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A photosystem II preparation from the thermophilic cyanobacterium Synechococcus elongatus, which is especially suitable for three-dimensional crystallization in a fully active form was developed. The efficient purification method applied here yielded 10 mg of protein of a homogenous dimeric complex of about 500 kDa within 2 days. Detailed characterization of the preparation demonstrated a fully active electron transport chain from the manganese cluster to plastoquinone in the Q B binding site. The oxygen-evolving activity, 5000 -6000 mol of O 2 /(h⅐mg of chlorophyll), was the highest so far reported and is maintained even at temperatures as high as 50°C. The crystals obtained by the vapor diffusion method diffracted to a resolution of 4.3 Å. The space group was determined to be P2 1 2 1 2 1 with four photosystem II dimers per unit cell. Analysis of the redissolved crystals revealed that activity, supramolecular organization, and subunit composition were maintained during crystallization. Photosystem II (PS II)1 functions as a water/plastoquinone oxidoreductase in the thylakoid membrane of chloroplasts and cyanobacteria yielding oxygen, protons, and reduced plastoquinone by the splitting of water. Powered by sunlight, this is the key reaction that created and still sustains an oxygenic atmosphere on our planet. As a pigment-protein complex, PS II consists of more than 20 different subunits (1), most of them being integral membrane proteins. The PS II reaction center (RC) itself is composed of the D1 and D2 proteins, which bind the cofactors for the light-driven electron transfer processes (2). Upon light absorption, an electron is transferred from the primary electron donor P680 to a nearby pheophytin molecule. This charge separation is stabilized by rapid electron transfer to a tightly bound plastoquinone molecule, Q A , and finally a mobile plastoquinone, Q B , on the stromal/cytoplasmic side of the membrane. After double reduction and protonation, the reduced Q B is released from its binding site on the D1 protein and replaced by an oxidized plastoquinone molecule. On the donor side, P680ϩ is reduced by a tyrosine residue of the D1 protein, tyrZ (3-5), which in turn is reduced by a cluster of four manganese atoms located on the luminal side of PS II. This manganese cluster is the place where water splitting occurs. In four successive charge separation steps, four positive charge equivalents are accumulated that yield, finally, one molecule of oxygen out of two molecules of water. Accordingly, the manganese cluster can assume five different oxidation states, S 0 -S 4 , with S 4 being the state in which oxygen is released. Because water is a very stable molecule, its oxidation requires a potential of ϩ1 V. Therefore, PS II is the most oxidizing enzyme in nature.Closely associated with the reaction center is cytochrome b 559 consisting of two subunits, ␣ and ␤. Each subunit of the cytochrome provides one histidine side chain for heme binding. The function of cytochrome b 559 is still not completely understood (fo...

Section: Isolation and Biochemical Characterization Of A Dimeric Pssupporting

confidence: 89%

Towards Structural Determination of the Water-splitting Enzyme

Kuhl¹,

Kruip²,

Seidler³

et al. 2000

120

“…The recent resolution of the threedimensional structure of the soluble form of cyt c 550 from three cyanobacteria, Synechocystis sp. PCC 6803 (9), Arthrospira maxima (10), and Thermosynechococcus elongatus (11) has confirmed a previously proposed bis-histidine coordinated heme that is very unusual for monoheme c-type cytochromes (8,11,12). Crystal structures of both isolated and PSII-bound forms of cyt c 550 show that the protein presents a hydrophobic inner core typical of monoheme cytochromes c, with three helices forming a nest for the prosthetic group and a fourth helical segment in the N-terminal domain protecting the heme from solvent, indicating that the heme structure is not very different from most c-type cytochromes (13).…”

supporting

confidence: 82%

“…Cyt c 550 , encoded by the psbV gene, is a monoheme protein with a molecular mass of Ϸ 15 kDa and an isoelectric point between 3.8 and 5.0 (7,8). The recent resolution of the threedimensional structure of the soluble form of cyt c 550 from three cyanobacteria, Synechocystis sp.…”

mentioning

confidence: 99%

See 1 more Smart Citation

A High Redox Potential Form of Cytochrome c550 in Photosystem II from Thermosynechococcus elongatus

