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Wim F J Vermaas - One of the best experts on this subject based on the ideXlab platform.
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Slr2013 Is a Novel Protein Regulating Functional Assembly of Photosystem II in Synechocystis sp. Strain PCC 6803
Journal of Bacteriology, 2003Co-Authors: Galyna I. Kufryk, Wim F J VermaasAbstract: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.
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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.
Biochemistry, 2001Co-Authors: Anna T. Keilty, Dmitrii V. Vavilin, Wim F J VermaasAbstract: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.
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Targeted Random Mutagenesis To Identify Functionally Important Residues in the D2 Protein of Photosystem II in Synechocystis sp. Strain PCC 6803
Journal of bacteriology, 2001Co-Authors: Svetlana Ermakova-gerdes, Wim F J VermaasAbstract:To identify important residues in the D2 Protein of photosystem II (PSII) in the cyanobacterium Synechocystis sp. strain PCC 6803, we randomly mutagenized a region of psbDI (coding for a 96-residue-long C-terminal part of D2) with sodium bisulfite. Mutagenized plasmids were introduced into a Synechocystis sp. strain PCC 6803 mutant that lacks both psbD genes, and mutants with impaired PSII function were selected. Nine D2 residues were identified that are important for PSII stability and/or function, as their mutation led to impairment of photoautotrophic growth. Five of these residues are likely to be involved in the formation of the QA-binding niche; these are Ala249, Ser254, Gly258, Ala260, and His268. Three others (Gly278, Ser283, and Gly288) are in transmembrane α-helix E, and their alteration leads to destabilization of PSII but not to major functional alterations of the remaining centers, indicating that they are unlikely to interact directly with cofactors. In the C-terminal lumenal tail of D2, only one residue (Arg294) was identified as functionally important for PSII. However, from the number of mutants generated it is likely that most or all of the 70 residues that are susceptible to bisulfite mutagenesis have been altered at least once. The fact that mutations in most of these residues have not been picked up by our screening method suggests that these mutations led to a normal photoautotrophic phenotype. A novel method of intragenic complementation in Synechocystis sp. strain PCC 6803 was developed to facilitate genetic analysis of psbDI mutants containing several amino acid changes in the targeted domain. Recombination between genome copies in the same cell appears to be much more prevalent in Synechocystis sp. strain PCC 6803 than was generally assumed.
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Mutations in the CD-loop region of the D2 Protein in Synechocystis sp. PCC 6803 modify charge recombination pathways in photosystem II in vivo.
Biochemistry, 2000Co-Authors: Dmitrii V. Vavilin, Wim F J VermaasAbstract: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 YD. 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 QA- with the donor side was faster in the majority of mutants (t1/2 = 45−140 ms) than in the control (t1/2 = 180 ms). However, in one mutant (named C7-3), the decay of QA- was 2 times slower than in the control (t1/2 = 360 ms). The decay half-time of each mutant correlated with the yield of the Q-band of thermoluminescence (TL) emitted due to S2QA- charge recombination. The C...
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Probing the CD Lumenal Loop Region of the D2 Protein of Photosystem II in Synechocystis sp. Strain PCC 6803 by Combinatorial Mutagenesis
Journal of bacteriology, 2000Co-Authors: Anna T. Keilty, Svetlana Ermakova-gerdes, Wim F J VermaasAbstract: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.
Svetlana Ermakova-gerdes - One of the best experts on this subject based on the ideXlab platform.
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Targeted Random Mutagenesis To Identify Functionally Important Residues in the D2 Protein of Photosystem II in Synechocystis sp. Strain PCC 6803
Journal of bacteriology, 2001Co-Authors: Svetlana Ermakova-gerdes, Wim F J VermaasAbstract:To identify important residues in the D2 Protein of photosystem II (PSII) in the cyanobacterium Synechocystis sp. strain PCC 6803, we randomly mutagenized a region of psbDI (coding for a 96-residue-long C-terminal part of D2) with sodium bisulfite. Mutagenized plasmids were introduced into a Synechocystis sp. strain PCC 6803 mutant that lacks both psbD genes, and mutants with impaired PSII function were selected. Nine D2 residues were identified that are important for PSII stability and/or function, as their mutation led to impairment of photoautotrophic growth. Five of these residues are likely to be involved in the formation of the QA-binding niche; these are Ala249, Ser254, Gly258, Ala260, and His268. Three others (Gly278, Ser283, and Gly288) are in transmembrane α-helix E, and their alteration leads to destabilization of PSII but not to major functional alterations of the remaining centers, indicating that they are unlikely to interact directly with cofactors. In the C-terminal lumenal tail of D2, only one residue (Arg294) was identified as functionally important for PSII. However, from the number of mutants generated it is likely that most or all of the 70 residues that are susceptible to bisulfite mutagenesis have been altered at least once. The fact that mutations in most of these residues have not been picked up by our screening method suggests that these mutations led to a normal photoautotrophic phenotype. A novel method of intragenic complementation in Synechocystis sp. strain PCC 6803 was developed to facilitate genetic analysis of psbDI mutants containing several amino acid changes in the targeted domain. Recombination between genome copies in the same cell appears to be much more prevalent in Synechocystis sp. strain PCC 6803 than was generally assumed.
