Functional phycobilisome core structures in a phycocyanin-less mutant of cyanobacterium Synechococcus sp. PCC 7942

We have constructed a mutant Synechococcus sp. PCC 7942, termed R2HECAT, in which the entire phycobilisome rod operon has been deleted. In the whole cell absorption spectra of R2HECAT, the peak corresponding to phycocyanin (PC), max620 nm, could not be detected. However, a single pigment-protein fraction with max=654 nm could be isolated on sucrose gradients from R2HECAT. Analysis of this pigment-protein fraction by non-denaturing PAGE indicates an apparent molecular mass of about 1200–1300 kDa. On exposure to low temperature, the isolated pigment-protein complex dissociated to a protein complex with a molecular mass of about 560 kDa. When analysed by SDS-PAGE, the pigment-protein fraction was found to consist of the core polypeptides but lacked PC, 27, 33, 30, and the 9 kDa polypeptides which are a part of the rods. All the chromophore bearing polypeptides of the core were found to be chromophorylated. CD as well as absorption spectra showed the expected maxima around 652 and 675 nm from allophycocyanin (APC) and allophycocyanin B (APC-B) chromophores. Low temperature fluorescence and excitation spectra also showed that the core particles were fully functional with respect to the energy transfer between the APC chromophores. We conclude that PC and therefore the rods are dispensable for the survival of Synechococcus sp. PCC 7942. The results indicate that stable and functional core can assemble in absence of the rods. These rod-less phycobilisome core is able to transfer energy to Photosystem II.Abbreviations PS II Photosystem II - PC phycocyanin - APC allophycocyanin - APC-B allophycocyanin B - PAGE polyacrylamide gel electrophoresis - Cml chloramphenicol - kbp kilobase pairs     

Structural and compositional analyses of the phycobilisomes of Synechococcus sp. PCC 7002. Analyses of the wild-type strain and a phycocyanin-less mutant constructed by interposon mutagenesis

The phycobilisomes and phycobiliproteins of Synechococcus sp. PCC 7002 wild-type strain PR6000 have been isolated and characterized. The hemidiscoidal phycobilisomes of strain PR6000 are composed of eleven different polypeptides: phycocyanin and subunits; allophycocyanin and subunits; subunit of allophycocyanin B; the allophycocyanin -subunit-like polypeptide of M_r 18 000; the linker phycobiliprotein of M_r 99 000; and non-chromophore-carrying linker polypeptides of M_r 33 000, 29 000, 9000, and 8000. Several of these polypeptides were purified to homogeneity and their amino acid compositions and amino-terminal amino acid sequences were determined. Analyses of the phycobiliproteins of Synechococcus sp. PCC 7002 were greatly facilitated by comparative studies performed with a mutant strain, PR6008, constructed to be devoid of the phycocyanin and subunits by recombinant DNA techniques and transformation of strain PR6000. The absence of phycocyanin did not greatly affect the allophycocyanin content of the mutant strain but caused the doubling time to increase 2–7-fold depending upon the light intensity at which the cells were grown. Although intact phycobilisome cores could not be isolated from this mutant, it is probable that functionally intact cores do exist in vivo.Abbreviations used SDS-PAGE polyacrylamide gel electrophoresis in the presence of sodium dodecylsulfate - 2D-PAGE two-dimensional gel electrophoresis in which the first dimension consisted of isoelectric focusing in the presence of 8.0 M urea in the pH range 4–6 and the second dimension consisted of electrophoresis in the presence of sodium dodecylsulfate. The nomenclature employed for the phycobiliprotein subunits and linker polypeptides is that defined by Glazer (1985)     

Characterization of a Synechococcus sp. strain PCC 7002 mutant lacking Photosystem I. Protein assembly and energy distribution in the absence of the Photosystem I reaction center core complex

