant strains, termed psaA::NmR and psaB::NmR, were blue because of a high ratio of phycobilin to chlorophyll and were unable to grow in light. These mutant cells also lacked chemically reducible P700 (the reaction-centre chlorophylls of PSI) and as a consequence did not exhibit any PSI-mediated photochemical activity. However, their photosystem II (PSII) complexes were fully active. The loss of the PsaA and PsaB proteins and their associated chlorophyll molecules resulted in a five- to sevenfold decrease in the chlorophyll/PSII ratio in the mutant cells relative to the wild-type cells. Interestingly, the psaB::NmR and not the psaA::NmR mutant strain retained a small fluorescence peak (77K) at 721 nm originating from chlorophyll molecule(s) presumably bound to a small amount of the PsaA protein present in the psaB mutant. These results demonstrate that this organism is suitable for the manipulation of PSI reaction-centre proteins.

Site-directed mutagenesis has been used to change conserved histidine residues in hydrophobic regions of the photosystem II chlorophyll-binding protein CP47 in the cyanobacterium Synechocystis sp. PCC 6803. Nine mutants with one, four mutants with two, and four mutants with three His mutations in CP47 have been generated and characterized. Mutation of any one of seven different His residues to Tyr leads to slower photoautotrophic growth and apparent destabilization of the PS II complex. Mutations introduced into multiple His residues in one mutant exhibited a cumulative effect. Replacing His by Asn leads to a much smaller effect than observed upon mutation to Tyr. This is consistent with the hypothesis that the mutated His residues are chlorophyll ligands: Asn can substitute as chlorophyll ligand, whereas Tyr cannot. Further evidence supporting a role of the mutated His residues in chlorophyll binding comes from measurements of the light intensity needed to half-saturate oxygen evolution. All His mutants with impaired PS II function needed higher light intensities for half-saturation than wild type. A possible explanation for this decrease in antenna efficiency in the mutants is a loss of the Mg in the chlorophyll due to a loss of the fifth ligand, and thus the formation of a pheophytin molecule in the antenna. We conclude that conserved His residues in hydrophobic regions of CP47 indeed are chlorophyll ligands and that these ligands are important for PS II stability as well as efficient antenna function.

The D2 protein contains an extended loop (the D-de loop) between helices D and de at the reducing side of photosystem II (PS II). Characterization of D2 mutants of the cyanobacterium Synechocystis sp. PCC 6803 has indicated that the length and amino acid composition of the D-de loop are not critical for basic PS II functions, although most of the residues in that region are conserved phylogenetically. Here we show using herbicide binding and electron-flow inhibition measurements that drastic modifications in the D-de loop of the D2 protein modify the interaction of some PS II-directed herbicides with their binding niche. The stability of (semi)-reduced QB in its binding pocket is altered in at least two of the mutants, as indicated by a shifted peak temperature of the thermoluminescence signal originating from charge recombination involving QB. These results suggest a close functional association between the D-de loop of the D2 protein and the QB/herbicide-binding environment, which is viewed as being coordinated mostly by residues of the D1 protein. This represents one of the first examples of modification of the QB/herbicide-binding domain by mutations in the D2 protein.

The preferred binding orientations for the herbicide DCMU within the QB-binding of the D1 protein model from a photosystem II reaction centre have been determined. Calculation of the intermolecular energy between the herbicide and the binding site has been instrumental in obtaining optimum positions reinforced by experimental results from mutation studies and herbicide binding to analogous bacterial reaction centres. We have shown that two binding sites are possible, one involving a hydrogen bond to the His 215 residue of the QB-binding site and the other to the Ser 264 residue. In both cases, it appears to be the van der Waals forces which are more important for the stabilization of the interactions.

Previous experiments have shown that a Synechocystis sp. PCC 6803 mutant (delta psbO) lacking the extrinsic manganese-stabilizing protein (MSP) exhibits impaired, but significant levels of H2O-splitting activity [Burnap, R., & Sherman, L.A. (1991) Biochemistry 30, 440-446]. [14C]DCMU-binding experiments now show that the number and affinity of DCMU-binding sites (normalized to chlorophyll) are equivalent in delta psbO and the wild type, suggesting equal concentrations of assembled reaction centers. A similar conclusion is reached on the basis of measurements of PSII electron transport (DPC-supported DCPIP reduction) by mutant and wild-type thylakoids. The pattern of flash O2 yield by delta psbO cells measured with a bare platinum electrode exhibits a period four oscillation (with a maximum on the third flash), indicating that the H2O-splitting enzyme in delta psbO retains the basic mechanistic features found in normal cells. However, the amplitude of these signals is smaller and more highly damped than those obtained from wild-type cells, suggesting the absence of MSP results in a higher miss probability and/or a reduction in the number of centers competent in oxygen evolution. Analysis of the rise kinetics of the ampermeric signal on the bare platinum electrode indicates that the S3-[S4]-S0 transition is retarded by at least a factor of 5 in the mutant. Thermoluminescence emission peak temperatures indicate that the S2QA-, S2QB-, and S3QB-charge pairs are significantly more stable with respect to recombination in the mutant. The intensities of the thermoluminescence emissions are also significantly reduced in the mutant.(ABSTRACT TRUNCATED AT 250 WORDS)

The ureas and phenolics are two major classes of herbicides that act on Photosystem II (PSII) and are normally inactive in the photosynthetic reaction centers of purple bacteria. However, the triazine-resistant mutant T4 from Rhodopseudomonas (Rps.) viridis, which has the tyrosine residue at position 222 on the L subunit substituted for phenylalanine (TyrL222Phe), is sensitive to both ureas and phenolics. Since for the first time structural data on urea binding are available, T4 is a particularly interesting model for the herbicide-binding site of PSII.