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Shutova, Tatiana
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Publications (10 of 20) Show all publications
Carius, A. B., Rogne, P., Duchoslav, M., Wolf-Watz, M., Samuelsson, G. & Shutova, T. (2019). Dynamic pH‐induced conformational changes of the PsbO protein in the fluctuating acidity of the thylakoid lumen. Physiologia Plantarum: An International Journal for Plant Biology, 166(1), 288-299
Open this publication in new window or tab >>Dynamic pH‐induced conformational changes of the PsbO protein in the fluctuating acidity of the thylakoid lumen
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2019 (English)In: Physiologia Plantarum: An International Journal for Plant Biology, ISSN 0031-9317, E-ISSN 1399-3054, Vol. 166, no 1, p. 288-299Article in journal (Refereed) Published
Abstract [en]

The PsbO protein is an essential extrinsic subunit of photosystem II, the pigment–protein complex responsible for light‐driven water splitting. Water oxidation in photosystem II supplies electrons to the photosynthetic electron transfer chain and is accompanied by proton release and oxygen evolution. While the electron transfer steps in this process are well defined and characterized, the driving forces acting on the liberated protons, their dynamics and their destiny are all largely unknown. It was suggested that PsbO undergoes proton‐induced conformational changes and forms hydrogen bond networks that ensure prompt proton removal from the catalytic site of water oxidation, i.e. the Mn4CaO5 cluster. This work reports the purification and characterization of heterologously expressed PsbO from green algae Chlamydomonas reinhardtii and two isoforms from the higher plant Solanum tuberosum (PsbO1 and PsbO2). A comparison to the spinach PsbO reveals striking similarities in intrinsic protein fluorescence and CD spectra, reflecting the near‐identical secondary structure of the proteins from algae and higher plants. Titration experiments using the hydrophobic fluorescence probe ANS revealed that eukaryotic PsbO proteins exhibit acid–base hysteresis. This hysteresis is a dynamic effect accompanied by changes in the accessibility of the protein's hydrophobic core and is not due to reversible oligomerization or unfolding of the PsbO protein. These results confirm the hypothesis that pH‐dependent dynamic behavior at physiological pH ranges is a common feature of PsbO proteins and causes reversible opening and closing of their β‐barrel domain in response to the fluctuating acidity of the thylakoid lumen.

Place, publisher, year, edition, pages
John Wiley & Sons, 2019
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:umu:diva-157462 (URN)10.1111/ppl.12948 (DOI)000466108300023 ()30793329 (PubMedID)
Funder
The Kempe FoundationsKnut and Alice Wallenberg Foundation, KAW2011.0055
Note

Special Issue: SI

Available from: 2019-03-21 Created: 2019-03-21 Last updated: 2019-06-10Bibliographically approved
Benlloch, R., Shevela, D., Hainzl, T., Grundström, C., Shutova, T., Messinger, J., . . . Sauer-Eriksson, E. (2015). Crystal structure and functional characterization of Photosystem II-associated carbonic anhydrase CAH3 in Chlamydomonas reinhardtii. Plant Physiology, 167(3), 950-962
Open this publication in new window or tab >>Crystal structure and functional characterization of Photosystem II-associated carbonic anhydrase CAH3 in Chlamydomonas reinhardtii
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2015 (English)In: Plant Physiology, ISSN 0032-0889, E-ISSN 1532-2548, Vol. 167, no 3, p. 950-962Article in journal (Refereed) Published
Abstract [en]

In oxygenic photosynthesis, light energy is stored in the form of chemical energy by converting CO2 and water into carbohydrates.The light-driven oxidation of water that provides the electrons and protons for the subsequent CO2 fixation takes place inphotosystem II (PSII). Recent studies show that in higher plants, HCO3– increases PSII activity by acting as a mobile acceptor ofthe protons produced by PSII. In the green alga Chlamydomonas reinhardtii, a luminal carbonic anhydrase, CrCAH3, was suggested toimprove proton removal from PSII, possibly by rapid reformation of HCO3– from CO2. In this study, we investigated the interplaybetween PSII and CrCAH3 by membrane inlet mass spectrometry and x-ray crystallography. Membrane inlet mass spectrometrymeasurements showed that CrCAH3 was most active at the slightly acidic pH values prevalent in the thylakoid lumen underillumination. Two crystal structures of CrCAH3 in complex with either acetazolamide or phosphate ions were determined at 2.6- and2.7-Å resolution, respectively. CrCAH3 is a dimer at pH 4.1 that is stabilized by swapping of the N-terminal arms, a feature notpreviously observed in a-type carbonic anhydrases. The structure contains a disulfide bond, and redox titration of CrCAH3 functionwith dithiothreitol suggested a possible redox regulation of the enzyme. The stimulating effect of CrCAH3 and CO2/HCO3– on PSIIactivity was demonstrated by comparing the flash-induced oxygen evolution pattern of wild-type and CrCAH3-less PSIIpreparations. We showed that CrCAH3 has unique structural features that allow this enzyme to maximize PSII activity at lowpH and CO2 concentration.

