Purification and Characterization of Oxygen-Evolving Photosystem II Core Complexes from the Green Alga Chlamydomonas reinhardtii

Bumann, Dirk; Oesterhelt, Dieter

doi:10.1021/bi00202a009

Cited by 19 publications

(12 citation statements)

References 46 publications

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“…Besides Z, cytochrome b559, car, and Chl are species known to be oxidized by long-living P680+ (46,50). Cytochrome b559 is already oxidized in our intact particles (28) and further oxidation of already oxidized cytochrome b559 has not been observed (50). The results obtained here show that car destruction is not the cause of inhibition (Fig.…”

Section: Discussionsupporting

confidence: 55%

“…Water-splitting PSII core complexes containing about 40 Chl a were isolated from the green alga Chiamydomonas reinhardtii as described (28). These complexes have a somewhat altered QB side.…”

Section: Methodsmentioning

confidence: 99%

“…However, since the electron transfer from Z to P680+ is already strongly impaired in freshly isolated PSII-RC (25)(26)(27), these particles cannot be used for the investigation of the first irreversibly inactivating reaction of photoinhibition. We therefore used PSII core complexes, which have an intermediate pigment content and are capable of water splitting (28). With these particles it is possible to observe the destruction of less than one Chl per PSII and to measure the loss of water-splitting activity (including the electron transfer from Z to P680+) in parallel.…”

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confidence: 99%

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Destruction of a single chlorophyll is correlated with the photoinhibition of photosystem II with a transiently inactive donor side.

Bumann

Oesterhelt

1995

Proc. Natl. Acad. Sci. U.S.A.

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Pigments destroyed during photoinhibition of water-splitting photosystem II core complexes from the green alga Chiamydomonas reinhardtii were studied. Under conditions of a transiently inactivated donor side, illumination leads to an irreversible inhibition of the electron transfer at the donor side that is paralleled by the destruction of chlorophylls a absorbing maximally around 674 and 682 nm. The observed stochiometry of 1 + 0.1 destroyed chlorophyll per inhibited photosystem II suggests that chlorophyll destruction could be the primary photodamage causing the inhibition of photosystem II under these conditions.In photosynthesis light energy is converted to chemical energy. However, side reactions can lead to considerable destruction of the photosynthetic apparatus and a concomitant loss of photosynthetic activity in a process called photoinhibition (1). In oxygenic photosynthesis the main target for photoinhibition is photosystem II (PSII) (2). In intact PSII the excited primary donor P680 reduces plastoquinone. The oxidized primary donor P680+ is then reduced by water via an redoxactive tyrosine "Z" (3). In case of a transient malfunction of the water-splitting reaction, P680+ has an extended lifetime during which it can degrade (4) or damage other PSII components, including carotenoids, chlorophylls (Chl), possibly Z, the manganese binding sites, and other amino acids of the PSII proteins (5-10). One or several of these damages result in an irreversible inhibition of the electron transfer from Z to P680+ and hence in an irreversible loss of the water-splitting activity (6,7,(10)(11)(12)(13)(14). Despite many investigations it is still not clear which of the various damages actually causes this inhibition. We investigated this problem by correlating pigment destruction with the loss of oxygen evolving activity.Quantitative observations of pigment destruction in vivo or in grana membranes are difficult due to the high number of pigments per PSII (200-700) (15, 16). PSII reaction center preparations (PSII-RC) contain only 4-6 Chl, 2 pheophytins (pheo), and 1-2 carotenoids (car) per PSII (17-21). Hence, destruction of less than one pigment per PSII can be easily observed in these particles (4,(22)(23)(24). However, since the electron transfer from Z to P680+ is already strongly impaired in freshly isolated PSII-RC (25-27), these particles cannot be used for the investigation of the first irreversibly inactivating reaction of photoinhibition. We therefore used PSII core complexes, which have an intermediate pigment content and are capable of water splitting (28). With these particles it is possible to observe the destruction of less than one Chl per PSII and to measure the loss of water-splitting activity (including the electron transfer from Z to P680+) in parallel.The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact. Experimental ProceduresWater-s...

