Tatiana Shutova

A photosystem II‐associated carbonic anhydrase regulates the efficiency of photosynthetic oxygen evolution

We show for the first time that Cah3, a carbonic anhydrase associated with the photosystem II (PSII) donor side in Chlamydomonas reinhardtii, regulates the water oxidation reaction. The mutant cia3, lacking Cah3 activity, has an impaired water splitting capacity, as shown for intact cells, thylakoids and PSII particles. To compensate this impairment, the mutant overproduces PSII reaction centres (1.6 times more than wild type). We present compelling evidence that the mutant has an average of two manganese atoms per PSII reaction centre. When bicarbonate is added to mutant thylakoids or PSII particles, the O2 evolution rates exceed those of the wild type by up to 50%. The donor side of PSII in the mutant also exhibits a much higher sensitivity to overexcitation than that of the wild type. We therefore conclude that Cah3 activity is necessary to stabilize the manganese cluster and maintain the water‐oxidizing complex in a functionally active state. The possibility that two manganese atoms are enough for water oxidation if bicarbonate ions are available is discussed.

The photosystem II-associated Cah3 in Chlamydomonas enhances the O2 evolution rate by proton removal.

Water oxidation in photosystem II (PSII) is still insufficiently understood and is assumed to involve HCO3−. A Chlamydomonas mutant lacking a carbonic anhydrase associated with the PSII donor side shows impaired O2 evolution in the absence of HCO3−. The O2 evolution for saturating, continuous illumination (RO2) was slower than in the wild type, but was elevated by HCO3− and increased further by Cah3. The RO2 limitation in the absence of Cah3/HCO3− was amplified by H2O/D2O exchange, but relieved by an amphiphilic proton carrier, suggesting a role of Cah3/HCO3− in proton translocation. Chlorophyll fluorescence indicates a Cah3/HCO3− effect at the donor side of PSII. Time‐resolved delayed fluorescence and O2‐release measurements suggest specific effects on proton‐release steps but not on electron transfer. We propose that Cah3 promotes proton removal from the Mn complex by locally providing HCO3−, which may function as proton carrier. Without Cah3, proton removal could become rate limiting during O2 formation and thus, limit water oxidation under high light. Our results underlie the general importance of proton release at the donor side of PSII during water oxidation.

A Metabolomic Approach to Study Major Metabolite Changes during Acclimation to Limiting CO2 in Chlamydomonas reinhardtii

Using a gas chromatography-mass spectrometry-time of flight technique, we determined major metabolite changes during induction of the carbon-concentrating mechanism in the unicellular green alga Chlamydomonas reinhardtii. In total, 128 metabolites with significant differences between high- and low-CO2-grown cells were detected, of which 82 were wholly or partially identified, including amino acids, lipids, and carbohydrates. In a 24-h time course experiment, we show that the amino acids serine and phenylalanine increase transiently while aspartate and glutamate decrease after transfer to low CO2. The biggest differences were typically observed 3 h after transfer to low-CO2 conditions. Therefore, we made a careful metabolomic examination at the 3-h time point, comparing low-CO2 treatment to high-CO2 control. Five metabolites involved in photorespiration, 11 amino acids, and one lipid were increased, while six amino acids and, interestingly, 21 lipids were significantly lower. Our conclusion is that the metabolic pattern during early induction of the carbon-concentrating mechanism fit a model where photorespiration is increasing.

Importance of post-translational modifications for functionality of a chloroplast-localized carbonic anhydrase (CAH1) in Arabidopsis thaliana

Background The Arabidopsis CAH1 alpha-type carbonic anhydrase is one of the few plant proteins known to be targeted to the chloroplast through the secretory pathway. CAH1 is post-translationally modified at several residues by the attachment of N-glycans, resulting in a mature protein harbouring complex-type glycans. The reason of why trafficking through this non-canonical pathway is beneficial for certain chloroplast resident proteins is not yet known. Therefore, to elucidate the significance of glycosylation in trafficking and the effect of glycosylation on the stability and function of the protein, epitope-labelled wild type and mutated versions of CAH1 were expressed in plant cells. Methodology/Principal Findings Transient expression of mutant CAH1 with disrupted glycosylation sites showed that the protein harbours four, or in certain cases five, N-glycans. While the wild type protein trafficked through the secretory pathway to the chloroplast, the non-glycosylated protein formed aggregates and associated with the ER chaperone BiP, indicating that glycosylation of CAH1 facilitates folding and ER-export. Using cysteine mutants we also assessed the role of disulphide bridge formation in the folding and stability of CAH1. We found that a disulphide bridge between cysteines at positions 27 and 191 in the mature protein was required for correct folding of the protein. Using a mass spectrometric approach we were able to measure the enzymatic activity of CAH1 protein. Under circumstances where protein N-glycosylation is blocked in vivo, the activity of CAH1 is completely inhibited. Conclusions/Significance We show for the first time the importance of post-translational modifications such as N-glycosylation and intramolecular disulphide bridge formation in folding and trafficking of a protein from the secretory pathway to the chloroplast in higher plants. Requirements for these post-translational modifications for a fully functional native protein explain the need for an alternative route to the chloroplast.