A Synechococcus sp. strain PCC 7002 psaAB::cat mutant has been constructed by deletional interposon mutagenesis of the psaA and psaB genes through selection and segregation under low-light conditions. This strain can grow photoheterotrophically with glycerol as carbon source with a doubling time of 25 h at low light intensity (10 E m^–2 s^–1). No Photosystem I (PS I)-associated chlorophyll fluorescence emission peak was detected in the psaAB::cat mutant. The chlorophyll content of the psaAB::cat mutant was approximately 20% that of the wild-type strain on a per cell basis. In the absence of the PsaA and PsaB proteins, several other PS I proteins do not accumulate to normal levels. Assembly of the peripheral PS I proteins PsaC,PsaD, PsaE, and PsaL is dependent on the presence of the PsaA and PsaB heterodimer core. The precursor form of PsaF may be inserted into the thylakoid membrane but is not processed to its mature form in the absence of PsaA and PsaB. The absence of PS I reaction centers has no apparent effect on Photosystem II (PS II) assembly and activity. Although the mutant exhibited somewhat greater fluorescence emission from phycocyanin, most of the light energy absorbed by phycobilisomes was efficiently transferred to the PS II reaction centers in the absence of the PS I. No light state transition could be detected in the psaAB::cat strain; in the absence of PS I, cells remain in state 1. Development of this relatively light-tolerant strain lacking PS I provides an important new tool for the genetic manipulation of PS I and further demonstrates the utility of Synechococcus sp. PCC 7002 for structural and functional analyses of the PS I reaction center.Abbreviations ATCC American type culture collection - Chl chlorophyll - DCMU 3-(3,4-dichlorophyl)-1,1-dimethylurea - DBMIB 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone - HEPES N-[2-hydroxyethyl]piperazine-N-[2-ethanesulfonic acid] - PCC Pasteur culture collection - PS I Photosystem I - PS II Photosystem II - SDS sodium dodecyl sulfate     

Form II of D1 is important during transition from standard to high light intensity in Synechococcus sp. strain PCC 7942

In Synechococcus sp. strain PCC 7942 the D1 protein of Photosystem II is encoded by a multigene family; psbAI encodes Form I of D1 whereas both psbAII and psbAIII encode Form II. The psbA genes are differentially regulated in response to changes in light intensity, such that psbAI expression and Form I predominate at standard light intensity, whereas psbAII and psbAIII are induced at high light intensity, causing insertion of Form II into the thylakoids. The present study addressed whether high-light induced Form II is important for Synechococcus cells during adaptation to high light intensity. Wild-type Synechococcus, and mutants which produce only Form I (R2S2C3) or only Form II (R2K1), were co-cultured at standard light (130 E · m^–2 · s^–1) and then shifted to high light (750 E·m^–2·s^–1). Measurement of the proportion of each cell type at various time intervals revealed that the growth of R2S2C3, which has psbAII and psbAIII inactive, and thus lacks Form II, is transiently impaired upon shift to high light. Both mutants R2S2C3 and R2K1 maintained normal levels of psbA messages and D1 protein under standard and high light through an unknown mechanism that compensates for the inactive psbA genes. Thus, the impairment of R2S2C3 at high light is not due to a deficiency of D1 protein, but results from lack of Form II. We discounted the influence of possible secondary mutations by re-creating the psbA-inactivated mutants and testing the newly isolated strains. We conclude that Form II of D1 is intrinsically important for Synechococcus cells during a critical transition period after exposure to high light intensities.     