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Section: Discussionsupporting

confidence: 55%

Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 99%

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Destruction of a single chlorophyll is correlated with the photoinhibition of photosystem II with a transiently inactive donor side.

Bumann

Oesterhelt

1995

Proc. Natl. Acad. Sci. U.S.A.

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“…The light-saturated rates of oxygen evolution in PSII core complexes were measured in a buffer containing 25 mM MES-KOH (pH 6.5), 300 mM sucrose, 10 mM NaCl, and 50 mM CaCl 2 , in the presence of 1 mM DCBQ and 1 mM K 3 Fe(CN) 6 as described by [55].…”

Section: Methodsmentioning

confidence: 99%

Phosphorylation Controls the Localization and Activation of the Lumenal Carbonic Anhydrase in Chlamydomonas reinhardtii

et al. 2012

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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.

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“…Previously, oxygen-evolving PSII particles have been purified from the green alga Chlamydomonas reinhardtii, but the PSII did not bind the extrinsic 23 kDa and 17 kDa proteins (Diner and Wollman 1980, de Vitry et al 1991, Bumann and Oesterhelt 1994. Sugiura et al (1998) and purified the oxygen-evolving PSII from Chlamydomonas reinhardtii with a His-tag at the C terminus of the D2 protein by a single-step Ni…”

Section: Introductionmentioning

confidence: 99%

Binding and Functional Properties of the Extrinsic Proteins in Oxygen-Evolving Photosystem II Particle from a Green Alga, Chlamydomonas reinhardtii having His-tagged CP47

Suzuki

Minagawa

Tomo

et al. 2003

Plant and Cell Physiology

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;Oxygen-evolving photosystem II (PSII) particles were purified from Chlamydomonas reinhardtii having His-tag extension at the C terminus of the CP47 protein, by a single-step Ni 2+ -affinity column chromatography after solubilization of thylakoid membranes with sucrose monolaurate. The PSII particles consisted of, in addition to intrinsic proteins, three extrinsic proteins of 33, 23 and 17 kDa. The preparation showed a high oxygen-evolving activity of 2,300-2,500 mmol O 2 (mg Chl) and Cl -when 2,6-dichloro-p-benzoquinone was used as the acceptor. These activities were scarcely inhibited by DCMU. The kinetics of flash-induced fluorescence decay revealed that the electron transfer from Q A -to Q B was significantly inhibited, and the electron transfer from Q A -to ferricyanide was largely stimulated in the presence of Ca 2+ . These results indicate that the acceptor side, Q B site, was altered in the PSII particles but its donor side remained intact. Release-reconstitution experiments revealed that the extrinsic 23 and 17 kDa proteins were released only partially by NaCl-wash, while most of the three extrinsic proteins were removed when treated with urea/NaCl, alkaline Tris or CaCl 2 . The 23 and 17 kDa proteins directly bound to PSII independent of the other extrinsic proteins, and the 33 kDa protein functionally re-bound to CaCl 2 -treated PSII which had been reconstituted with the 23 and 17 kDa proteins. These binding properties were largely different from those of the extrinsic proteins in higher plant PSII, and suggest that each of the three extrinsic proteins has their own binding sites independent of the others in the green algal PSII.

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Purification and Characterization of Oxygen-Evolving Photosystem II Core Complexes from the Green Alga Chlamydomonas reinhardtii

Cited by 19 publications

References 46 publications

Destruction of a single chlorophyll is correlated with the photoinhibition of photosystem II with a transiently inactive donor side.

Destruction of a single chlorophyll is correlated with the photoinhibition of photosystem II with a transiently inactive donor side.

Phosphorylation Controls the Localization and Activation of the Lumenal Carbonic Anhydrase in Chlamydomonas reinhardtii

Binding and Functional Properties of the Extrinsic Proteins in Oxygen-Evolving Photosystem II Particle from a Green Alga, Chlamydomonas reinhardtii having His-tagged CP47

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