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Probing the CD Lumenal Loop Region of the D2 Protein of Photosystem II in Synechocystis sp. Strain PCC 6803 by Combinatorial Mutagenesis
Journal of bacteriology, 2000Co-Authors: Anna T. Keilty, Svetlana Ermakova-gerdes, Wim F J VermaasAbstract: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.
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Tryptophan at position 181 of the D2 Protein of photosystem II confers quenching of variable fluorescence of chlorophyll: implications for the mechanism of energy-dependent quenching.
Biochemistry, 1999Co-Authors: Dmitrii V. Vavilin, Svetlana Ermakova-gerdes, And Anna T. Keilty, Wim F J VermaasAbstract:The lumenal CD loop region of the D2 Protein of photosystem II contains residues that interact with a reaction center chlorophyll and the redox-active TyrD. Using combinatorial mutagenesis, photoautotrophic mutants of Synechocystis sp. PCC 6803 have been generated with multiple amino acid changes in this region. The CD loop mutations were transferred into a photosystem I-less Synechocystis strain to facilitate characterization of photosystem II properties in the mutants. Most of the combinatorial photosystem I-less mutants obtained had a high yield of variable fluorescence, FV. However, in three mutants, which shared a replacement of Phe181 by Trp, the FV yield was dramatically reduced although a high rate of oxygen evolution was maintained. A site-directed F181W D2 mutant shared similar properties. Picosecond time-resolved fluorescence measurements revealed that in the combinatorial F181W mutants the fluorescence lifetimes in closed and open photosystem II centers were essentially identical and were simi...
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Mobility of the primary electron-accepting plastoquinone QA of photosystem II in a Synechocystis sp. PCC 6803 strain carrying mutations in the D2 Protein.
Biochemistry, 1998Co-Authors: Svetlana Ermakova-gerdes, Willem VermaasAbstract: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.
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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
Plant Molecular Biology, 1996Co-Authors: Svetlana Ermakova-gerdes, Sergey Shestakov, Wim VermaasAbstract: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 psb DI 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 psb DI 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.
Willem Vermaas - One of the best experts on this subject based on the ideXlab platform.
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Association of His117 in the D2 Protein of photosystem II with a chlorophyll that affects excitation-energy transfer efficiency to the reaction center.
European journal of biochemistry, 1998Co-Authors: Michael T. Lince, Willem VermaasAbstract:His117 of the D2 Protein of photosystem II (PS II) is a conserved residue in the second transmembrane region of the Protein and has been suggested to bind chlorophyll. Nine site-directed mutations were introduced at residue 117, using both photosystem I (PS I)-containing and PS I-less background strains of the cyanobacterium Synechocystis sp. PCC 6803. Of these nine, four (H117C, H117M, H117N, and H117T) were photoautotrophic in the PS I-containing background. The other mutants (H117F, H117L, H117P, H117R, and H117Y) did not accumulate appreciable amounts of PS II in their thylakoids. The type of residues that can functionally replace His117 support the notion of His117 serving as a chlorophyll ligand. The properties of the H117N and H117T mutants were characterized in more detail. Whereas the properties of the H117N mutant were close to those of wild type, in the H117T mutant the 77-K fluorescence emission spectrum shows a much smaller amplitude at 695 nm than expected on the basis of the amount of PS II that is present. Moreover, in H117T, the amount of light needed to half-saturate O2-evolution rates was twofold higher than in the control strain, and the variable fluorescence yield was quenched. However, O2 evolution rates at saturating light intensity and electron-transport kinetics were normal in the mutant. Also, the radical accessory chlorophyll (Chl.es+.rb.eiZ.rb) formed by donation of an electron to the PS-II reaction center could be generated normally by illumination at low temperature in the H117T mutant. We conclude that the chlorophyll associated with residue 117 of the D2 Protein is important for efficient excitation transfer between the proximal antenna and the PS II reaction center. A possible mechanism involving a chlorophyll cation to explain the quenching in the H117T mutant is discussed.
