Mutational studies on conserved histidine residues in the chlorophyll-binding protein CP43 of photosystem II

Two chlorophyll-binding antenna proteins in the photosystem II core, CP43 and CP47, are structurally similar and are thought to have evolved from a common ancestor. Several conserved histidine residues in hydrophobic regions of CP47 have been shown to be important for photosystem II structure, function, and energy transfer. The purpose of this study was to determine whether similarly located histidine residues in CP43 function in a similar way. Three conserved histidine residues in presumed membrane-spanning regions of CP43, His40, His105, and His119, were mutated to glutamine (Q) and tyrosine (Y). The strains H105Q, H119Q, and H119Y were photoautotrophs whereas H40Q, H40Y, and H105Y were obligate photoheterotrophs. The H40Y and H105Y strains lacked detectable amounts of photosystem II reaction centers and hence could not evolve oxygen whereas H40Q retained a significant amount of photosystem II and oxygen evolution capacity. The observation that mutation of histidine residues to tyrosine has more drastic effects than mutation of these residues to glutamine is in agreement with results obtained for CP47 and suggests the involvement of these residues in chlorophyll binding. The drastic functional changes observed upon mutating His40 and His105 of CP43 are similar to those observed when mutating the corresponding histidine residues in CP47, thus suggesting that the similarity between CP43 and CP47 extends to the relative importance of functionally relevant residues. Interestingly, the His40-->Gln mutation in CP43 had significant effects on photosystem II electron transfer in that it affected the thermodynamics of Q(A)- oxidation by Q(B) and increased the charge recombination rate between Q(A)- and donor side components. This indicates that relatively minor changes in CP43 can significantly impact the properties of the photosystem II reaction center. The implications of this finding are discussed.     

Role of the RCII-D1 protein in the reversible association of the oxygen-evolving complex proteins with the lumenal side of photosystem II

The nuclear-encoded proteins of the oxygen-evolving complex (OEC) of photosystem II are bound on the lumenal side of the thylakoid membrane and stabilize the manganese ion cluster forming the photosystem II electron donor side. The OEC proteins are released from their binding site(s) following light-induced degradation of reaction center II (RCII)-D1 protein in Chlamydomonas reinhardtii. The kinetics of OEC proteins release correlates with that of RCII-D1 protein degradation. Only a limited amount of RCII-D2 protein is degraded during the process, and no loss of the core proteins CP43 and CP47 is detected. The release of the OEC proteins is prevented when the photoinactivated RCII-D1 protein degradation is retarded by addition of 3-(3,5-dichlorophenyl)-1,1-dimethylurea or by a high PQH2/PQ ratio prevailing in membranes of the plastocyanin-deficient mutant Ac208. The released proteins are not degraded but persist in the thylakoid lumen for up to 8 h and reassociate with photosystem II when new D1 protein is synthesized in cells exposed to low light, thus allowing recovery of photosystem II function. Reassociation also occurs following D1 protein synthesis in darkness when RCII activity is only partially recovered. These results indicate that (i) the D1 protein participates in the formation of the lumenal OEC proteins binding site(s) and (ii) the photoinactivation of RCII-D1 protein does not alter the conformation of the donor side of photosystem II required for the binding of the OEC proteins.     

Comparison of the functional properties of the monomeric and dimeric forms of the isolated CP47-reaction center complex

