Effect of the manganese complex on the binding of the extrinsic proteins (17, 23 and 33 kDa) of Photosystem II

Selective extraction-reconstitution experiments with the extrinsic Photosystem II polypeptides (33 kDa, 23 kDa and 17 kDa) have demonstrated that the manganese complex and the 33 kDa polypeptide are both necessary structural elements for the tight binding of the water soluble 17 and 23 kDa species. When the manganese complex is intact the 33 kDa protein interacts strongly with the rest of the photosynthetic complex. Destruction of the Mn-complex has two dramatic effects: i) The binding of the 33 kDa polypeptide is weaker, since it can be removed by exposure of the PS II system to 2 M NaCl, and ii) the 17 and 23 kDa species do not rebind to Mn-depleted Photosystem II membranes that retain the 33 kDa protein.Abbreviations Chl chlorophyll - HQ hydroquinone - MES 2(N-morpholino)ethanesulfonic acid - PS II Photosystem II - Tris 2-amino-2-hydroxymethylpropane-1,3-diol     

Effect of linolenic acid on release of the 43 kDa polypeptide and manganese from photosystem II membranes depleted of the 33 kDa extrinsic polypeptide

The effect of linolenic acid (18:3) on release of the 43 kDa polypeptide and manganese from photosystem II ( PS II ) membranes depleted of extrinsic polypeptides was studied. In both control and NaCl-washed particles which were depleted of the extrinsic 23 and 16 kDa polypeptides, the 18:3 treatment caused a 20% release of the 33 and 43 kDa polypeptides. In CaCl₂, (or urea + NaCl)-washed particles, which were depleted of the 33 kDa polypeptide in addition to the 23 and 16 kDa polypeptides, the release of the 43 kDa polypeptide increased to 70%, whereas only 25% of the 47 kDa polypeptide was removed. These findings suggest (i) that the 33 and the 43 kDa polypeptides are neighbows in the photosynthetic membrane and (ii) that the 33 kDa polypeptide shields the 43 kDa polypeptide against the action of 18:3. Incubation of CaCl₂, or (urea + NaCI)-treated PSII particles in the presence or absence of 18:3 resulted in the loss of only 2 of the 4 Mn atoms present per reaction center. this indicates that the 2 Mn atoms more firmly associated with PSII are not affected by the removal of the extrinsic 16, 23 and 33 kDa polypeptides, and the intrinsic 43 kDa polypeptide. nor by the treatment with linolenic acid.     

Low pH-Induced Dissociation of Three Extrinsic Proteins from O2-Evolving Photosystem II

Three extrinsic proteins involved in oxygen evolution reversiblydissociated from Photo-system II (PS II) membranes at acidicpHs showing distinctly different pH dependencies. The pHs forhalf dissociation of 17, 23 and 33 kDa extrinsic proteins weredetermined to be 5.0, 4.1 and 3.6, respectively. The half dissociationpHs of 17 and 23 kDa proteins were much lower than their respectiveisoelectric points, while that for 33 kDa protein was closeto its isoelectric point. It was suggested that protonationof the negatively charged binding domain on membrane proteinscauses dissociation of the former two extrinsic proteins, whereasprotonation of the extrinsic protein itself is responsible forthe dissociation of 33 kDa protein. Based on these, featuresof low pH-induced dissociation of extrinsic proteins and Mnfrom PS II were discussed. (Received September 25, 1990; Accepted February 13, 1991)     

Stoichiometric association of extrinsic cytochrome c550 and 12 kDa protein with a highly purified oxygen-evolving photosystem II core complex from Synechococcus vulcanus.

A highly purified, native photosystem II (PS II) core complex was isolated from thylakoids of Synechococcus vulcanus, a thermophilic cyanobacterium by lauryldimethylamine N-oxide (LDAO) and dodecyl beta-D-maltoside solubilization. This native PS II core complex contained, in addition to the proteins that have been well characterized in the core complex previously purified by LDAO and Triton X-100, two more extrinsic proteins with apparent molecular weights of 17 and 12 kDa. These two proteins were associated with the core complex in stoichiometric amounts and could be released by treatment with 1 M CaCl2 or 1 M alkaline Tris but not by 2 M NaCl or low-glycerol treatment, indicating that they are the real components of PS II of this cyanobacterium. N-Terminal sequencing revealed that the 17 and 12 kDa proteins correspond to the apoprotein of cytochrome c550, a low potential c-type cytochrome, and the 9 kDa extrinsic protein previously found in a partially purified PS II preparation from Phormidium laminosum, respectively. In spite of retention of these two extrinsic proteins, no homologues of higher plant 23 and 17 kDa extrinsic proteins could be detected in this cyanobacterial PS II core complex.     

