Manganese-stabilizing Protein Research Articles

Interaction of Soluble and Immobilized Manganese-Stabilizing PsbO Protein with Manganese Ions and Isolated D1/D2/cyt b559 Complex of the PSII Reaction Center

Interaction of water-soluble and immobilized (on BrCN-activated agarose) manganese-stabilizing protein PsbO with Mn2+ and Mn3+ cations and with preparations of D1/D2/cyt b559 reaction center (RC) of photosystem II was studied. By native electrophoresis, the formation of dimeric and aggregated forms of PsbO protein were found in the presence of Mn2+, Mg2+, or Fe2+ ions. The dimerization of PsbO occurred after ultraviolet irradiation of the protein preparation. The presence of protein-bound Mn3+ cations increased the electrostatic interaction of the immobilized PsbO with the RC. This was evidenced to by higher amounts of CaCl2 that were required for dissociation of the PsbORC complex. It was first demonstrated that the protein exhibited superoxide dismutase (SOD) activity after an electrophoresis in PAAG upon incubation of the gel in an Mn2+-containing solution. Tetrazolium-reductase activity was also ascertained in the protein after its electrophoresis in a mixture with preparations of the oxygen-evolving complex (OEC). It is suggested that the protein interaction with Mn ions and superoxide radicals, as well as short-term UV irradiation, reduces tyrosine and a disulfide bond in the PsbO protein. This yields tyrosil radical and SH-groups participating in redox reactions with ETC components. The interactions of PsbO with Mn cations and UV light, taking place in the chloroplast thylakoids, may regulate the protein binding to RC, modify structural organization of the protein, and promote its participation in alternative pathways of electron transport under the influence of stress factors. The hypothetical scheme of interaction of the immobilized PsbO protein with Mn ions and RC is discussed.

Root cultures of potato mutant lacking MSPI isoform, indispensable for photosynthetic light reactions, exhibit characteristics similar to intact plant roots

Potato (Solanum tuberosum) mutant (ST) lacking one isoform of manganese-stabilizing protein (MSPI) of photosystem II exhibited besides spontaneous tuberization also growth changes with strongly impaired root system development. Previous studies revealed marked changes in carbohydrate levels and allocation within ST plant body. To verify causal relationship between changed carbohydrate balance and root growth restriction we engaged dark grown sucrose-supplied root organ-cultures of ST plants to exclude/confirm shoot effects. Unexpectedly, in ST root cultures we observed large alterations in growth and architecture as well as saccharide status similar to those found in the intact plant roots. The gene expression analysis, however, proved PsbO1 transcript (coding MSPI protein) neither in ST nor in WT root-organ cultures. Therefore, the results point to indirect effects of PsbO1 allele absence connected possibly with some epigenetic modulations.

Effects of PSII Manganese-Stabilizing Protein Succinylation on Photosynthesis in the Model Cyanobacterium Synechococcus sp. PCC 7002

Lysine succinylation is a newly identified protein post-translational modification and plays important roles in various biological pathways in both prokaryotes and eukaryotes, but its extent and function in photosynthetic organisms remain largely unknown. Here, we performed the first systematic studies of lysine succinylation in cyanobacteria, which are the only prokaryotes capable of oxygenic photosynthesis and the established model organisms for studying photosynthetic mechanisms. By using mass spectrometry analysis in combination with the enrichment of succinylated peptides from digested cell lysates, we identified 1,704 lysine succinylation sites on 691 proteins in a model cyanobacterium Synechococcus sp. PCC 7002. Bioinformatic analysis revealed that a large proportion of the succinylation sites were present on proteins in photosynthesis and metabolism. Among all identified succinylated proteins involved in photosynthesis, the PSII manganese-stabilizing protein (PsbO) was found to be succinylated on Lys99 and Lys234. Functional studies of PsbO were performed by site-directed mutagenesis, and mutants mimicking either constitutively succinylated (K99E and K234E) or non-succinylated states (K99R and K234R) were constructed. The succinylation-mimicking K234E mutant exhibited a decreased oxygen evolution rate of the PSII center and the efficiency of energy transfer during the photosynthetic reaction. Molecular dynamics simulations suggested a mechanism that may allow succinylation to influence the efficiency of photosynthesis by altering the conformation of PsbO, thereby hindering the interaction between PsbO and the PSII core. Our findings suggest that reversible succinylation may be an important regulatory mechanism during photosynthesis in Synechococcus, as well as in other photosynthetic organisms.