Chlorophyll fluorescence, thermoluminescence, and EPR spectroscopy have been used to investigate the functional properties of the monomeric and dimeric forms of the photosystem II CP47-reaction center (CP47-RC) subcore complex that was isolated (Zheleva, D., Sharma, J., Panico, M., Morris, H. R., and Barber, J. (1998) J. Biol. Chem. 273, 16122-16127). Chlorophyll fluorescence yield changes induced either by the initiation of continuous actinic light or by repetitive light flashes indicated that the dimeric, but not the monomeric, form of the CP47-RC complex showed secondary electron transport properties indicative of QA reduction. Thermoluminescence measurements also clearly distinguished the monomer from the dimer in that the latter showed a ZV band, which appeared at -55 degreesC, following illumination at -80 degreesC. This band has been determined to be an indicator of the photoaccumulation of QA-. The ability of the dimeric CP47-RC to show secondary electron transport properties was clearly demonstrated by EPR studies. The dimer was characterized by organic radical signals at about g = 2 induced either by illumination or by the addition of dithionite. The dithionite-induced signal was attributed to QA-, but there was no indication of any interaction with non-heme iron. The signal induced by light was more complex, being composed not only of the QA- radical but also of radicals generated on the donor side. Difference analyses indicated that one of these radicals is likely to be due to a D1 tyrosine 161 or D2 tyrosine 161. In contrast, the monomeric CP47-RC complex did not show similar EPR-detectable radicals and instead was dominated by a high yield of the spin-polarized triplet signal generated by recombination reactions between the oxidized primary reductant, pheophytin, and the primary donor, P680. It is also concluded from EPR analyses that both the monomeric and dimeric forms of the CP47-RC subcore complex contain one cytochrome b559 per reaction center. Overall the results suggest that photosystem II normally functions as a dimer complex and that monomerization at the level of the CP47-RC subcore complex leads to destabilization of the bound plastoquinone, which functions as QA.     

Functional analysis of combinatorial mutants altered in a conserved region in loop E of the CP47 protein in Synechocystis sp. PCC 6803

Regions in the large lumenally exposed region (loop E) of CP47 affect properties of the watersplitting system in photosystem II (PS II). To investigate the role of these regions, we developed a method for functional complementation of obligate photoheterotrophic mutants carrying a deletion in one such region. Using an obligate photoheterotrophic mutant that carries a short deletion (delta (D440-P447) in loop E of CP47, completely degenerate sequences of eight codons in length were introduced at the site of the deletion. Transformants that were complemented to photoautotrophic growth were selected, and 20 such mutants were studied. Sequence analysis revealed that, as expected, in each of them CP47 had been restored to its wild-type length. However, none of the amino acid residues in the deleted region were found to be critical for function. A negatively charged residue at position 440 and a positively charged one at position 444 were favored but not required. Photoautotrophic growth of mutants obtained varied from almost normal to significantly impaired. The mutants contained 20-100% of the amount of PS II present in the wild type, with PS II amounts correlating with the initial rates of oxygen evolution. The mutants had a high rate of photoinactivation, and many mutants showed an up to 1000-fold increase in chloride requirement for photoautotrophic growth. These phenotypic effects were a direct consequence of the CP47 mutations and were not caused by altered binding of one of the extrinsic proteins. No particular amino acid residues in positions 440-447 of CP47 were found to be indispensable for photoautotrophic growth, and many amino acid combinations in this region support PS II function. However, the mutagenized region is shown to interact with the oxygen-evolving site of PS II and appears to have a direct role in chloride binding.     

The nature of the excited state of the reaction center of photosystem II of green plants: a high-resolution fluorescence spectroscopy study

We studied the electronically excited state of the isolated reaction center of photosystem II with high-resolution fluorescence spectroscopy at 5 K and compared the obtained spectral features with those obtained earlier for the primary electron donor. The results show that there is a striking resemblance between the emitting and charge-separating states in the photosystem II reaction center, such as a very similar shape of the phonon wing with characteristic features at 19 and 80 cm-1, almost identical frequencies of a number of vibrational modes, a very similar double-Gaussian shape of the inhomogeneous distribution function, and relatively strong electron-phonon coupling for both states. We suggest that the emission at 5 K originates either from an exciton state delocalized over the inactive branch of the photosystem or from a fraction of the primary electron donor that is long-lived at 5 K. The latter possibility can be explained by a distribution of the free energy difference of the primary charge separation reaction around zero. Both possibilities are in line with the idea that the state that drives primary charge separation in the reaction center of photosystem II is a collective state, with contributions from all chlorophyll molecules in the central part of the complex.     