Over-production of the D1 protein of photosystem II reaction centre in the cyanobacterium Synechococcus sp. PCC 7942

The unicellular cyanobacterium Synechococcus sp. PCC 7942 has three psbA genes encoding two different forms of the photosystem II reaction centre protein D1 (D1:1 and D1:2). The level of expression of these psbA genes and the synthesis of D1:1 and D1:2 are strongly regulated under varying light conditions. In order to better understand the regulatory mechanisms underlying these processes, we have constructed a strain of Synechococcus sp. PCC 7942 capable of over-producing psbA mRNA and D1 protein. In this study, we describe the over-expression of D1:1 using a tac-hybrid promoter in front of the psbAI gene in combination with lacI ^Q repressor system. Over-production of D1:1 was induced by growing cells for 12 h at 50 mol photons m^-2 s^-1 in the presence of 40 or 80 g/ml IPTG. The amount of psbAI mRNA and that of D1:1 protein in cells grown with IPTG was three times and two times higher, respectively. A higher concentration of IPTG (i.e., 150 g/ml) did not further increase the production of the psbAI message or D1:1. The over-production of D1:1 caused a decrease in the level of D1:2 synthesised, resulting in most PSII reaction centres containing D1:1. However, the over-production of D1:1 had no effect on the pigment composition (chlorophyll a or phycocyanin/number of cells) or the light-saturated rate of photosynthesis. This and the fact that the total amounts of D1 and D2 proteins were not affected by IPTG suggest that the number of PSII centres within the membranes remained unchanged. From these results, we conclude that expression of psbAI can be regulated by using the tac promoter and lacI ^Q system. However, the accumulation of D1:1 protein into the membrane is regulated by the number of PSII centres.     

Pigment orientation changes accompanying the light state transition in Synechococcus sp. PCC 6301

Low temperature (77 K) linear dichroism spectroscopy was used to characterize pigment orientation changes accompanying the light state transition in the cyanobacterium, Synechococcus sp. PCC 6301 and those accompanying chromatic acclimation in Porphyridium cruentum in samples stabilized by glutaraldehyde fixation. In light state 2 compared to light state 1 intact cells of Synechococcus showed an increased alignment of allophycocyanin parallel to the cells' long axis whereas the phycobilisomethylakoid membrane fragments exhibited an increased allophycocyanin alignment parallel to the membrane plane. The phycobilisome-thylakoid membrane fragments showed less alignment of a short wave-length chlorophyll a (Chl a) Q_y transition dipole parallel to the membrane plane in state 2 relative to state 1.To aid identification of the observed Chl a orientation changes in Synechococcus, linear dichroism spectra were obtained from phycobilisome-thylakoid membrane fragments isolated from red light-grown (increased number of PS II centres) and green light-grown (increased number of PS I centres) cells of the red alga Porphyridium cruentum. An increased contribution of short wavelength Chl a Q_y transition dipoles parallel to the long axis of the membrane plane was directly correlated with increased levels of PS II centres in red light-grown P. cruentum.Our results indicate that the transition to state 2 in cyanobacteria is accompanied by an increase in the orientation of allophycocyanin and a decrease in the orientation of Chl a associated with PS II with respect to the thylakoid membrane plane.Abbreviations APC - allophycocyanin - Chl a - chlorophyll a - DCMU - 3-(3,4-dichlorophenyl)-1,1-dimethylurea - LD - linear dichroism - LD/A - linear dichroism divided by absorbance - LHC - light-harvesting complex - PBS - phycobilisome - PC - phycocyanin - PS - Photosystem     

Isolation of two thioredoxins from the cyanobacterium Synechococcus 6301

Two different thioredoxins designated as thioredoxin A and B have been isolated from the cyanobacterium Synechococcus 6301. Methods for large scale purification of these thioredoxins were developed. Thioredoxin B has been purified to homogeneity; it has a molecular weight of 11,800 and an isoelectric point of 4.6. The following K _m data were obtained for this thioredoxin; a) in the PAPS-sulfotransferase assay of Synechococcus 6301: 10.7 M; b) in the fructose-1-6-bisphosphatase assay of Synechococcus 6301: 1.7 M; c) in the APS-sulfotransferase assay of Chroococcidiopsis 7203: 5.4M. Thioredoxin A has an isoelectric point of 4.1 and it is active in the PAPS-sulfotransferase and fructose-1-6-bisphosphatase of Synechococcus 6301; it is not active in the APS-sulfotransferase of Chroococcidiopsis 7203.Dedicated to Professor Dr. O. Kandler on the occasion of his 60th birthday     