Lysine Acetylome Analysis Reveals Photosystem II Manganese-stabilizing Protein Acetylation is Involved in Negative Regulation of Oxygen Evolution in Model Cyanobacterium Synechococcus sp. PCC 7002.

Nε-Acetylation of lysine residues represents a frequently occurring post-translational modification widespread in bacteria that plays vital roles in regulating bacterial physiology and metabolism. However, the role of lysine acetylation in cyanobacteria remains unclear, presenting a hurdle to in-depth functional study of this post-translational modification. Here, we report the lysine acetylome of Synechococcus sp. PCC 7002 (hereafter Synechococcus) using peptide prefractionation, immunoaffinity enrichment, and coupling with high-precision liquid chromatography-tandem mass spectrometry analysis. Proteomic analysis of Synechococcus identified 1653 acetylation sites on 802 acetylproteins involved in a broad range of biological processes. Interestingly, the lysine acetylated proteins were enriched for proteins involved in photosynthesis, for example. Functional studies of the photosystem II manganese-stabilizing protein were performed by site-directed mutagenesis and mutants mimicking either constitutively acetylated (K99Q, K190Q, and K219Q) or nonacetylated states (K99R, K190R, and K219R) were constructed. Mutation of the K190 acetylation site resulted in a distinguishable phenotype. Compared with the K190R mutant, the K190Q mutant exhibited a decreased oxygen evolution rate and an enhanced cyclic electron transport rate in vivo Our findings provide new insight into the molecular mechanisms of lysine acetylation that involved in the negative regulation of oxygen evolution in Synechococcus and creates opportunities for in-depth elucidation of the physiological role of protein acetylation in photosynthesis in cyanobacteria.

Cellular capacities for high-light acclimation and changing lipid profiles across life cycle stages of the green alga Haematococcus pluvialis.

The unicellular microalga Haematococcus pluvialis has emerged as a promising biomass feedstock for the ketocarotenoid astaxanthin and neutral lipid triacylglycerol. Motile flagellates, resting palmella cells, and cysts are the major life cycle stages of H. pluvialis. Fast-growing motile cells are usually used to induce astaxanthin and triacylglycerol biosynthesis under stress conditions (high light or nutrient starvation); however, productivity of biomass and bioproducts are compromised due to the susceptibility of motile cells to stress. This study revealed that the Photosystem II (PSII) reaction center D1 protein, the manganese-stabilizing protein PsbO, and several major membrane glycerolipids (particularly for chloroplast membrane lipids monogalactosyldiacylglycerol and phosphatidylglycerol), decreased dramatically in motile cells under high light (HL). In contrast, palmella cells, which are transformed from motile cells after an extended period of time under favorable growth conditions, have developed multiple protective mechanisms—including reduction in chloroplast membrane lipids content, downplay of linear photosynthetic electron transport, and activating nonphotochemical quenching mechanisms—while accumulating triacylglycerol. Consequently, the membrane lipids and PSII proteins (D1 and PsbO) remained relatively stable in palmella cells subjected to HL. Introducing palmella instead of motile cells to stress conditions may greatly increase astaxanthin and lipid production in H. pluvialis culture.

A novel P700 redox kinetics probe for rapid, non-intrusive and whole-tissue determination of photosystem II functionality, and the stoichiometry of the two photosystems in vivo.

We sought a rapid, non-intrusive, whole-tissue measure of the functional photosystem II (PS II) content in leaves. Summation of electrons, delivered by a single-turnover flash to P700(+) (oxidized PS I primary donor) in continuous background far-red light, gave a parameter S in absorbance units after taking into account an experimentally determined basal electron flux that affects P700 redox kinetics. S was linearly correlated with the functional PS II content measured by the O(2) yield per single-turnover repetitive flash in Arabidopsis thaliana expressing an antisense construct to the PsbO (manganese-stabilizing protein in PS II) proteins of PS II (PsbO mutants). The ratio of S to z(max) (total PS I content in absorbance units) was comparable to the PS II/PS I reaction-center ratio in wild-type A. thaliana and in control Spinacea oleracea. Both S and S/z(max) decreased in photoinhibited spinach leaf discs. The whole-tissue functional PS II content and the PS II/photosystem I (PS I) ratio can be non-intrusively monitored by S and S/z(max), respectively, using a quick P700 absorbance protocol compatible with modern P700 instruments.