Structural organization of the oxidizing side of photosystem II. Exogenous reductants reduce and destroy the Mn-complex in photosystems II membranes depleted of the 17 and 23 kDa polypeptides

Removal of 23 and 17 kDa water-soluble polypeptides from PS II membranes causes a marked decrease in oxygen-evolution activity, exposes the oxidizing side of PS II to exogenous reductants (Ghanotakis, D.F., Babcock, G.T. and Yocum, C.F. (1984) Biochim. Biophys. Acta 765, 388–398) and alters a high-affinity binding site for Ca²⁺ in the oxygen-evolving complex (Ghanotakis, D.F., Topper, J.N., Babcock, G.T. and Yocum, C.F. (1984) FEBS Lett. 170, 169–173). We have examined further the state of the functional Mn complex in PS II membranes from which the 17 and 23 kDa species have been removed by high-salt treatment. These membranes contain a structurally altered Mn complex which is sensitive to destruction by low concentrations of NH₂OH which cannot, in native PS II membranes, cause extraction of functional Mn. In addition to NH₂OH, a wide range of other small (H₂O₂, NH₂NH₂, Fe²⁺) and bulky (benzidine, hydroquinone) electron donors extract Mn (up to 80%) from the polypeptide-depleted PS II preparations. This extraction is due to reduction of the functional Mn complex since light, which would generate higher oxidation states within the Mn complex, prevents Mn release by reductants. Release of Mn by reductants does not extract the 33 kDa water-soluble protein implicated in Mn binding to the oxidizing side of PS II, although the protein can be partially or totally extracted from Mn-depleted preparations by exposure to high ionic strength or to high (0.8 M) concentrations of Tris. We view our results as evidence for a shield around the Mn complex of the oxygen-evolving complex comprised of the 33 kDa polypeptide along with the 23 and 17 kDa proteins and tightly bound Ca²⁺.     

The status of cysteine residues in the extrinsic 33 kDa protein of spinach photosystem II complexes

Two cysteine residues of the extrinsic 33 kDa protein in the oxygen-evolving photosystemII (PS II) complexes were found to exist as cystine residues in situ. The 33 kDa protein, when reduced by 2-mercaptoethanol in either the presence or the absence of 6 M guanidine-HCl (Gdn-HCl), could not rebind with the CaCl₂-treated PS II complexes, from which the 33 kDa protein was removed, and evolve any oxygen. Two sulfhydryl (SH) groups of the 33 kDa protein were easily reoxidized to a disulfide (S-S) bond by stirring under aerobic conditions with the concomitant regaining of both the binding ability to the CaCl₂-treated PS II complexes and the oxygen-evolving activity.The molecular conformation of the 33 kDa protein was examined by circular dichroic (CD) spectrometry in the UV regions to reveal that the conformation in the reduced state was completely different from those of the untreated and reoxidized states. The disulfide (S-S) bond of the 33 kDa protein is thus essential to maintain the molecular conformation required to function.Abbreviations CD circular dichroism - Chl chlorophyll - DMQ 2,5-dimethyl-p-benzoquinone - DTNB 5,5-dithio-bis (2-nitrobenzoic acid) - EDTA ethylendiamine-tetraacetic acid - Gdn-HCl guanidine-hydrochloric acid - PS II photosystem II - SDS sodium dodecylsulfate This paper was presented at the U.S.-Japan Binational Seminar on Solar Energy Conversion, Okazaki, Japan, March 17–21, 1987     

Photoactivation of Oxygen-Evolving Enzyme in Dark-Grown Pine Cotyledons: Relationship between Assembly of Photosystem II Proteins and Integration of Manganese and Calcium