Is the manganese stabilizing 33 kDa protein of photosystem II attaining a ‘natively unfolded’ or ‘molten globule’ structure in solution?

This study compares the properties of the extrinsic 33 kDa subunit acting as ‘manganese stabilizing protein’ (MSP) of the water oxidizing complex with characteristic features of proteins that are known to attain a ‘natively unfolded’ or a ‘molten globule’ structure. The analysis leads to the conclusion that the MSP in solution is most likely a ‘molten globule’ with well defined compact regions of β structure. The possible role of these structural peculiarities of MSP in solution for its function as important constituent of the WOC is discussed.

Phosphorylation controls the localization and activation of the lumenal carbonic anhydrase in Chlamydomonas reinhardtii.

Background Cah3 is the only carbonic anhydrase (CA) isoform located in the thylakoid lumen of Chlamydomonas reinhardtii. Previous studies demonstrated its association with the donor side of the photosystem II (PSII) where it is required for the optimal function of the water oxidizing complex. However this enzyme has also been frequently proposed to perform a critical function in inorganic carbon acquisition and CO2 fixation and all mutants lacking Cah3 exhibit very poor growth after transfer to low CO2 conditions. Results/Conclusions In the present work we demonstrate that after transfer to low CO2, Cah3 is phosphorylated and that phosphorylation is correlated to changes in its localization and its increase in activity. When C. reinhardtii wild-type cells were acclimated to limiting CO2 conditions, the Cah3 activity increased about 5–6 fold. Under these conditions, there were no detectable changes in the level of the Cah3 polypeptide. The increase in activity was specifically inhibited in the presence of Staurosporine, a protein kinase inhibitor, suggesting that the Cah3 protein was post-translationally regulated via phosphorylation. Immunoprecipitation and in vitro dephosphorylation experiments confirm this hypothesis. In vivo phosphorylation analysis of thylakoid polypeptides indicates that there was a 3-fold increase in the phosphorylation signal of the Cah3 polypeptide within the first two hours after transfer to low CO2 conditions. The increase in the phosphorylation signal was correlated with changes in the intracellular localization of the Cah3 protein. Under high CO2 conditions, the Cah3 protein was only associated with the donor side of PSII in the stroma thylakoids. In contrast, in cells grown at limiting CO2 the protein was partly concentrated in the thylakoids crossing the pyrenoid, which did not contain PSII and were surrounded by Rubisco molecules. Significance This is the first report of a CA being post-translationally regulated and describing phosphorylation events in the thylakoid lumen.

Mobile hydrogen carbonate acts as proton acceptor in photosynthetic water oxidation

Significance Photosynthesis by cyanobacteria, algae, and plants sustains life on Earth by oxidizing water to the O2 we breathe and by converting CO2 into biomass we eat, burn, or use otherwise. Although O2 production and CO2 reduction are functionally and structurally well separated in photosynthetic organisms, there is a long debated role of CO2/ in water oxidation. Here we demonstrate that acts as mobile acceptor and transporter of protons produced by photosystem II, and that depletion of leads to a reversible down-regulation of O2 production. These findings add a previously unidentified component to the regulatory networks in higher plants, algae, and cyanobacteria and conclude the long quest for the function of CO2/ in photosynthetic water oxidation. Cyanobacteria, algae, and plants oxidize water to the O2 we breathe, and consume CO2 during the synthesis of biomass. Although these vital processes are functionally and structurally well separated in photosynthetic organisms, there is a long-debated role for CO2/ in water oxidation. Using membrane-inlet mass spectrometry we demonstrate that acts as a mobile proton acceptor that helps to transport the protons produced inside of photosystem II by water oxidation out into the chloroplast’s lumen, resulting in a light-driven production of O2 and CO2. Depletion of from the media leads, in the absence of added buffers, to a reversible down-regulation of O2 production by about 20%. These findings add a previously unidentified component to the regulatory network of oxygenic photosynthesis and conclude the more than 50-y-long quest for the function of CO2/ in photosynthetic water oxidation.