Evaluation of abiotic stress tolerance in transgenic potato plants with reduced expression of PSII manganese stabilizing protein

Manganese stabilizing protein (MSP) is an important component of the Photosystem II (PSII) oxygen evolving complex. In our previous work, transgenic potato plants with reduced expression of MSP (MSP-As) were developed and their physiological and biochemical responses were studied. In this report, we address the response of MSP-As plants toward salinity, heavy metal and osmotic stresses. MSP-As plants treated with NaCl, ZnCl2 or mannitol solution showed significant level of tolerance under all the stress conditions. Specific enzyme activities of major ROS-scavenging enzymes were found significantly higher in MSP-As plants than the control plants. MSP-As plants accumulated increased levels of proline and low molecular weight metabolites such as ascorbate and α-tocopherol, which indicated that these plants were much more resistant to stress compared to the corresponding control plants. The primary photochemical efficiencies and the OJIP kinetics analyses further confirmed that MSP-As plants were in better optimal health under stress compared to the control plants. Although the exact reason behind the increased stress tolerance in stressed MSP-As plants is unclear, our results strongly indicate the role of MSP of unknown function in abiotic stress tolerance.

Physiological and biochemical responses of transgenic potato plants with altered expression of PSII manganese stabilizing protein

Manganese-stabilizing protein (MSP) represents a key component of the oxygen-evolving complex (OEC). Transgenic potato plants with both enhanced (sense) and reduced (anti-sense) MSP expression levels were generated to investigate the possible physiological role of MSP in overall plant growth, particularly in tuber development. MSP antisense plants exhibited both higher tuberization frequency and higher tuber yield with increased total soluble carbohydrates. The photosynthetic efficiencies of the plants were examined using the OJIP kinetics; MSP-antisense plants were photosynthetically more active than the MSP-sense and UT (untransformed) control plants. The oxygen measurements indicated that the relative oxygen evolution was directly proportional to the MSP expression, as MSP-antisense plants showed much lower oxygen evolution compared to MSP-sense as well as UT plants. MSP-sense plants behaved like the UT plants with respect to morphology, tuber yield, and photosynthetic performance. Chlorophyll a fluorescence analyses indicate a possible lack of intact Oxygen Evolving Complexes (OECs) in MSP antisense plants, which allow access to internal non-water electron donors (e.g., ascorbate and proline) and consequently increase the Photosystem II (PSII) activity of those plants. These findings further indicate that this altered photosynthetic machinery may be associated with early tuberization and increased tuberization frequency.

Probing the N-terminal sequence of spinach PsbO: evidence that essential threonine residues bind to different functional sites in eukaryotic photosystem II

The N-terminal ¹E-⁶L domain of the manganese-stabilizing protein (PsbO) from spinach prevents non-specific binding of the subunit to photosystem II (PSII) and deletions of the ¹E-⁷T or ¹E-¹⁵T sequences from the PsbO N-terminus reduce or impair, respectively, functional binding of PsbO to PSII (Popelkova et al., Biochemistry 42:6193-6200, 2003). The work presented here provides deeper insights into the interaction of PsbO with PSII. The data show that a single mutation, ¹⁵T → A in mature PsbO from spinach reduces the stoichiometry of its functional binding from two to one subunit per PSII and decreases reconstitution of activity to about 45 % of the wild-type control. Replacement of the ¹E-⁶L domain with ⁶M in the T15A PsbO mutant has no additional negative effect on recovery of O₂ evolution activity, but it significantly weakens both functional and nonspecific binding of the truncated mutant to PSII. These results suggest that the ¹⁵T side-chain by itself is essential for binding of one of two PsbO subunits to eukaryotic PSII and that specific PSII-binding sites for PsbO are distinguishable; one PSII-binding site does not require PsbO-¹⁵T and probably interacts with the other N-terminal domain of PsbO. Identity of the latter domain is revealed by a requirement for the presence of the ¹E-⁶L sequence that is shown here to be necessary for high-affinity binding of PsbO to PSII. When combined with previous results, the data presented here lead to a more detailed model for PsbO binding in eukaryotic PSII.