Place, publisher, year, edition, pages
American Society of Plant Biologists, 2015
National Category
Botany Biochemistry and Molecular Biology
Research subject
biological chemistry; Biochemistry
Identifiers
urn:nbn:se:umu:diva-103651 (URN)10.1104/pp.114.253591 (DOI)000354413900027 ()
Funder
Knut and Alice Wallenberg FoundationSwedish Research Council
Available from: 2015-05-26 Created: 2015-05-26 Last updated: 2018-06-07Bibliographically approved
Koroidov, S., Shevela, D., Shutova, T., Samuelsson, G. & Messinger, J. (2014). Mobile hydrogen carbonate acts as proton acceptor in photosynthetic water oxidation. Proceedings of the National Academy of Sciences of the United States of America, 11(17), 6299-6304
Open this publication in new window or tab >>Mobile hydrogen carbonate acts as proton acceptor in photosynthetic water oxidation
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2014 (English)In: Proceedings of the National Academy of Sciences of the United States of America, ISSN 0027-8424, E-ISSN 1091-6490, Vol. 11, no 17, p. 6299-6304Article in journal (Refereed) Published
Abstract [en]

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/HCO3 in water oxidation. Using membrane-inlet mass spectrometry we demonstrate that HCO3 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 HCO3 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/ HCO3 in photosynthetic water oxidation.

Place, publisher, year, edition, pages
National Academy of Sciences, 2014
Keywords
carbon dioxide, bicarbonate, proton release, oxygen evolution, water splitting
National Category
Biophysics Physical Chemistry
Identifiers
urn:nbn:se:umu:diva-86300 (URN)10.1073/pnas.1323277111 (DOI)000335199000053 ()
Note

Included in thesis in manuscript form.

Available from: 2014-02-21 Created: 2014-02-21 Last updated: 2018-06-08Bibliographically approved
Shevela, D., Nöring, B., Koroidov, S., Shutova, T., Samuelsson, G. & Messinger, J. (2013). Efficiency of photosynthetic water oxidation at ambient and depleted levels of inorganic carbon. Photosynthesis Research, 117(1-3), 401-412
Open this publication in new window or tab >>Efficiency of photosynthetic water oxidation at ambient and depleted levels of inorganic carbon
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2013 (English)In: Photosynthesis Research, ISSN 0166-8595, E-ISSN 1573-5079, Vol. 117, no 1-3, p. 401-412Article in journal (Refereed) Published
Abstract [en]

Over 40 years ago, Joliot et al. (Photochem Photobiol 10:309-329, 1969) designed and employed an elegant and highly sensitive electrochemical technique capable of measuring O2 evolved by photosystem II (PSII) in response to trains of single turn-over light flashes. The measurement and analysis of flash-induced oxygen evolution patterns (FIOPs) has since proven to be a powerful method for probing the turnover efficiency of PSII. Stemler et al. (Proc Natl Acad Sci USA 71(12):4679-4683, 1974), in Govindjee's lab, were the first to study the effect of "bicarbonate" on FIOPs by adding the competitive inhibitor acetate. Here, we extend this earlier work by performing FIOPs experiments at various, strictly controlled inorganic carbon (Ci) levels without addition of any inhibitors. For this, we placed a Joliot-type bare platinum electrode inside a N2-filled glove-box (containing 10-20 ppm CO2) and reduced the Ci concentration simply by washing the samples in Ci-depleted media. FIOPs of spinach thylakoids were recorded either at 20-times reduced levels of Ci or at ambient Ci conditions (390 ppm CO2). Numerical analysis of the FIOPs within an extended Kok model reveals that under Ci-depleted conditions the miss probability is discernibly larger (by 2-3 %) than at ambient conditions, and that the addition of 5 mM HCO3 (-) to the Ci-depleted thylakoids largely restores the original miss parameter. Since a "mild" Ci-depletion procedure was employed, we discuss our data with respect to a possible function of free or weakly bound HCO3 (-) at the water-splitting side of PSII.