Primary structure of the Synechococcus PCC 7942 PAPS reductase gene

The structural gene encoding a thioredoxin-dependent 5-phosphoadenylyl sulphate (PAPS) reductase (EC 1.8.4.-) from cyanobacterium Synechococcus PCC 7942 (Anacystis nidulans) was detected by heterologous hybridization with the cysH gene from Escherichia coli K12. The cyanobacterial gene (further called par gene) comprised 696 nt which are 57.8% homologous to the enterobacterial gene. The putative open reading frame encoded a polypeptide consisting of 232 amino acid residues (deduced molecular weight 26635) which showed significant homologies to the polypeptide from E. coli (50.8%) and to the polypeptide from Saccharomyces cerevisiae (30.3%). A single cysteine located at the C-terminus of the polypeptide of E. coli (Cys₂₃₉) was conserved in Synechococcus. Conservation of this cysteinyl residue seems indispensable for catalysis. Complementation of a cysH-deficient mutant of E. coli by the cyanobacterial gene indicated that the cloned DNA is the structural gene of the PAPS reductase.Abbreviations IPTG isopropyl-1-thio--D-galactoside - PAPS 3-phosphoadenosine-5-phosposulphate     

Integration of Apo-α-Phycocyanin into Phycobilisomes and Its Association with FNRL in the Absence of the Phycocyanin α-Subunit Lyase (CpcF) in Synechocystis sp. PCC 6803

Phycocyanin is an important component of the phycobilisome, which is the principal light-harvesting complex in cyanobacteria. The covalent attachment of the phycocyanobilin chromophore to phycocyanin is catalyzed by the enzyme phycocyanin lyase. The photosynthetic properties and phycobilisome assembly state were characterized in wild type and two mutants which lack holo-α-phycocyanin. Insertional inactivation of the phycocyanin α-subunit lyase (ΔcpcF mutant) prevents the ligation of phycocyanobilin to α-phycocyanin (CpcA), while disruption of the cpcB/A/C2/C1 operon in the CK mutant prevents synthesis of both apo-α-phycocyanin (apo-CpcA) and apo-β-phycocyanin (apo-CpcB). Both mutants exhibited similar light saturation curves under white actinic light illumination conditions, indicating the phycobilisomes in the ΔcpcF mutant are not fully functional in excitation energy transfer. Under red actinic light illumination, wild type and both phycocyanin mutant strains exhibited similar light saturation characteristics. This indicates that all three strains contain functional allophycocyanin cores associated with their phycobilisomes. Analysis of the phycobilisome content of these strains indicated that, as expected, wild type exhibited normal phycobilisome assembly and the CK mutant assembled only the allophycocyanin core. However, the ΔcpcF mutant assembled phycobilisomes which, while much larger than the allophycocyanin core observed in the CK mutant, were significantly smaller than phycobilisomes observed in wild type. Interestingly, the phycobilisomes from the ΔcpcF mutant contained holo-CpcB and apo-CpcA. Additionally, we found that the large form of FNR (FNR_L) accumulated to normal levels in wild type and the ΔcpcF mutant. In the CK mutant, however, significantly less FNR_L accumulated. FNR_L has been reported to associate with the phycocyanin rods in phycobilisomes via its N-terminal domain, which shares sequence homology with a phycocyanin linker polypeptide. We suggest that the assembly of apo-CpcA in the phycobilisomes of ΔcpcF can stabilize FNR_L and modulate its function. These phycobilisomes, however, inefficiently transfer excitation energy to Photosystem II.     

