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Structural properties of the isolated extrinsic regulatory 33 kDa protein of the water-oxidizing complex were analyzed at different pH values. It was found that (a) titrations of the buffer capacity reveal a characteristic hysteresis effect that is unique for the 33 kDa subunit and is not observed for the other extrinsic proteins, (b) changes of the emission from the fluorescence probe 1,8-ANS are indicative of an increased accessibility of the hydrophobic core of the 33 kDa protein to the dye at lower pH, (c) the near-UV circular dichroism spectrum of the polypeptide is altered owing to a pH decrease from 6.8 to 3.8 and becomes drastically changed at pH 2.8, and (d) the content of secondary structure elements remains virtually constant in the range 3.8 < pH < 6.8, with the following values gathered from far-UV CD spectra: approximately 8% alpha-helix, approximately 33% beta-strand, approximately 15% turns, and approximately 44% random coil. Further acidification down to pH 2.8 gives rise to a decreased alpha-helix and increased beta-strand and random coil content. A theoretical model [Ptitsyn, O., & Finkelstein, A. (1983) Biopolymers 2, 15-22] was used to predict the probability and location of secondary structure elements within the protein sequence. On the basis of these calculations, an extended hydrophobic beta-sheet domain could exist in the center of the protein and an alpha-helix in the C-terminal region. From these data, the 33 kDa protein is inferred to change its tertiary structure in vitro upon acidification of the aqueous environment. Possible implications of these features are discussed.

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.

Cyanobacteria, algae, and plants oxidize water to the O 2 we breathe, and consume CO 2 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 CO 2 /HCO − 3 in water oxidation. Using membrane-inlet mass spectrometry we demonstrate that HCO − 3 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 O 2 and CO 2 . Depletion of HCO − 3 from the media leads, in the absence of added buffers, to a reversible down-regulation of O 2 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 CO 2 / HCO − 3 in photosynthetic water oxidation.O xygenic photosynthesis in cyanobacteria, algae, and higher plants leads to the reduction of atmospheric CO 2 to energy-rich carbohydrates. The electrons needed for this process are extracted in a cyclic, light-driven process from water that is split into dioxygen (O 2 ) and protons. This reaction is catalyzed by a penta-μ-oxo bridged tetra-manganese calcium cluster (Mn 4 CaO 5 ) within the oxygen-evolving complex (OEC) of photosystem II (PSII) (1-4). The possible roles of inorganic carbon, C i ðC i = CO 2 ; H 2 CO 3 ; HCO − 3 ; CO 2− 3 Þ, in this process have been a controversial issue ever since Otto Warburg and Günter Krippahl (5) reported in 1958 that oxygen evolution by PSII strictly depends on CO 2 and therefore has to be based on the photolysis of H 2 CO 3 ("Kohlensäure") and not of water. These first experiments were indirect and, as became apparent later, were wrongly interpreted (6-8). Several research groups followed up on these initial results and identified two possible sites of C i interaction within PSII (reviewed in refs. 9-12). Functional and spectroscopic studies showed that HCO − 3 facilitates the reduction of the secondary plastoquinone electron acceptor (Q B ) of PSII by participating in the protonation of Q 2− B . Binding of HCO − 3 (or CO 2− 3 ) to the nonheme Fe between the quinones Q A and Q B was recently confirmed by X-ray crystallography (3,13,14). Despite this functional role at the acceptor side, the very tight binding of HCO − 3 to this site makes it impossible for the activity of PSII to be affected by changing the C i level of the medium; instead inhibitors such as formate need to be added to induce the acceptor-side effect (15). Consequently, the watersplitting electron-donor side of PSII has also been studied intensively (for recent reviews, see refs. 11 and 12). Although a tight binding of C i near the Mn 4 CaO 5 cluster is excluded on the basis of X-ray crystallography (3, 14), FTIR spectroscopy (16), and mass spectrometry (17, 18), the possibility that a weakly bound HCO − 3 affects the activity of PSII at the donor side remains a viable option (reviewed i...

Besides an essential role in optimizing water oxidation in photosystem II (PSII), it has been reported that the spinach PsbO protein binds GTP [C. Spetea, T. Hundal, B. Lundin, M. Heddad, I. Adamska, B. Andersson, Proc. Natl. Acad. Sci. U.S.A. 101 (2004) 1409-1414]. Here we predict four GTP-binding domains in the structure of spinach PsbO, all localized in the beta-barrel domain of the protein, as judged from comparison with the 3D-structure of the cyanobacterial counterpart. These domains are not conserved in the sequences of the cyanobacterial or green algae PsbO proteins. MgGTP induces specific changes in the structure of the PsbO protein in solution, as detected by circular dichroism and intrinsic fluorescence spectroscopy. Spinach PsbO has a low intrinsic GTPase activity, which is enhanced fifteen-fold when the protein is associated with the PSII complex in its dimeric form. GTP stimulates the dissociation of PsbO from PSII under light conditions known to also release Mn(2+) and Ca(2+) ions from the oxygen-evolving complex and to induce degradation of the PSII reaction centre D1 protein. We propose the occurrence in higher plants of a PsbO-mediated GTPase activity associated with PSII, which has consequences for the function of the oxygen-evolving complex and D1 protein turnover.

(J.A.R.) 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-CO 2 -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 CO 2 . The biggest differences were typically observed 3 h after transfer to low-CO 2 conditions. Therefore, we made a careful metabolomic examination at the 3-h time point, comparing low-CO 2 treatment to high-CO 2 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.

BackgroundCah3 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/ConclusionsIn 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.SignificanceThis is the first report of a CA being post-translationally regulated and describing phosphorylation events in the thylakoid lumen.

BackgroundThe 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 FindingsTransient 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/SignificanceWe 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.