Charge separation in the reaction center of photosystem II studied as a function of temperature

In photosystem II of green plants the key photosynthetic reaction consists of the transfer of an electron from the primary donor called P680 to a nearby pheophytin molecule. We analyzed the temperature dependence of this reaction by subpicosecond transient absorption spectroscopy over the temperature range 20-240 K using isolated photosystem II reaction centers from spinach. After excitation in the red edge of the Qy absorption band, the decay of the excited state can conveniently be described by two kinetic components that both accelerate with temperature. This temperature behavior differs remarkably from that observed in purple bacterial reaction centers. We attribute the first component, which accelerates from 2.6 ps at 20 K to 0.4 ps at 240 K, to charge separation after direct excitation of P680, and explain its temperature dependence by an intermediate that lies in energy above the singlet-excited P680 and that possibly has charge-transfer character. The second component accelerates from 120 ps at 20 K to 18 ps at 240 K and is attributed to charge separation after direct excitation of the "trap" state near-degenerate with P680 and subsequent slow energy transfer from this trap state to P680. We suggest that the slow energy transfer from the trap state to P680 plays an important role in the kinetics of radical pair formation at room temperature.     

Structure and thermal stability of photosystem II reaction centers studied by infrared spectroscopy

The secondary structure of photosystem II reaction centers isolated from pea has been deduced from quantitative analysis of the component bands of the infrared amide I spectral region, determined by FTIR spectroscopy. The analysis shows the isolated complex to consist of 40% alpha-helix, 10% beta-sheet, 14% beta-strands (or extended chains), 17% turns, 15% loops, and 3% nonordered segments. These structural protein elements were determined for samples in H2O, in D2O, and in dried films. The isolated reaction center, composed of proteins D1,D2,cytochrome b559, and PsbI, has been predicted to contain a total of 13 transmembrane alpha-helices, which conveys a percentage of this type of structure congruent with the structural determination deduced from FTIR spectra. The process of thermal destabilization of the reaction centers has also been studied by FTIR spectroscopy, showing a clear main conformational transition at 42 degrees C, which indicates a high thermal sensitivity of the secondary structure of this protein complex. Such thermal instability may correlate with the well-described high sensitivity of photosystem II to damage and may relate to the process of rapid protein degradation that photosystem II suffers during photoinhibition of plants.     

Heterogenous lipid distribution among chlorophyll-binding proteins of photosystem II in maize mesophyll chloroplasts

Photosystem II membrane fractions from dark-adapted mesophyll chloroplasts of maize were solubilized in different concentrations of dodecyl beta-D-maltoside. Chlorophyll-binding proteins from photosystem II were isolated either by ultracentrifugation on a sucrose gradient, or by flat bed isoelectric focusing and identified by gel electrophoresis analysis for their polypeptide composition. Lipid and fatty acid compositions were determined in complexes prepared by both methods and also in purified light-harvesting complex II, in minor chlorophyll a/b binding complexes 29, 26, 24, in photosystem II antennae (chlorophyll-protein complexes 43, 47) and in the photosystem II reaction centers chlorophyll-protein complexes. Comparative analysis of the results suggests that a true heterogeneity exists in the lipid class distribution among the different chlorophyll-protein complexes in this region of the photosynthetic membrane. Photosystem II core fractions prepared either by ultra-centrifugation on a sucrose gradient or by isoelectric focusing were found significantly enriched in monogalactosyldiacylglycerol; fractionation of the photosystem II core in its components showed that it was the chlorophyll-protein complexes 43 and 47 which were mainly responsible for this enrichment. One of them, the chlorophyll-protein complex 47, was found containing monogalactosyldiacylglycerol and having a very high level of saturated fatty acids. The minor chlorophyll a/b binding linkers (chlorophyll-protein complexes 24, 26 and 29) retain a largely higher amount of lipids than all other complexes and especially of highly unsaturated galactolipids. Concerning the main light-harvesting antenna (LHCII), it is demonstrated that phosphatidylglycerol is strongly linked to the complex if it cannot be detached at high detergent concentration, while many galactolipids (which nevertheless represent the major lipid classes) are lost. This main light-harvesting complex has been fractionated into several families by isoelectric focusing showing a marked difference in lipid and polypeptide composition. A spectacular increase in the phosphatidylglycerol content was observed in the fraction migrating near the anode and enriched in a 26-kDa polypeptide; but this result is difficult to interpret in physiological terms as it was shown that phosphatidylglycerol alone, because of its negative charge, also migrates toward the anode in isoelectric focusing.     