Genes of the R-phycocyanin II locus of marine Synechococcus spp., and comparison of protein-chromophore interactions in phycocyanins differing in bilin composition

R-phycocyanin II (RPCII) is a recently discovered member of the phycocyanin family of photosynthetic light-harvesting proteins. Genes encoding the and subunits of RPCII were cloned and sequenced from marine Synechococcus sp. strains WH8020 and WH8103. The deduced amino acid sequences of RPCII were compared to two other types of phycocyanin, C-phycocyanin (CPC) and phycoerythrocyanin (PEC). These three types vary in the composition of their covalently bound bilin prosthetic groups. In terms of amino acid sequence identity RPCII is highly homologous to CPC and PEC, suggesting that the known three-dimensional structures of the latter two are representative of RPCII. Thus the amino acid residues contacting the three bilins of RPCII could be inferred and compared to those in CPC and PEC. Certain residues were identified among the three phycocyanins as possibly correlating with specific bilin isomers. In overall sequence RPCII and CPC are more homologous to one another than either is to PEC. This probably reflects functional homology in the roles of RPCII and CPC in the transfer of light energy to the core of the phycobilisome, a function not attributed to PEC. The genomes of Synechococcus sp. strains WH8020, WH8103 and WH7803 share homologous open reading frames in the vicinity of RPCII genes. The nucleotide sequence extending 3 from RPCII genes in strain WH8020 revealed two open reading frames homologous to components of an CPC phycocyanobilin lyase. These open reading frames may encode a lyase specific for the attachment of phycoerythrobilin to RPCII.     

Nitrite reductase gene from Synechococcus sp. PCC 7942: homology between cyanobacterial and higher-plant nitrite reductases

The gene encoding nitrite reductase (nir) from the cyanobacterium Synechococcus sp. PCC 7942 has been identified and sequenced. This gene comprises 1536 nucleotides and would encode a polypeptide of 56506 Da that shows similarity to nitrite reductase from higher plants and to the sulfite reductase hemoprotein from enteric bacteria. Identities found at positions corresponding to those amino acids which in the above-mentioned proteins hold the Fe₄S₄-siroheme active center suggest that nitrite reductase from Synechococcus bears an active site much alike that present in those reductases. The fact that the Synechococcus and higher-plant nitrite reductases are homologous proteins gives support to the endosymbiont theory for the origin of chloroplasts.     

Inactivation of the water-oxidizing enzyme in manganese stabilizing protein-free mutant cells of the cyanobacteria Synechococcus PCC7942 and Synechocystic PCC6803 during dark incubation and conditions leading to photoactivation

The previously constructed MSP (manganese stabilizing protein-psbO gene product)-free mutant of Synechococcus PCC7942 (Bockholt R, Masepohl B and Pistorius E K (1991) FEBS Lett 294: 59–63) and a newly constructed MSP-free mutant of Synechocystis PCC6803 were investigated with respect to the inactivation of the water-oxidizing enzyme during dark incubation. O₂ evolution in the MSP-free mutant cells, when measured with a sequence of short saturating light flashes, was practically zero after an extended dark adaptation, while O₂ evolution in the corresponding wild type cells remained nearly constant. It could be shown that this inactivation could be reversed by photoactivation. With isolated thylakoid membranes from the MSP-free mutant of PCC7942, it could be demonstrated that photoactivation required illumination in the presence of Mn²⁺ and Ca²⁺, while Cl^– addition was not required under our experimental conditions. Moreover, an extended analysis of the kinetic properties of the water-oxidizing enzyme (kinetics of the S₃(S₄)S₀ transition, S-state distribution, deactivation kinetics) in wild type and mutant cells of Synechococcus PCC7942 and Synechocystis PCC6803 was performed, and the events possibly leading to the reversible inactivation of the water-oxidizing enzyme in the mutant cells are discussed. We could also show that the water-oxidizing enzyme in the MSP-free mutant cells is more sensitive to inhibition by added NH₄Cl-suggesting that NH₃ might be a physiological inhibitor of the water oxidizing enzyme in the absence of MSP.Abbreviations Chl chlorophyll - DCBQ 2,6-Dichloro-p-benzoquinone - MSP manganese stabilizing protein (psbO gene product) - PS II Photosystem II - WOE water oxidizing enzyme - WT wild type This paper is dedicated to Prof. Dr. Bernard Axelrod on the occasion of his 80th birthday     