Dark-grown cotyledons of pine (Pinus thunbergit) did not exhibitO₂ evolution, but this capability was rapidly activated by illuminationfor a short period (photoactivation). To examine the biochemicalchanges which accompany the process of photoactivation in gymnosperms,a method enabling the preparation of highly active O₂-evolvingphotosystem II (PS II) membranes was applied to light-grown,dark-grown, and photoactivated cotyledons. PS II membranes preparedfrom light-grown cotyledons exhibited high O₂-evolving activity,and contained all the intrinsic proteins as well as the threeextrinsic proteins (32, 23 and 17 kDa) associated with PS II.These membranes were also found to contain 4.4 Mn and 0.83 Ca/PSII reaction center. PS II membranes from dark-grown cotyledonscontained all the intrinsic proteins, but preserved only 32kDa extrinsic protein, and zero Mn and 0.85 Ca/PS II reactioncenter. The two extrinsic proteins (23 and 17 kDa) absent inthe PS II membranes from dark-grown cotyledons were, however,present as mature forms in whole thylakoid membranes from thecorresponding sample. The PS II membranes isolated from photoactivatedcotyledons showed a high activity of O₂ evolution and retainedthe three extrinsic proteins, 5.3 Mn and 1.1 Ca/PS II reactioncenter, respectively. The results indicated that Mn and thetwo extrinsic proteins were tightly integrated in the O₂-evolvingapparatusduring the process of photoactivation but integration of Capreceded the integration of Mn by photoactivation. (Received December 9, 1991; Accepted February 1, 1992)     

Identification of polypeptides associated with the 23 and 33 kDa proteins of photosynthetic oxygen evolution

An immunological approach was used for nearest-neighbor analyses for the 23 and 33 kDA proteins of the oxygen-evolving complex. Functional Photosystem II particles with a simple polypeptide composition were partly solubilized with detergent and incubated with monospecific antibodies against either the 23 or the 33 kDa protein. SDS-polyacrylamide gel electrophoresis revealed that the immunoprecipitates, apart from the antigenic proteins, also contained polypeptides at 24, 22 and 10 kDa. In contrast, polypeptides of the light-harvesting and Photosystem II core complexes showed very poor coprecipitation with the 23 and 33 kDa proteins. The 24, 22 and 10 kDa polypeptides were not precipitated by the antibodies if the 23 and 33 kDa proteins had been removed from the particles prior to solubilization. These observations demonstrate a close association between the 24, 22 and 10 kDa polypeptides and the 23 and 33 kDa proteins of the oxygen-evolving complex. None of these precipitated polypeptides contained any manganese. It is suggested that the 24, 22 and 10 kDa polypeptides are subunits of the oxygen-evolving complex and involved in the binding of the extrinsic 23 and 33 kDa proteins to the inner thylakoid surface.     

Evidence for an association between a 33 kDa extrinsic membrane protein,manganese and photosynthetic oxygen evolution. I. Correlation with the S2 multiline EPR signal

The removal of peripheral membrane proteins of a molecular mass of 17 and 23 kDa by washing of spinach Photosystem-II (PS II) membranes in 1 M salt between pH 4.5 and 6.5 produces a minimal loss of the S₁ → S₂ reaction, as seen by the multiline EPR signal for the S₂ state of the water-oxidizing complex, while reversibly inhibiting O₂ evolution. The multiline EPR signal simplifies from a ‘19-line’ spectrum to a ‘16-line’ spectrum, suggestive of partial uncoupling of a cluster of 3 or 4 to yield photo-oxidation of a binuclear Mn site. Alkaline salt washing progressively releases a 33 kDa peripheral protein between pH 6.5 and 9.5, in direct parallel with the loss of O₂ evolution and the S₂ multiline EPR signal. The 33 kDa protein can be partially removed (20%) at pH 8.0 prior to managanese release. Salt treatment releases four Mn ions between pH 8.0 and 9.5 with the first 2 or 3 Mn ions released cooperatively. A common binding site is thus suggested in agreement with earlier EPR spectroscopic data establishing a tetranuclear Mn site. At least two of these Mn ions bind directly at a site in the PS II complex for which photooxidation by the reaction center is controlled by the 33 kDa protein. The washing of PS II membranes with 1 M CaCl₂ to affect the release of the 33 kDa protein, while preserving Mn binding to the membrane (Ono, T.-A. and Inoue, Y. (1983) FEBS Lett. 164, 255–260), is found to leave some 33 kDa protein undissociated in proportion to the extent of O₂ evolution and S₂ multiline yield. These depleted membranes do not oxidize water or produce the normal S₂ state without the binding of the 33 kDa protein. A method for the accurate determination of relative concentrations of the peripheral membrane proteins using gel electrophoresis is presented.     