Involvement of active oxygen species in degradation of the D1 protein under strong illumination in isolated subcomplexes of photosystem II

The effects of strong illumination on the proteins in photosystem II (PSII) were investigated using three different isolated subcomplexes of PSII, namely, the PSII complex depleted of major light-harvesting proteins, the core complex, and the reaction center complex. Under illumination, not only the D1 protein of the reaction center but also other intrinsic proteins sustained some damage in all three subcomplexes: Coomassie blue-stained bands after polyacrylamide gel electrophoresis were smeared, and their migration distances on the gel were reduced with increasing duration of illumination. Such damage occurred first in the D1 and D2 proteins and subsequently in the 43- and 47-kDa proteins of the core antenna and the subunit of cytochrome b559. Immunoblot analysis using an antibody specific to the D1 protein showed that the D1 protein was degraded to major fragments of about 23 and 16 kDa during illumination. The smearing and changes in mobility of protein bands, as well as the fragmentation of the D1 protein, were greatly suppressed by scavengers of active oxygen species. From the effectiveness of scavengers, it appeared that superoxide anions participate in the protein damage in the PSII complex, hydrogen peroxide in the PSII and core complexes, and singlet oxygen, hydroxyl, and alkoxyl radicals in all three subcomplexes. We also found that fragments of the D1 protein of 23 and 16 kDa were formed even when PSII complexes that had been completely solubilized with sodium dodecyl sulfate were illuminated. This fragmentation was also suppressed by active oxygen scavengers.(ABSTRACT TRUNCATED AT 250 WORDS)     

Purification and determination of intact molecular mass by electrospray ionization mass spectrometry of the photosystem II reaction center subunits

A reverse phase high pressure liquid chromatography purification system for the rapid separation of photosystem II reaction center proteins free of salts and detergents is described. This procedure results in the isolation of the three small subunits: alpha- and beta-subunits of cytochrome b559 and PsbI protein, with near base-line resolution between each peak, although the D1 and D2 proteins were partially deconvoluted. The molecular masses obtained by electrospray ionization mass spectrometry for the purified beta-subunit of cytochrome b559, alpha-subunit of cytochrome b559, and the PsbI protein, 4,394.8 +/- 0.4, 9,283.7 +/- 0.8, and 4,209.5 +/- 0.4 Da, respectively, are in excellent agreement with values obtained from previous characterization studies (Sharma, J., Panico, M., Barber, J., and Morris, H. R. (1997) J. Biol. Chem. 272, 3935-3943). Direct electrospray analysis of the D1 and D2 proteins suggests that these components exist in heterogeneous forms. The molecular mass ascribed to a predominant form of the D1 protein, 38, 040.9 +/- 6.5 Da, and the D2 protein, 39,456.1 +/- 7.7, are also in agreement with those expected for the mature nonphosphorylated states of these subunits.     

Changes in the reaction mechanism of electron transfer from plastocyanin to photosystem I in the cyanobacterium Synechocystis sp. PCC 6803 as induced by site-directed mutagenesis of the copper protein

The kinetic mechanism of plastocyanin oxidation by photosystem I in the cyanobacterium Synechocystis sp. PCC 6803 is drastically changed by modifying the metalloprotein by site-directed mutagenesis. The mutations herein considered concern four specific residues, two in the east face and the other two in the hydrophobic patch of plastocyanin. The first set of mutants include D44A, D44K, D47A, and D47R, as well as the double mutants D44A/D47A and D44R/D47R; the second set consists of L12A and K33E. The kinetic efficiency of all these mutant plastocyanins has been analyzed by laser-flash absorption spectroscopy. The plastocyanin concentration dependence of the observed electron transfer rate constant (kobs) is linear with most mutant plastocyanins, as with wild-type plastocyanin, but exhibits a saturation plateau at high protein concentration with the double mutant D44R/D47R, which suggests the formation of a plastocyanin-PSI transient complex. The effect of ionic strength on kobs varies from the wild-type plastocyanin to some of the mutants, for instance D44K, for which the salt concentration dependence of kobs is just the reverse as compared to the wild-type protein. The ionic strength dependence of kobs with D44R/D47R exhibits a bell-shaped profile, which is similar to that of green algae and higher plants. These findings indicate that the double mutant D44R/D47R follows a reaction mechanism involving not only complex formation with PSI but also further reorientation to properly accommodate the redox centers prior to electron transfer, as is the case in most evolved species, whereas the wild-type copper protein reacts with PSI by following a simple collisional kinetic model.     