Molecular cloning and nucleotide sequence of the psaA and psaB genes of the cyanobacterium Synechococcus sp. PCC 7002

The psaA and psaB genes, which encode the P700 chlorophyll a apoproteins of the Photosystem I complex, have been cloned from the unicellular, transformable cyanobacterium Synechococcus sp. PCC 7002. The nucleotide sequence of these genes and of their flanking sequences have been determined by the chain termination method. As found in the chloroplast genomes of higher plants, the psaA gene lies 5 to the psaB gene; however, the cyanobacterial genes are separated by a greater distance (173 vs. 25–26 bp). The psaA gene is predicted to encode a polypeptide of 739 amino acid residues (81.7 kDa), and the psaB gene is predicted to encode a polypeptide of 733 residues (81.4 kDa). The cyanobacterial psa gene products are 76% to 81% identical to their higher plant homologues; moreover, because of conservative amino acid replacements, the cyanobacterial sequences are more than 95% homologous to those determined for higher plants. These results provide the basis for a genetic analysis of Photosystem I, and are discussed in relationship to structural and functional aspects of the Photosystem I complexes of both cyanobacteria and higher plants.     

Structure and mutation of a gene encoding a Mr 33000 phycocyanin-associated linker polypeptide

The gene encoding a phycocyanin-associated linker polypeptide of M_r 33000 from the cyanobacterium Synechococcus sp. PCC 7002 was found to be located adjacent and 3 to the genes encoding the and subunits of phycocyanin. The identity of this gene, designated cpcC, was proven by matching the amino-terminal sequence of the authentic polypeptide with that predicted by the nucleotide sequence. A cpcC mutant strain of this cyanobacterium was constructed. The effect of the mutation was to prevent assembly of half the total phycocyanin into phycobilisomes. By electron microscopy, phycobilisomes from this mutant were shown to contain rod substructures composed of a single disc of hexameric phycocyanin, as opposed to two discs in the wild type. It was concluded that the M_r 33000 linker polypeptide is required for attachment of the core-distal phycocyanin hexamer to the core-proximal one. Using absorption spectra of the wild type, CpcC^–, and phycocyanin-less phycobilisomes, the in situ absorbances expected for specific phycocyanin-linker complexes were calculated. These data confirm earlier findings on isolated complexes regarding the influence of linkers on the spectroscopic properties of phycocyanin.Abbreviations PC phycocyanin - PEC phycoerythrocyanin - AP allophycocyanin - SDS-PAGE polyacrylamide gel electrophoresis in the presence of sodium dodecylsulfate. Linker polypeptides are abbreviated according to Glazer (1985). L _infX ^supY refers to a linker having a mass Y, located at a position X in the phycobilisome, where X can be R (rod), RC (rod or core), C (core) or CM (core to membrane). When necessary, the abbreviation for a linker is appended with that of its associated phycobiliprotein. Thus, L _infR ^sup34.5PEC is a rod linker of M_r 34 500 that is associated with phycoerythrocyanin     

A thioredoxin-activated fructose-1,6-bisphosphatase from the cyanobacterium Synechococcus 6301

Fructose-1,6-bisphosphatase was isolated from the cyanobacterium Synechococcus 6301 by acid precipitation, ammonium-sulfate fractionation, and Sephadex gel chromatography. The purified enzyme needed thiols and MgCl₂ for activity. The following K_m-values were obtained: a) for fructose-1,6-bisphosphate: 1.7 mM; b) for MgCl₂: 12.5 mM; c) for dithiocrythritol: 0,56 mM; d) for glutathione: 14 mM; e) for mercaptoethanol: 22 mM; f) for cysteine: 50 mM. Thioredoxin B isolated from this organism will activate this fructose-1,6-bisphosphatase. The K_m of thioredoxin B for this fructose-1,6-bisphosphatase was determined to be 1.7 M, endicotiy that thioredoxin might activate the fructose-1,6-bisphosphatase in Synechococcus in vivo.     