Place, publisher, year, edition, pages
Springer, 2013
Keywords
Flash-induced oxygen evolution patterns, S states, An extended Kok model, Hydrogen carbonate (bicarbonate), Photosynthetic water oxidation
National Category
Chemical Sciences Biochemistry and Molecular Biology Botany
Identifiers
urn:nbn:se:umu:diva-83168 (URN)10.1007/s11120-013-9875-5 (DOI)000326604900028 ()23828399 (PubMedID)
Funder
Swedish Research CouncilKnut and Alice Wallenberg Foundation
Note

Special Issues on Photosynthesis Education Honoring Govindjee

Available from: 2013-11-20 Created: 2013-11-20 Last updated: 2018-06-08Bibliographically approved
Blanco-Rivero, A., Shutova, T., José Román, M., Villarejo, A. & Martinez, F. (2012). Phosphorylation Controls the Localization and Activation of the Lumenal Carbonic Anhydrase in Chlamydomonas reinhardtii. PLoS ONE, 7(11), Article ID e49063.
Open this publication in new window or tab >>Phosphorylation Controls the Localization and Activation of the Lumenal Carbonic Anhydrase in Chlamydomonas reinhardtii
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2012 (English)In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 7, no 11, article id e49063Article in journal (Refereed) Published
Abstract [en]

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.

Place, publisher, year, edition, pages
Public library science, 2012
National Category
Biophysics Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:umu:diva-104246 (URN)10.1371/journal.pone.0049063 (DOI)000311315300070 ()23139834 (PubMedID)
Available from: 2015-06-11 Created: 2015-06-09 Last updated: 2018-06-07Bibliographically approved
Shitov, A. V., Zharmukhamedov, S. K., Shutova, T., Allakhverdiev, S. I., Samuelsson, G. & Klimov, V. V. (2011). A carbonic anhydrase inhibitor induces bicarbonate-reversible suppression of electron transfer in pea photosystem 2 membrane fragments. Journal of Photochemistry and Photobiology. B: Biology, 104(1-2), 366-371
Open this publication in new window or tab >>A carbonic anhydrase inhibitor induces bicarbonate-reversible suppression of electron transfer in pea photosystem 2 membrane fragments
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2011 (English)In: Journal of Photochemistry and Photobiology. B: Biology, ISSN 1011-1344, E-ISSN 1873-2682, Vol. 104, no 1-2, p. 366-371Article in journal (Refereed) Published
Abstract [en]

The effects of suppression of the carbonic anhydrase (CA) activity by a CA-inhibitor, acetazolamide (AA), on the photosynthetic activities of photosystem II (PS II) particles from higher plants were investigated. AA along with CA-activity inhibits the PS II photosynthetic electron transfer and the AA-induced suppression is totally reversed by the addition of bicarbonate (3-5 mM). Similar effect of recovery in the PS II photosynthetic activity was also revealed upon the addition of known artificial electron donors (potassium ferrocyanide and TMPD). Significance and possible functions of CA for the PS II donor side are discussed.

Place, publisher, year, edition, pages
Elsevier, 2011
Keywords
Carbonic anhydrase, Bicarbonate, Photosystem 2, Inhibitors of carbonic anhydrase, Acetazolamide, Chlorophyll a fluorescence yield
Identifiers
urn:nbn:se:umu:diva-45947 (URN)10.1016/j.jphotobiol.2011.04.001 (DOI)21530302 (PubMedID)
Available from: 2011-08-23 Created: 2011-08-23 Last updated: 2018-06-08Bibliographically approved
Burén, S., Ortega-Villasante, C., Blanco-Rivero, A., Martínez-Bernardini, A., Shutova, T., Shevela, D., . . . Samuelsson, G. (2011). Importance of post-translational modifications for functionality of a chloroplast-localized carbonic anhydrase (CAH1) in Arabidopsis thaliana. PLoS ONE, 6(6), e21021
Open this publication in new window or tab >>Importance of post-translational modifications for functionality of a chloroplast-localized carbonic anhydrase (CAH1) in Arabidopsis thaliana
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2011 (English)In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 6, no 6, p. e21021-Article in journal (Refereed) Published
Abstract [en]

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.