Selective extraction of CP 26 and CP 29 proteins without affecting the binding of the extrinsic proteins (33, 23 and 17 kDa) and the DCMU sensitivity of a Photosystem II core complex

A highly purified oxygen evolving Photosystem II core complex was isolated from PS II membranes solubilized with the non-ionic detergent n-octyl--D-thioglucoside. The three extrinsic proteins (33, 23 and 17 kDa) were functionally bound to the PS II core complex. Selective extraction of the 22, 10 kDa, CP 26 and CP 29 proteins demonstrated that these species are not involved in the binding of the extrinsic proteins (33, 23 and 17 kDa) or the DCMU sensitivity of the Photosystem II complex.Abbreviations Chl chlorophyll - DCBQ 2,6-dichloro-p-benzoquinone - DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea - LHC light-harvesting complex - MES 2-(N-morpholino)ethanesulfonic acid - OGP n-octyl--d-glucoside - OTG n-octyl--d-thioglucoside - PAGE polyacrylamide gel electrophoresis - PS II Photosystem II - SDS sodium dodecyl sulfate     

Evidence from Crosslinking for Nearest-Neighbor Relationships among the Three Extrinsic Proteins of Spinach Photosystem II Complexes that are Associated with Oxygen Evolution

Treatment of oxygen-evolving Photosystem II complexes, whichlack light-harvesting chlorophyll a/b proteins, with a seriesof disuccinimidyl esters with different chain lengths yieldeda crosslinked product which consisted of one molecule each ofthe extrinsic 33 kDa and 23 kDa proteins. In addition, crosslinkingbetween the 33 kDa protein and the chlorophyll-carrying 47 kDaprotein and between the 23 kDa and 17 kDa proteins was confirmed.Thus, the three extrinsic proteins are closely associated witheach other to form a complex which is attached to the PS IIreaction center complexes. (Received December 1, 1989; Accepted May 2, 1990)     

Degradation and release from the thylakoid membrane of Photosystem II subunits after UV-B irradation of the liverwort Conocephalum conicum

The effect of ultraviolet-B (UV-B) radiation on the amount of various Photosystem (PS) II subunits has been studied in the thalloid liverwort Conocephalum conicum. UV-B irradiation led to a drastic decrease of the reaction center proteins D1 and D2 and the outer light harvesting antenna (LHC II). A minor reduction was found in the levels of the CP 43 polypeptide of the inner antenna and the 33, 23 and 16 kDa extrinsic polypeptides of PS II. During UV-B irradiation, the extrinsic polypeptides accumulated in the soluble protein fraction, but D1 and D2 were not dedectable. Streptomycin, but not cycloheximide inhibited the repair process of PS II, indicating that only protein synthesis in the chloroplast is necessary for recovery. This indicates that the extrinsic proteins of PS II dissociate from the membrane during UV-B treatment and reassociate with PS II in the course of the repair process. We conclude that the reaction center core is a target of UV-B radiation in C. concicum. The extrinsic proteins of PS II are not directly affected by UV-B, but their release is the consequence of UV-B-induced degradation of the D1 and D2 proteins.     

Binding and functional properties of the extrinsic proteins in oxygen-evolving photosystem II particle from a green alga,Chlamydomonas reinhardtii having his-tagged CP47

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 micro mol O(2) (mg Chl)(-1) h(-1) in the presence of Ca(2+) using ferricyanide as the electron acceptor, while its activity was 680-720 micro mol O(2) (mg Chl)(-1) h(-1) in the absence of Ca(2+) and Cl(-) ions. The activity was 710-820 micro mol O(2) (mg Chl)(-1) h(-1) independent of the presence or absence of Ca(2+) 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.     