Acetate uptake by the unicellular cyanobacteria Synechococcus and Aphanocapsa

Acetate uptake by strains of Synechococcus and Aphanocapsa in short experiments required light, and was strongly inhibited by m-dichlorocarbonyl cyanide phenylhydrazone and dichlorophenyl dimethyl urea. Acetate carbon was distributed in amino acids and in the acyl portion of lipids in the same way as during growth experiments when CO₂ was available, but the reduced incorporation in the absence of CO₂ was primarily into the lipid fraction. An apparent K _m for uptake by Synechococcus and for Aphanocapsa 6308 of 20 and 180 M at pH 7.4 was obtained; corresponding V _max values were 6 and 11 nmol x min^-1 x mg protein^-1. Uptake with Synechococcus was affected by pH, with affinity decreased and maximal rate increase with rising pH. Acetate uptake was not affected by propionate or butyrate when both were added at the same time, but a light and concentration dependent inhibition developed if suspensions were preincubated with propionate. Acetate carbon moved rapidly into acid insoluble material, but after 10–15 s 75% or more of the recovered intracellular counts were in acetyl CoA. Counts in this compound were reduced by preincubation with propionate.Kinetic measurements of acetyl CoA synthetase in fractionated cell extracts gave values for K _m of about 50 M for acetate, 5 mM for propionate, 100 M for CoA and 0.38 mM for ATP. The internal pool of free CoA was measured to be about 20 M, and was reduced by preincubation with propionate. This suggests that the activity of CoA-mediated reactions may be regulated by the availability of this cofactor.Abbreviations Used CCCP m-Dichlorocarbonyl cyanide phenyl hydrazone - DCMU dichlorophenyl dimethyl urea - TCA trichloroacetic acid - Tris trishydroxymethyl amino methane - HEPES N-2-hydroxyethylpiperazine-N-2-ethane-sulfonic acid     

Binding affinity of bicarbonate and formate in herbicide-resistant D1 mutants of Synechococcus sp. PCC 7942

We examined the effects of mutations at amino acid residues S264 and F255 in the D1 protein on the binding affinity of the stimulatory anion bicarbonate and inhibitory anion formate in Photosystem II (PS II) in Synechococcus sp. PCC 7942. Measurements on the rates of oxygen evolution in the wild type and mutant cells in the presence of different concentrations of formate with a fixed bicarbonate concentration and vice versa, analyzed in terms of an equilibrium activator-inhibitor model, led to the conclusion that the equilibrium dissociation constant for bicarbonate is increased in the mutants, while that of the formate remains unchanged (11±0.5 mM). The hierarchy of the equilibrium dissociation constant for bicarbonate (highest to lowest, ±2 M) was: D1-F255L/S264A (46 M)>D1-F255Y/ S264A (31 M)D1-S264A (34 M)D1-F255Y (33 M)>wild type (25 M). The data suggest the importance of D1-S264 and D1-F255 in the bicarbonate binding niche. A possible involvement of bicarbonate and these two residues in the protonation of Q_B ^-, the reduced secondary plastoquinone of PS II, in the D1 protein is discussed.Abbreviations Chl a chlorophyll a - DBMIB 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone - DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea - DMQ 2,5-dimethyl-p-benzoquinone - HEPES N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid - MES 2-[N-morpholino]ethanesulfonic acid - PSI Photosystem I - PS II Photosystem II - Q_A bound plastoquinone, a one-electron acceptor in Photosystem II - Q_B another bound plastoquinone, a two-electron acceptor in Photosystem II This paper is dedicated to the memory of my dear friend Robin Hill-Govindjee.