Loss of inhibition by formate in newly constructed photosystem II D1 mutants, D1-R257E and D1-R257M, of Chlamydomonas reinhardtii

Formate is known to cause significant inhibition in the electron and proton transfers in photosystem II (PSII); this inhibition is uniquely reversed by bicarbonate. It has been suggested that bicarbonate functions by providing ligands to the non-heme iron and by facilitating protonation of the secondary plastoquinone QB. Numerous lines of evidence indicate an intimate relationship of bicarbonate and formate binding of PSII. To investigate the potential amino acid binding environment of bicarbonate/formate in the QB niche, arginine 257 of the PSII D1 polypeptide in the unicellular green alga Chlamydomonas reinhardtii was mutated into a glutamate (D1-R257E) and a methionine (DQ-R257M). The two mutants share the following characteristics. (1) Both have a drastically reduced sensitivity to formate. (2) A larger fraction of QA- persists after flash illumination, which indicates an altered equilibrium constant of the reaction QA-QB<-->QA QB-, in the direction of [QA-], or a larger fraction of non-QB centers. However, there appears to be no significant difference in the rate of electron transfer from QA- to QB. (3) The overall rate of oxygen evolution is significantly reduced, most likely due to changes in the equilibrium constant on the electron acceptor side of PSII or due to a larger fraction in non-QB centers. Additional effects on the donor side cannot yet be excluded. (4) The binding affinity for the herbicide DCMU is unaltered. (5) The mutants grow photosynthetically, but at a decreased (approximately 70% of the wild type) level. (6) The Fo level was elevated (approximately 40-50%) which could be due to a decrease in the excitation energy transfer from the antenna to the PSII reaction center, and/or to an increased level of [QA-] in the dark. (7) A decreased (approximately 10%) ratio of F685 (mainly from CP43) and F695 (mainly from CP47) to F715 (mainly from PSI) emission bands at 77 K suggests a change in the antenna complex. Taken together these results lead to the conclusion that D1-R257 with the positively charged side chain is important for the fully normal functioning of PSII and of growth, and is specially critical for the in vivo binding of formate. Several alternatives are discussed to explain the almost normal functioning of the D1-R257E and D1-R257M mutants.     

S-state dependence of chloride binding affinities and exchange dynamics in the intact and polypeptide-depleted O2 evolving complex of photosystem II

The Cl- binding properties in the successive oxidation states of the O2 evolving complex of photosystem II were investigated by measurements of UV absorbance changes, induced by a series of saturating flashes, that monitor manganese oxidation state transitions. In dark-adapted, intact photosystem II, Cl- can be replaced by NO3- in minutes, in an exchange reaction that depends on the NO3- concentration and that is not rate-limited by dissociation of Cl- from its binding site. Preillumination of dark-adapted photosystem II by one or two flashes accelerated the NO3- substitution reaction by an order of magnitude. A quantitative analysis of the Cl- concentration dependence of UV absorbance changes, measured in photosystem II preparations depleted of extrinsic 17 and 23 kDa polypeptides, shows that the Cl- binding properties of photosystem II change with the oxidation state of the oxygen evolving complex. Although the affinity for the individual S-states could not be determined with precision, it is shown that the affinity is an order of magnitude lower in the S2 state than in the S1 state. Comparison of the results obtained using intact photosystem II and preparations depleted of the 17 and 23 kDa extrinsic polypeptides suggests that these proteins constitute a diffusion barrier, which prevents fast equilibration of the Cl- binding site with the medium, but does not change the Cl- affinity of the binding site.     

Investigation of the structure of spinach photosystem II reaction center complex

The photosystem II (PSII) reaction center (RC) complex was isolated from spinach and characterized by gel electrophoresis, gel filtration and analytical ultracentrifugation. The purified complex contained the PsbA, PsbD, PsbE, PsbF and PsbI subunits. Gel filtration and analytical ultracentrifugation indicated the presence of a homogeneous complex. The mass of the RC complexes was found to be 107 kDa by analytical ultracentrifugation and 132 kDa by scanning transmission electron microscopy (STEM). The mass obtained showed the isolated complex to exist as a monomer and only one cytochrome b559 (cyt b559) to be associated with the RC complex. Digital images of negatively stained RC complexes were recorded by STEM and analyzed by single-particle averaging. The complex was 9 nm long and 5 nm wide, and exhibited a pronounced quasi-twofold symmetry. This supports the symmetric organization of the PSII complex, with the PsbA and the PsbD proteins in the center and symmetrically arranged PsbB and PsbC proteins at the periphery of the monomeric complex.     