Carbonic Anhydrase Activities in Pea Thylakoids

Pea thylakoids with high carbonic anhydrase (CA) activity (average rates of 5000 µmol H+ (mg Chl)−1 h−1 at pH 7.0) were prepared. Western blot analysis using antibodies raised against the soluble stromal β-CA from spinach clearly showed that this activity is not a result of contamination of the thylakoids with the stromal CA but is derived from a thylakoid membrane-associated CA. Increase of the CA activity after partial membrane disintegration by detergent treatment, freezing or sonication implies the location of the CA in the thylakoid interior. Salt treatment of thylakoids demonstrated that while one part of the initial enzyme activity is easily soluble, the rest of it appears to be tightly associated with the membrane. CA activity being measured as HCO3− dehydration (dehydrase activity) in Photosystem II particles (BBY) was variable and usually low. The highest and most reproducible activities (approximately 2000 µmol H+ (mg Chl)−1 h−1) were observed in the presence of detergents (Triton X-100 or n-octyl-β-D-glucopyranoside) in low concentrations. The dehydrase CA activity of BBY particles was more sensitive to the lipophilic CA inhibitor, ethoxyzolamide, than to the hydrophilic CA inhibitor, acetazolamide. CA activity was detected in PS II core complexes with average rate of 13,000 µmol H+ (mg Chl)−1 h−1 which was comparable to CA activity in BBY particles normalized on a PS II reaction center basis.

Importance of a single disulfide bond for the PsbO protein of photosystem II: protein structure stability and soluble overexpression in Escherichia coli

PsbO protein is an important constituent of the water–oxidizing complex, located on the lumenal side of photosystem II. We report here the efficient expression of the spinach PsbO in E. coli where the solubility depends entirely on the formation of the disulfide bond. The PsbO protein purified from a pET32 system that includes thioredoxin fusion is properly folded and functionally active. Urea unfolding experiments imply that the reduction of the single disulfide bridge decreases stability of the protein. Analysis of inter-residue contact density through the PsbO molecule shows that Cys51 is located in a cluster with high contact density. Reduction of the Cys28–Cys51 bond is proposed to perturb the packing interactions in this cluster and destabilize the protein as a whole. Taken together, our results give evidence that PsbO exists in solution as a compact highly ordered structure, provided that the disulfide bridge is not reduced.

Carbonic anhydrase activity of different photosystem II preparations

The requirement of the inorganic carbon (IC) for the proper electron transport activity of thylakoids has been notified far ago. The generally accepted view on the site of its action implies an involvement of IC in electron transfer on the acceptor side of Photosystem II [1]. However, there were some works demonstrating the involvement of IC in the maintaining of photochemistry at the donor side of PSII [2–6]. There were a number of works settled to reveal the «active species» of IC-effect on photochemistry, either bicarbonate ion or CO2. The principally new approach had originated from work of Stemler [7] putting together the scheme of both IC forms participation in the development of the «bicarbonate effect». This concept implies a coupling of an electron transport event with an act of conversion between forms of inorganic carbon, i.e. the participation of CA reaction in the performance of primary photosynthetic machinery. Added CA has been found to activate the Hill reaction maintained by chloroplast fragments [8]. Effects of light on the CA activity of thylakoids, both inhibitory or stimulatory in different conditions [9–11], dependency of this enzyme activity on the surrounding redox-potential [12], and on proton gradient in thylakoids both in the dark [13] and in the light [14] have been found providing additional basis to the existence of such coupling. Concerning an existence of specific CA in thylakoids, the corresponding activity was found in number of algae cells and in mesophyll of both C3- [15] and C4- [9] plants. This activity is believed to be associated with Photosystem II [15,9] and is different from the known soluble form of CA [12,13]. The isolation and sequencing of a form of CA localized in PSII of Chlamydomonas reinhardtii was performed recently [16]. However, the question on the nature and function of CA-activity in thylakoids and PSII particles in higher plants remains open. We show here that CA-activity of PSII fragments is associated with such a deep structure as PSII core complexes.