PH Optimum of the Photosystem II H2O Oxidation Reaction: Effects of PsbO, the Manganese-Stabilizing Protein, Cl– Retention, and Deprotonation of a Component Required for O2 Evolution Activity

Hydroxide ion inhibits Photosystem II (PSII) activity by extracting Cl(-) from its binding site in the O(2)-evolving complex (OEC) under continuous illumination [Critchley, C., et al. (1982) Biochim. Biophys. Acta 682, 436]. The experiments reported here examine whether two subunits of PsbO, the manganese-stabilizing protein, bound to eukaryotic PSII play a role in protecting the OEC against OH(-) inhibition. The data show that the PSII binding properties of PsbO affect the pH optimum for O(2) evolution activity as well as the Cl(-) affinity of the OEC that decreases with an increasing pH. These results suggest that PsbO functions as a barrier against inhibition of the OEC by OH(-). Through facilitation of efficient retention of Cl(-) in PSII [Popelkova, H., et al. (2008) Biochemistry 47, 12593], PsbO influences the ability of Cl(-) to resist OH(-)-induced release from its site in the OEC. Preventing inhibition by OH(-) allows for normal (short) lifetimes of the S(2) and S(3) states in darkness [Roose, J. L., et al. (2011) Biochemistry 50, 5988] and for maximal steady-state activity by PSII. The data presented here indicate that activation of H(2)O oxidation occurs with a pK(a) of ∼6.5, which could be a function of deprotonation of one or more amino acid residues that reside near the OEC active site on the D1 and CP43 intrinsic subunits of the PSII reaction center.

Expression pattern of the psbO gene and its involvement in acclimation of the photosynthetic apparatus during abiotic stresses in Festuca arundinacea and F. pratensis

PsbO, the manganese-stabilizing protein, plays a crucial role in oxygen-evolving complex functioning and stabilization, by maintaining optimal manganese, calcium and chloride concentrations at the active state of PSII. In this paper we present a study focused on recognizing the relationship between psbO gene activity and acclimation of the photosynthetic apparatus under abiotic stresses in the grasses Festuca arundinacea and F. pratensis. PsbO expression was compared between two distinct genotypes within each species which differed in their levels of stress tolerance (drought and frost, respectively) during drought treatment (F. arundinacea) and cold acclimation (F. pratensis). The research involved: (1) the analyses of psbO gene expression profiles using real-time PCR, and (2) the analyses of PsbO protein accumulation profiles using protein gel blot hybridization. The results indicate that PsbO plays a protective function with respect to the photosynthetic apparatus during abiotic stresses. In cold-treated F. pratensis plants the accumulation of PsbO seems to be responsible for differences in the PSII photochemical efficiency. Higher stability of PSII during drought, observed in the high-drought tolerant F. arundinacea genotype, is not associated with PsbO accumulation, although the degradation of this protein affects destabilization of the oxygen-evolving complex in drought.

Probing the topography of the photosystem II oxygen evolving complex: PsbO is required for efficient calcium protection of the manganese cluster against dark-inhibition by an artificial reductant

The photosystem II (PSII) manganese-stabilizing protein (PsbO) is known to be the essential PSII extrinsic subunit for stabilization and retention of the Mn and Cl(-) cofactors in the oxygen evolving complex (OEC) of PSII, but its function relative to Ca(2+) is less clear. To obtain a better insight into the relationship, if any, between PsbO and Ca(2+) binding in the OEC, samples with altered PsbO-PSII binding properties were probed for their potential to promote the ability of Ca(2+) to protect the Mn cluster against dark-inhibition by an exogenous artificial reductant, N,N-dimethylhydroxylamine. In the absence of the PsbP and PsbQ extrinsic subunits, Ca(2+) and its surrogates (Sr(2+), Cd(2+)) shield Mn atoms from inhibitory reduction (Kuntzleman et al., Phys Chem Chem Phys 6:4897, 2004). The results presented here show that PsbO exhibits a positive effect on Ca(2+) binding in the OEC by facilitating the ability of the metal to prevent inhibition of activity by the reductant. The data presented here suggest that PsbO may have a role in the formation of the OEC-associated Ca(2+) binding site by promoting the equilibrium between bound and free Ca(2+) that favors the bound metal.

Binding Stoichiometry and Affinity of the Manganese-Stabilizing Protein Affects Redox Reactions on the Oxidizing Side of Photosystem II

It has been reported previously that the two subunits of PsbO, the photosystem II (PSII) manganese stabilizing protein, have unique functions in relation to the Mn, Ca(2+), and Cl(-) cofactors in eukaryotic PSII [Popelkova; (2008) Biochemistry 47, 12593]. The experiments reported here utilize a set of N-terminal truncation mutants of PsbO, which exhibit altered subunit binding to PSII, to further characterize its role in establishing efficient O(2) evolution activity. The effects of PsbO binding stoichiometry, affinity, and specificity on Q(A)(-) reoxidation kinetics after a single turnover flash, S-state transitions, and O(2) release time have been examined. The data presented here show that weak rebinding of a single PsbO subunit to PsbO-depleted PSII repairs many of the defects in PSII resulting from the removal of the protein, but many of these are not sustainable, as indicated by low steady-state activities of the reconstituted samples [Popelkova; (2003) Biochemistry 42 , 6193]. High affinity binding of PsbO to PSII is required to produce more stable and efficient cycling of the water oxidation reaction. Reconstitution of the second PsbO subunit is needed to further optimize redox reactions on the PSII oxidizing side. Native PsbO and recombinant wild-type PsbO from spinach facilitate PSII redox reactions in a very similar manner, and nonspecific binding of PsbO to PSII has no significance in these reactions.