Site-directed mutagenesis of basic arginine residues 305 and 342 in the CP 43 protein of photosystem II affects oxygen-evolving activity in Synechocystis 6803

The intrinsic chlorophyll protein CP 43, a component of photosystem II (PS II) in higher plants, green algae, and cyanobacteria, is encoded by the psbC gene. Oligonucleotide-directed mutagenesis was employed to introduce mutations into a segment of psbC that encodes the large extrinsic loop E of CP 43 in the cyanobacterium Synechocystis 6803. Two mutations, R305S and R342S, each produced a strain with impaired photosystem II activity. The R305S mutant strain grew photoautotrophically at rates comparable to the control strain. Immunological analyses of a number of PSII components indicated that this mutant accumulated normal quantities of PSII proteins. However, this mutant evolved oxygen to only 70% of control rates at saturating light intensities. Measurements of total variable fluorescence yield indicated that this mutant assembled approximately 70% of the PSII centers found in the control strain. The R342S mutant failed to grow photoautotrophically and exhibited no capacity for oxygen evolution. However, when grown photoheterotrophically in medium containing both glucose and 3-(3, 4-dichlorophenyl)-1,1-dimethylurea (DCMU), oxygen-evolving activity was observed in the R342S mutant, but at a low level of approximately 10% of the control rate. Immunological analysis of isolated thylakoid membranes from this mutant also indicated that this strain accumulated normal amounts of PSII core proteins. Total variable fluorescence yields for the R342S mutant indicated that it assembled a severely reduced number of fully functional PSII centers. R305S and R342S mutant strains exhibited, respectively, 2.7- and 4-fold increased sensitivity to photoinactivation. The fluorescence rise times for both mutants were comparable to the control when hydroxylamine was used as electron donor. However, both strains exhibited an increase (2.5- and 8-fold, respectively, for R305S and R342S) in fluorescence rise times with water as an electron donor. These results suggest that the mutations R305S and R342S each produce a defect associated with the oxygen-evolving complex of photosystem II. These are the first site-directed mutations in CP 43 to show such an effect.     

Characterization of the light-induced cross-linking of the alpha-subunit of cytochrome b559 and the D1 protein in isolated photosystem II reaction centers

Illumination of the isolated reaction center of photosystem II generates a protein of 41 kDa molecular mass. Using immunoblotting, it is confirmed that the protein is an adduct of the D1 protein and the alpha-subunit of cytochrome b559. Its formation seems to be photochemically induced, being independent of temperature between 4 and 20 degrees C and unaffected by a mixture of protease inhibitors. The maximum levels are detected when the pH is in the region 6.5-8.5 and when illumination intensities are moderate. Although higher light intensities induce a higher rate of formation, the accumulation of elevated levels of the 41-kDa protein does not occur due to light-induced degradation. This degradation is also unaffected by the presence of protease inhibitors. Proteolytic mapping and N-terminal sequencing indicates that the cross-linking process involves the N-terminal serine of the alpha-subunit of cytochrome b559 and D1 residues in the 239-244 FGQEEE motif close to the QB binding site. In conclusion, the results indicate that the N terminus of the alpha-subunit is exposed on the stromal side of photosystem II in such a way as to undergo light-induced cross-linking in the QB region of the D1 protein. They also suggest that the 41-kDa adduct may be an intermediate before the light-induced cleavage of the D1 protein in the FGQEEE region.     

Effects of CeCl3 on Energy Transfer and Oxygen Evolution in Spinach Photosystem Ⅱ

Due to 4f electron characteristics and alternation valence, cerium involved in an oxidation-reduction reaction in plant, closely relating to photosynthesis. Our studies proved that cerium could promote photosynthesis and greatly improve spinach growth. However, the mechanism of promoting energy transfer and conversion by cerium remains unclear. Here we reported that the effects of Ce^3＋ on energy transfer and oxygen evolution in photosystem Ⅱ （PS Ⅱ ） isolated from spinach, which was related to 4f electron characteristics and alternation valence in Ce molecule. The methods of absorption spectrum, fluorescence spectrum were used in the research. Results showed that Ce^3＋ treatment at low concentration could suitably change PS Ⅱ mieroenvironment, increase the absorbance of visible light, improve the energy transfer among amino acids within PS Ⅱ protein-pigment complex, and accelerate energy transport from tyrosine residue to chlorophyll a. In summary, the photochemical activity of PS Ⅱ （fluorescence quantum yield） and its oxygen evolving rate were enhanced by Ce^3＋.     