Place, publisher, year, edition, pages
Public Library of Science, 2011
Identifiers
urn:nbn:se:umu:diva-45474 (URN)10.1371/journal.pone.0021021 (DOI)21695217 (PubMedID)
Note

This work was supported by the Swedish Research Council (VR), the Kempe Foundations and Carl Tryggers Foundation to GS, and grant numbers BIO2006-08946 and BIO2009-11340 from the Spanish Ministerio de Ciencia e Innovacio´n (MICINN) to AV. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Available from: 2011-07-05 Created: 2011-07-05 Last updated: 2018-06-08Bibliographically approved
Aksmann, A., Shutova, T., Samuelsson, G. & Tukaj, Z. (2011). The mechanism of anthracene interaction with photosynthetic apparatus: A study using intact cells, thylakoid membranes and PS II complexes isolated from Chlamydomonas reinhardtii. Aquatic Toxicology, 104(3-4), 205-210
Open this publication in new window or tab >>The mechanism of anthracene interaction with photosynthetic apparatus: A study using intact cells, thylakoid membranes and PS II complexes isolated from Chlamydomonas reinhardtii
2011 (English)In: Aquatic Toxicology, ISSN 0166-445X, E-ISSN 1879-1514, Vol. 104, no 3-4, p. 205-210Article in journal (Refereed) Published
Abstract [en]

Intact cells of Chlamydomonas reinhardtii as well as isolated thylakoid membranes and photosystem II complexes were used to examine a possible mechanism of anthracene (ANT) interaction with the photosynthetic apparatus. Since ANT concentrations above 1 mM were required to significantly inhibit the rate of oxygen evolution in PS II membrane fragments it may indicate that the toxicant did not directly interact with this photosystem. On the other hand, stimulation of oxygen uptake by ANT-treated thylakoids suggested that ANT could either act as an artificial electron acceptor in the photosynthetic electron transport chain or function as an uncoupler. Electron transfer from excited chlorophyll to ANT is impossible due to the very low reduction potential of ANT and therefore we propose that toxic concentrations of ANT increase the thylakoid membrane permeability and thereby function as an uncoupler, enhancing electron transport in vitro. Hence, its unspecific interference with photosynthetic membranes in vitro suggests that the inhibitory effect observed on intact cell photosynthesis is caused by uncoupling of phosphorylation. 

Keywords
Chlamydomonas, Anthracene, Toxicity, Photosystems, Phosphorylation
National Category
Botany
Identifiers
urn:nbn:se:umu:diva-104833 (URN)10.1016/j.aquatox.2011.04.017 (DOI)000293042100006 ()21632024 (PubMedID)
Available from: 2015-06-17 Created: 2015-06-15 Last updated: 2018-06-07Bibliographically approved
Renberg, L., Johansson, A. I., Shutova, T., Stenlund, H., Aksmann, A., Raven, J. A., . . . Samuelsson, G. (2010). A metabolomic approach to study major metabolite changes during acclimation to limiting CO2 in chlamydomonas reinhardtii. Plant Physiology, 154(1), 187-196
Open this publication in new window or tab >>A metabolomic approach to study major metabolite changes during acclimation to limiting CO2 in chlamydomonas reinhardtii
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2010 (English)In: Plant Physiology, ISSN 0032-0889, E-ISSN 1532-2548, Vol. 154, no 1, p. 187-196Article in journal (Refereed) Published
Abstract [en]

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.

Identifiers
urn:nbn:se:umu:diva-37355 (URN)10.1104/pp.110.157651 (DOI)000281570000015 ()
Available from: 2010-10-28 Created: 2010-10-28 Last updated: 2018-06-08Bibliographically approved
Nikitina, J., Shutova, T., Melnik, B., Chernyshov, S., Marchenkov, V., Semisotnov, G., . . . Samuelsson, G. (2008). Importance of a single disulfide bond for the PsbO protein of photosystem II: protein structure stability and soluble overexpression in Escherichia coli.. Photosynthesis Research, 98, 391-403
Open this publication in new window or tab >>Importance of a single disulfide bond for the PsbO protein of photosystem II: protein structure stability and soluble overexpression in Escherichia coli.
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2008 (English)In: Photosynthesis Research, ISSN 0166-8595, E-ISSN 1573-5079, Vol. 98, p. 391-403Article in journal (Refereed) Published
Abstract [en]

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.

National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:umu:diva-10932 (URN)10.1007/s11120-008-9327-9 (DOI)000261577900034 ()18709441 (PubMedID)
Available from: 2009-01-13 Created: 2009-01-13 Last updated: 2020-02-05Bibliographically approved
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