Structural Investigation of PsbO from Plant and Cyanobacterial Photosystem II

The manganese-stabilizing protein PsbO is associated with the luminal side of thylakoids close to the redox-active Mn4Ca cluster at the catalytically active site of photosystem II (PSII). PsbO is believed to increase the efficiency of oxygen evolution and to stabilize the Mn4Ca cluster against photoinhibition. Using small-angle X-ray scattering, we investigated the low-resolution structure of wild-type spinach PsbO and that of chimeric spinach PsbO fused with maltose-binding protein. Small-angle X-ray scattering data revealed that both proteins are monomeric in solution, and that plant and cyanobacterial PsbO have similar structures. We show a highly efficient expression system that allows recombinant production of the active wild type and the chimeric PsbO from spinach and cyanobacteria, with yields compatible with biophysical and structural studies. The binding of spinach PsbO fused with maltose-binding protein to PSII depleted of extrinsic subunits (PSII-ΔpsbO,P,Q) was confirmed by matrix-assisted laser desorption ionization time-of-flight mass spectrometry. The reconstituted PSII was shown to have similar oxygen evolution rates as obtained with wild-type spinach PsbO.

The double mutation ΔL6MW241F in PsbO, the photosystem II manganese stabilizing protein, yields insights into the evolution of its structure and function

The W241F mutation in spinach manganese-stabilizing protein (PsbO) decreases binding to photosystem II (PSII); its thermostability is increased and reconstituted activity is lower [Wyman et al. (2008) Biochemistry 47, 6490–6498]. The results reported here show that W241F cannot adopt a normal solution structure and fails to reconstitute efficient Cl − retention by PSII. An N-terminal truncation of W241F, producing the ΔL6MW241F double mutant that resembles some features of cyanobacterial PsbO, significantly repairs the defects in W241F. Our data suggest that the C-terminal F → W mutation likely evolved in higher plants and green algae in order to preserve proper PsbO folding and PSII binding and assembly, which promotes efficient Cl − retention in the oxygen-evolving complex.

Function of PsbO, the Photosystem II Manganese-Stabilizing Protein: Probing the Role of Aspartic Acid 157

The D157N, D157E, and D157K mutations in the psbO gene encoding the photosystem II (PSII) manganese-stabilizing protein from spinach, exhibit near-wild-type PSII binding but are significantly impaired in O(2) evolution activity and Cl(-) retention by PSII [Popelkova et al. (2009) Biochemistry 48, 11920-11928]. To better characterize the role of PsbO-Asp157 in eukaryotic PSII, the effect of mutations in Asp157 on heat-induced changes in PsbO solution structure, O(2) release kinetics, and PSII redox reactions both within and outside the oxygen-evolving complex (OEC) have been examined. The data presented here show that Asn, Glu, or Lys mutations in PsbO-Asp157 modify PsbO thermostability in solution, which is consistent with the previously reported perturbation of the functional assembly of PsbO-Asp157 mutants into PSII that caused inefficient Cl(-) retention by PSII. Fluorescence decay signals from PSII reconstituted with Asp157 mutants indicate that that the Q(A)(-) to Q(B) transition on the PSII reducing side is unaffected, but complex alterations are detected on the PSII oxidizing side that affect the recombination of Q(A)(-) with the O(2)-evolving complex. In addition, oxygen yield on the first flash is increased, which indicates an impaired ability of mutant-reconstituted PSII samples to decay back to the S(1) state in the dark.

The evolution of Photosystem II: insights into the past and future

This article attempts to address the molecular origin of Photosystem II (PSII), the central component in oxygenic photosynthesis. It discusses the possible evolution of the relevant cofactors needed for splitting water into molecular O2 with respect to the following functional domains in PSII: the reaction center (RC), the oxygen evolving complex (OEC), and the manganese stabilizing protein (MSP). Possible ancestral sources of the relevant cofactors are considered, as are scenarios of how these components may have been brought together to produce the intermediate steps in the evolution of PSII. Most importantly, the driving forces that maintained these intermediates for continued adaptation are considered. We then apply our understanding of the evolution of PSII to the bioengineering of a water oxidizing catalyst for utilization of solar energy.