Guerrero

Sedoud

Kirilovsky

et al. 2011

Cytochrome c 550 (cyt c 550 ) is a component of photosystem II (PSII) from cyanobacteria, red algae, and some other eukaryotic algae. Its physiological role remains unclear. In the present work, measurements of the midpoint redox potential (E m ) were performed using intact PSII core complexes preparations from a histidine-tagged PSII mutant strain of the thermophilic cyanobacterium Thermosynechococcus (T.) elongatus. When redox titrations were done in the absence of redox mediators, an E m value of ؉200 mV was obtained for cyt c 550 . This value is ϳ300 mV more positive than that previously measured in the presence of mediators (E m ‫؍‬ ؊80 mV). The shift from the high potential form (E m ‫؍‬ ؉200 mV) to the low potential form (E m ‫؍‬ ؊80 mV) of cyt c 550 is attributed to conformational changes, triggered by the reduction of a component of PSII that is sequestered and out of equilibrium with the medium, most likely the Mn 4 Ca cluster. This reduction can occur when reduced low potential redox mediators are present or under highly reducing conditions even in the absence of mediators. Based on these observations, it is suggested that the E m of ؉200 mV obtained without mediators could be the physiological redox potential of the cyt c 550 in PSII. This value opens the possibility of a redox function for cyt c 550 in PSII.In all photosynthetic oxygen-evolving organisms, the primary steps of light conversion take place in a large pigmentprotein complex named PSII, 2 which drives light-induced electron transfer from water to plastoquinone with the concomitant production of molecular oxygen (for review, see Ref.1). The reaction center of PSII is made up of two membranespanning polypeptides, D1 and D2, which bind four chlorophylls, two pheophytins, two quinones, Q A and Q B (the primary and secondary quinone acceptors of the reaction centre of PSII), a non-heme iron atom, and a cluster made up of four manganese ions and one calcium ion. In green algae and higher plants, three extrinsic proteins are associated to reaction center in water-splitting active PSII complexes: 23-24, 16 -18, and 33 kDa proteins, whereas in cyanobacteria, red algae and some other eukaryotic algae, cyt c 550 , 12 kDa and 33 kDa proteins are found. The three-dimensional structure of PSII confirmed that cyt c 550 binds on the lumenal membrane surface in the vicinity of the D1 and CP43 (2-6).Cyt c 550 , encoded by the psbV gene, is a monoheme protein with a molecular mass of Ϸ 15 kDa and an isoelectric point between 3.8 and 5.0 (7, 8). The recent resolution of the threedimensional structure of the soluble form of cyt c 550 from three cyanobacteria, Synechocystis sp. PCC 6803 (9), Arthrospira maxima (10), and Thermosynechococcus elongatus (11) has confirmed a previously proposed bis-histidine coordinated heme that is very unusual for monoheme c-type cytochromes (8,11,12). Crystal structures of both isolated and PSII-bound forms of cyt c 550 show that the protein presents a hydrophobic inner core typical of monoheme cytochromes c, with three helices...

“…The low midpoint redox potential (EЈ m ) values of the purified cyt c 550 (from Ϫ250 to Ϫ314 mV (1,12,13)) seems incompatible with a redox function in PS II electron transfer. However, we have recently demonstrated that the cyt c 550 from the thermophilic cyanobacteria Thermosynechococcus elongatus has a significantly higher EЈ m value when it is bound to the PS II (Ϫ80/Ϫ100 mV) compared with its soluble form after its extraction from PS II (Ϫ240 mV at pH 6) (14).…”

mentioning

confidence: 99%

Cytochrome c550 in the Cyanobacterium Thermosynechococcus elongatus

Kirilovsky

Roncel

Boussac

et al. 2004

Cytochrome c 550 is one of the extrinsic Photosystem II subunits in cyanobacteria and red algae. To study the possible role of the heme of the cytochrome c 550 we constructed two mutants of Thermosynechococcus elongatus in which the residue His-92, the sixth ligand of the heme, was replaced by a Met or a Cys in order to modify the redox properties of the heme. The H92M and H92C mutations changed the midpoint redox potential of the heme in the isolated cytochrome by ؉125 mV and ؊30 mV, respectively, compared with the wild type. The binding-induced increase of the redox potential observed in the wild type and the H92C mutant was absent in the H92M mutant. Both modified cytochromes were more easily detachable from the Photosystem II compared with the wild type. The Photosystem II activity in cells was not modified by the mutations suggesting that the redox potential of the cytochrome c 550 is not important for Photosystem II activity under normal growth conditions. A mutant lacking the cytochrome c 550 was also constructed. It showed a lowered affinity for Cl ؊ and Ca 2؉ as reported earlier for the cytochrome c 550 -less Synechocystis 6803 mutant, but it showed a shorter lived S 2 Q B Ϫ state, rather than a stabilized S 2 state and rapid deactivation of the enzyme in the dark, which were characteristic of the Synechocystis mutant. It is suggested that the latter effects may be caused by loss (or weaker binding) of the other extrinsic proteins rather than a direct effect of the absence of the cytochrome c 550 .Cytochrome c 550 (cyt c 550 ), 1 present in cyanobacteria and red algae, was first discovered by Holton and Myers (1) as a soluble monoheme c-type cytochrome. The cyt c 550 has a molecular mass of about 15 kDa, His/His coordination and a very low redox potential around Ϫ260 mV (for review see Ref.2). showed that cyt c 550 is stoichiometrically bound to the Photosystem II (PS II), activates oxygen evolving activity and allows the binding of the 12 kDa protein, another extrinsic component of the cyanobacterial PS II involved in oxygen evolution. The three-dimensional structure of the PS II from two thermophilic cyanobacteria strains confirmed that cyt c 550 binds to the luminal PS II surface in the vicinity of the D1 and CP43 proteins (6 -8). The phenotype of the ⌬psbV (cyt c 550 -less) mutant of Synechocystis PCC 6803 was already characterized with respect to PS II activity (9 -11). The ⌬psbV and the double ⌬psbV/⌬psbU (encoding the 12 kDa protein) mutants were unable to grow in the absence of Ca 2ϩ and Cl Ϫ ions, their PS II activity decreased to 40% of the wild type, and they showed a very rapid inactivation of the enzyme in the dark (after 2 h, the activity decreased about 90% whereas in the wild type only 25% of the activity was lost). A slight retardation in O 2 release from the S 3 state was also observed in these mutants. Another effect observed in these Synechocystis PCC 6803 mutants was a large intensity decrease of the B band of thermoluminescence (TL) and an upshift in the temperature maxima of the ...