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Mobility of the primary electron-accepting plastoquinone QA of photosystem II in a Synechocystis sp. PCC 6803 strain carrying mutations in the D2 Protein.
Biochemistry, 1998Co-Authors: Svetlana Ermakova-gerdes, Willem VermaasAbstract: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.
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Truncation of the D2 Protein in Synechocystis sp. PCC 6803: a role of the C-terminal domain of D2 in photosystem II function and stability.
Biochemistry, 1993Co-Authors: Beth Eggers, Willem VermaasAbstract:Termination and deletion mutations were introduced near the C-terminal end of the D2 Protein in the cyanobacterium Synechocystis sp. PCC 6803 in order to determine the role of the large hydrophilic C-terminal domain of D2 in the function and stability of photosystem II (PS II). The loss of 57 residues from the C-terminal end of D2 (most of the hydrophilic tail) resulted in the loss of D2 and PS II reaction centers from thylakoids. Truncation of 16, 15, 14, or 13 amino acid residues from the C-terminus of D2 resulted in a virtual disappearance of oxygen evolution, a loss of photoautotrophic growth, and a decrease in the number of PS II centers in thylakoids. The loss of 11 C-terminal amino acid residues led to a photoautotrophic mutant that grew at one-half the rate of the wild type under photoautotrophic conditions and that showed a progressive loss of oxygen evolution at high light intensity. Truncation of 9 residues from D2 led to a virtual loss of CP43, presumably because of interference of the mutation with the overlapping ribosome-binding site for psbC translation. To delete smaller portions of D2 and yet not interfere with psbC expression, various deletions were made between the tenth and twentieth amino acid residues from the C-terminal end of D2, resulting in the loss of 8, 7, 4, 3, and 2 residues.(ABSTRACT TRUNCATED AT 250 WORDS)
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Modified EPR spectra of the tyrosineD radical in photosystem II in site-directed mutants of Synechocystis sp. PCC 6803: identification of side chains in the immediate vicinity of tyrosineD on the D2 Protein.
Biochemistry, 1993Co-Authors: Cecilia Tommos, Lars Davidsson, Bengt Svensson, Cathy Madsen, Willem Vermaas, Stenbjörn StyringAbstract:The oxidizing side of photosystem II contains two redox-active tyrosyl side chains, TyrZ and TyrD, and a cluster of Mn atoms involved in water oxidation. The structural environment of these components is unknown, and with computer-assisted modeling we have created a three-dimensional model for the structures around TyrZ and TyrD [Svensson et al. (1990) EMBO J. 9, 2051-2059]. Both tyrosines are proposed to form hydrogen bonds to nearby histidine residues (for Synechocystis 6803, these are His190 on the D1 and His189 on the D2 Proteins). We have tested this proposal by electron paramagnetic resonance (EPR) spectroscopy of TyrDox in mutants of the cyanobacterium Synechocystis 6803 carrying site-directed mutations in the D2 Protein. In two mutants, where His189 of the D2 Protein is changed to either Tyr or Leu, the normal EPR spectrum from TyrDox is replaced by narrow, structureless radical signals with g-values similar to that of TyrDox (g approximately 2.0050). The new radicals copurify with photosystem II, are dark-stable, destabilized by elevated pH, and light-inducible, and originate from radicals formed by oxidation. These properties are similar to those of normal TyrDox, and we assign the new spectra to TyrDox in an altered environment induced by the point mutation in His189. In a third mutant, where Gln164 of the D2 Protein was mutated to Leu, we also observed a modified EPR spectrum from TyrDox. This is also consistent with the model in which this residue is found in the immediate vicinity of TyrDox. Thus the results provide experimental evidence supporting essential aspects of the structural model.
Miwa Sugiura - One of the best experts on this subject based on the ideXlab platform.
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variants of photosystem ii d1 Protein in thermosynechococcus elongatus
Research on Chemical Intermediates, 2014Co-Authors: Miwa Sugiura, Alain BoussacAbstract:Cyanobacteria have several psbA genes encoding PsbA, the D1 reaction center Protein of the photosystem II (PSII) complex which bears, with PsbD, the D2 Protein, most of the cofactors involved in electron-transfer reactions. The thermophilic cyanobacterium Thermosynechococcus elongatus has three psbA genes differently expressed depending on the environmental conditions. Among the 344 residues constituting each of the three possible PsbA variants there are 21 substitutions between PsbA1 and PsbA3, 31 between PsbA1 and PsbA2, and 27 between PsbA2 and PsbA3. In this review, we briefly summarize the changes already identified in the properties of the redox cofactors depending on the D1 variant constituting PSII in T. elongatus.