Expeditious synthesis of a new hexasaccharide using transglycosylation reaction catalyzed by Bacillus (1-->3),(1-->4)-Beta-D-glucan 4-glucanohydrolase

In this study, we report the structural characterization of photosystem II complexes obtained from partially solubilized photosystem II membranes. Direct observation by electron microscopy, within a few minutes after a mild disruption of the membranes with the detergent n-dodecyl-alpha,D-maltoside, revealed the presence of several large supramolecular complexes. Images of these complexes were subjected to multivariate statistical analysis and classification procedures, resolving a new complex consisting of the previously characterized dimeric supercomplex of photosystem II and light-harvesting complex II [Boekema et al., Proc. Natl. Acad. Sci. USA 92 (1995) 175-179] and two additional, symmetrically organized protein masses each containing a second type of trimeric light-harvesting II complex. We conclude that large and labile integral membrane proteins, such as photosystem II, can be quickly structurally characterized without extensive purification.     

Modification of pigment composition in the isolated reaction center of photosystem II

The pigment content of isolated reaction centers of photosystem II was modified using an exchange protocol similar to that used for purple bacterial reaction centers. With this method, which is based on incubation of reaction centers at elevated temperature with an excess of chemically modified pigments, it was possible to incorporate [3-acetyl]-chlorophyll a and [Zn]-chlorophyll a into photosystem II reaction centers. Pigment exchange has been verified by absorption, circular dichroism and fluorescence spectroscopy, and quantitated by HPLC analysis of pigment extracts.     

Electrically evoked compound action potentials of guinea pig and cat: responses to monopolar, monophasic stimulation

On the basis of sequence comparison with the M subunit of the reaction center of purple bacteria, no residues in photosystem II can be clearly identified that may be predicted to correspond to the His residue that binds one of the accessory bacteriochlorophylls in the purple bacterial reaction center. However, the Arg180 residue of the D2 protein is close to where this residue is predicted to be and could conceivably serve as a chlorophyll ligand. To analyze the function of Arg180, it was changed to nine different amino acids in the cyanobacterium Synechocystis sp. PCC 6803. Except for the Arg180-->Gln (R180Q) mutant, the resulting strains were no longer photoautotrophic. The properties of photosystem II upon mutation of Arg180 were probed in strains from which photosystem I had been deleted genetically. Mutations at the Arg180 residue affected oxygen evolution capacity and the amount of photosystem II that was present in thylakoids. Surprisingly, in the Arg180 mutants, EPR signals that may originate from the oxidized redoxactive Tyr160 of the D2 protein (Y(D)ox) were small and generally did not resemble the usual signal IIs, signifying an effect of the Arg180 mutations on the environment surrounding Tyr160. In addition, in most mutants, the charge recombination kinetics between the primary electron-accepting quinone in photosystem II (Q(A)-) and oxidized species on the donor side were faster upon introducing mutations at Arg180 suggesting an increased steady-state concentration of P680+ in the mutants. However, Arg180 mutations also affected Q(A)- oxidation by the secondary electron-accepting quinone (Q(B)). HPLC analysis showed that, in the Arg180 mutants that were assayed, the pheophytin/chlorophyll ratio of photosystem II had not changed, indicating that the mutations did not lead to a pheophytinization of one of the chlorophyll molecules. Even though the results presented do not provide positive evidence that Arg180 of the D2 protein corresponds in function to the ligand to the central Mg in an accessory bacteriochlorophyll in reaction centers of purple bacteria, it is clear that changes in Arg180 greatly affect Tyr160 and P680. Various scenarios are discussed that are compatible with the data presented, and include an apparently close interaction between Arg180, His189, and Tyr160, and the possibility of the involvement of multiple chlorophylls to together form P680.