Interaction of bicarbonate with the manganese-stabilizing protein of photosystem II

The effect of reversible removal of HCO 3 - on structural re-arrangements in the Mn-stabilizing protein (MSP) of photosystem II, isolated from pea leaves, was studied using measurements of characteristic alterations in fluorescence of hydrophobic probe 8-anilino-1-naphthalene-sulfonic acid (ANS). It was shown that the treatments capable of removal of HCO 3 - (or CO 2) from possible binding sites in MSP (pH lowering from 6.5 to 3.5, addition of a structurally similar anion HCO 3 - in concentration 1–20 mM or air evacuation at pH 3.5) result in a significant (up to 370%) increase of ANS fluorescence (indicative of structural changes in MSP), whereas HCO 3 - lowers the ANS fluorescence to the initial level observed in untreated protein at pH 6.5. Since the effects are revealed at (sub)micromolar concentrations of HCO 3 - , the specific high-affinity binding of HCO 3 - (or CO 2) to MSP (required for its native structure preservation) is proposed. Possible bicarbonate binding sites and its physiological role within the water-oxidizing complex of photosystem II are discussed.

Asp157 Is Required for the Function of PsbO, the Photosystem II Manganese Stabilizing Protein

PsbO, the photosystem II manganese stabilizing protein, contains an aspartate residue [Asp157 (spinach numbering)], which is highly conserved in eukaryotic and prokaryotic PsbOs. The homology model of the PSII-bound conformation of spinach PsbO presented here positions Asp157 in the large flexible loop of the protein. We have characterized site-directed mutants (D157N, D157E, and D157K) of spinach PsbO that were rebound to PsbO-depleted PSII to probe the role of Asp157. Structural data revealed that PsbO Asp157 mutants exhibit near-wild-type solution structure at 25 degrees C, but functional analyses of the mutants showed that these are the first genetically modified PsbO proteins from spinach that combine wild-type PSII binding behavior with significantly impaired O(2) evolution activity; all of the mutants reconstituted approximately 30% of control O(2) evolution activity. PsbO Asp157 has been proposed to be a part of a putative H(2)O/H(+) channel that links the active site of the oxygen-evolving complex with the lumen [De Las Rivas, J., and Barber, J. (2004) Photosynth. Res. 81, 329-343]. Unsuccessful attempts to use chemical rescue to enhance the activity restored by Asp157 mutants could indicate that this residue is not involved in a proton transfer network. It is shown, however, that these mutants are deficient in restoring efficient Cl(-) retention by PSII.

Inorganic Cofactor Stabilization and Retention: The Unique Functions of the Two PsbO Subunits of Eukaryotic Photosystem II

Eukaryotic PsbO, the photosystem II (PSII) manganese-stabilizing protein, has two N-terminal sequences that are required for binding of two copies of the protein to PSII [Popelkova, H., et al. (2002) Biochemistry 41, 10038-10045; Popelkova, H., et al. (2003) Biochemistry 42, 6193-6200]. In the work reported here, a set of selected N-terminal truncation mutants of PsbO that affect subunit binding to PSII were used to determine the effects of PsbO stoichiometry on the Mn, Ca(2+), and Cl(-) cofactors and to characterize the roles of each of the PsbO subunits in PSII function. Results of the experiments with the PsbO-depleted PSII membranes reconstituted with the PsbO deletion mutants showed that the presence of PsbO does not affect Ca(2+) retention by PSII in steady-state assays of activity, nor is it required for Ca(2+) to protect the Mn cluster against reductive inhibition in darkness. In contrast to the results with Ca(2+), PsbO increases the affinity of Cl(-) for the active site of the O(2)-evolving complex (OEC) as expected. These results together with other data on activity retention suggest that PsbO can stabilize the Mn cluster by facilitating retention of Cl(-) in the OEC. The data presented here indicate that each of two copies of PsbO has a distinctive function in PSII. Binding of the first PsbO subunit fully stabilizes the Mn cluster and enhances Cl(-) retention, while binding of the second subunit optimizes Cl(-) retention, which in turn maximizes O(2) evolution activity. Nonspecific binding of some PsbO truncation mutants to PSII has no functional significance.