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some photosystem ii properties depending on the d1 Protein variants in thermosynechococcus elongatus
Biochimica et Biophysica Acta, 2014Co-Authors: Miwa Sugiura, Alain BoussacAbstract:Abstract Cyanobacteria have multiple psbA genes encoding PsbA, the D1 reaction center Protein of the Photosystem II complex which bears together with PsbD, the D2 Protein, most of the cofactors involved in electron transfer reactions. The thermophilic cyanobacterium Thermosynechococcus elongatus has three psbA genes differently expressed depending on the environmental conditions. Among the 344 residues constituting each of the 3 possible PsbA variants there are 21 substitutions between PsbA1 and PsbA3, 31 between PsbA1 and PsbA2 and 27 between PsbA2 and PsbA3. In this review, we summarize the changes already identified in the properties of the redox cofactors depending on the D1 variant constituting Photosystem II in T. elongatus . This article is part of a Special Issue entitled: Photosynthesis Research for Sustainability: Keys to Produce Clean Energy.
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Properties of Chlamydomonas Photosystem II Core Complex with a His-Tag at the C-Terminus of the D2 Protein
Plant and Cell Physiology, 1999Co-Authors: Miwa Sugiura, Jun Minagawa, Yorinao InoueAbstract:A His-tagged PSII core complex was purified from recombinant Chlamydomonas reinhardtii D2-H thylakoids by single-step Ni2+-affinity column chromatography and its properties were partially characterized in terms of their PSII functions and chemical compositions. The PSII core complex that has a His-tag extension at the C-terminus of the D2 Protein evolved oxygen at a high rate of 2,400 ^mol (mg Chi)"1 h"1 at the optimum pH of 6.5 with ferricyanide and 2,6-dichlorobenzoquinone as electron acceptors in the presence of Ca 2+ as an essential cofactor, and approximately 90% of the activity was blocked by 10 /M DCMU. The core complex exhibited the thermoluminescence Q-band but not the B-band regardless of the presence or absence of DCMU, although both bands were observed in the His-tagged thylakoids. The core complex was free from PSI and contained one YD, Tyr 160 of the D2 Protein, four Mn atoms, two cytochrome b-559, about 46 Chi a molecules, and probably one QA, the primary acceptor quinone of PSII. It was inferred from these results that His-tagging at the C-terminus of the D2 Protein does not affect the functional and structural integrity of the PSII core complex, and that the 'His-tag strategy' is highly useful for biochemical, physicochemical, and structural studies of Chlamydomonas PSII.
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Rapid and discrete isolation of oxygen-evolving His-tagged photosystem II core complex from Chlamydomonas reinhardtii by Ni2+ affinity column chromatography
FEBS letters, 1998Co-Authors: Miwa Sugiura, Yorinao Inoue, Jun MinagawaAbstract:Abstract We have developed a simple and rapid procedure to isolate an oxygen-evolving photosystem II (PS II) core complex from Chlamydomonas reinhardtii. A His-tag made of six consecutive histidine residues was genetically attached at the carboxy terminus of D2 Protein to create a metal binding site on the PS II supramolecular complex. The recombinant cells producing the His-tagged variant of D2 Protein grew photoautotrophically as well as the wild-type cells. Characterization of the oxygen evolution and the thermoluminescence properties revealed that the His-tagging did not affect the functional integrity of the PS II reaction center. A PS II core complex was isolated from the detergent-solubilized thylakoids of the recombinant cells in 4 h by a single one-step Ni2+ affinity column chromatography. This preparation consists of D1, D2, CP43, CP47, 33 kDa, and a few low molecular weight Proteins, and retains a high rate of oxygen-evolving activity (=1000 μmol/mg Chl/h).
Anna T. Keilty - One of the best experts on this subject based on the ideXlab platform.
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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.
Biochemistry, 2001Co-Authors: Anna T. Keilty, Dmitrii V. Vavilin, Wim F J VermaasAbstract: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.
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Probing the CD Lumenal Loop Region of the D2 Protein of Photosystem II in Synechocystis sp. Strain PCC 6803 by Combinatorial Mutagenesis
Journal of bacteriology, 2000Co-Authors: Anna T. Keilty, Svetlana Ermakova-gerdes, Wim F J VermaasAbstract: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.