Properties of a peripheral 34 kDa protein in Synechococcus vulcanus Photosystem II particles. Its exchangeability with spinach 33 kDa protein in reconstitution of O2 evolution

Photosystem II (PS II) particles retaining a high rate of O₂ evolution were prepared from a thermophilic cyanobacterium, Synechococcus vulcanus Copeland, and the composition and properties of their peripheral proteins were investigated. The following results were obtained. (1) The O₂-evolving PS II particles of S. vulcanus contained only one peripheral protein with a molecular mass of 34000 which corresponded to the 33 kDa protein in higher plant PS II particles, but no other peripheral proteins corresponding to the 24 and 16 kDa proteins of higher plant PS II particles. (2) The cyanobacterial peripheral 34 kDa protein was removed from the particles by 1 M CaCl₂-washing concomitant with total inactivation of O₂ evolution, and the inactivated O₂ evolution was reconstituted to 75% of the original activity by rebinding of this protein back to the washed particles. (3) The cyanobacterial peripheral 34 kDa protein rebound to CaCl₂-washed spinach PS II particles and restored O₂ evolution to an appreciable extent (28%). (4) The spinach peripheral 33 kDa protein rebound to CaCl₂-washed PS II particles of S. vulcanus and partially restored O₂ evolution (60%). These results suggested that the peripheral 34 kDa protein of S. vulcanus possesses the determinants for both binding and activity reconstitution identical with those of the peripheral 33 kDa protein of spinach.     

Selective extraction of 22 kDa and 10 kDa polypeptides from Photosystem II without removal of 23 kDa and 17 kDa extrinsic proteins

Selective solubilization of Photosystem II membranes with the non-ionic detergent octyl thioglucopyranoside has allowed the isolation of a PS II system which has been depleted of the 22 and 10 kDa polypeptides but retains all three extrinsic proteins (33, 23 and 17 kDa). The PS II membranes which have been depleted of the 22 and 10 kDa species show high rates of oxygen evolution activity, external calcium is not required for activity and the manganese complex is not destroyed by exogenous reductants. When we compared this system to control PS II membranes, we observed a minor modification of the reducing side, and a conversion of the high-potential to the low-potential form of cytochrome b ₅₅₉.Abbreviations Chl- chlorophyll - DCBQ- 2,5-dichloro-p-benzoquinone - DCMU- 3-(3,4-dichlorophenyl)-1,1-dimethylurea - ESR- electron spin resonance - MES- 2-(N-morpholino)ethanesulfonic acid - OTG- octyl--d-thioglucopyranoside - PS II- Photosystem II - PEG- polyethylene glycol, M_r=6000 - Tris- 2-amino-2-hydroxyethylpropane-1,3-diol     

Extrinsic proteins of photosystem II: an intermediate member of PsbQ protein family in red algal PS II.

The oxygen-evolving photosystem II (PS II) complex of red algae contains four extrinsic proteins of 12 kDa, 20 kDa, 33 kDa and cyt c-550, among which the 20 kDa protein is unique in that it is not found in other organisms. We cloned the gene for the 20-kDa protein from a red alga Cyanidium caldarium. The gene consists of a leader sequence which can be divided into two parts: one for transfer across the plastid envelope and the other for transfer into thylakoid lumen, indicating that the gene is encoded by the nuclear genome. The sequence of the mature 20-kDa protein has low but significant homology with the extrinsic 17-kDa (PsbQ) protein of PS II from green algae Volvox Carteri and Chlamydomonas reinhardtii, as well as the PsbQ protein of higher plants and PsbQ-like protein from cyanobacteria. Cross-reconstitution experiments with combinations of the extrinsic proteins and PS IIs from the red alga Cy. caldarium and green alga Ch. reinhardtii showed that the extrinsic 20-kDa protein was functional in place of the green algal 17-kDa protein on binding to the green algal PS II and restoration of oxygen evolution. From these results, we conclude that the 20-kDa protein is the ancestral form of the extrinsic 17-kDa protein in green algal and higher plant PS IIs. This provides an important clue to the evolution of the oxygen-evolving complex from prokaryotic cyanobacteria to eukaryotic higher plants. The gene coding for the extrinsic 20-kDa protein was named psbQ' (prime).     

Isolation and characterization of oxygen-evolving photosystem II complexes retaining the PsbO, P and Q proteins from Euglena gracilis

Oxygen-evolving photosystem II (PSII) complexes of Euglena gracilis were isolated and characterized. (1) The PSII complexes contained three extrinsic proteins of 33 kDa (PsbO), 23 kDa (PsbP) and 17 kDa (PsbQ), and showed oxygen-evolving activity of around 700 micromol O2 (mg Chl)(-1) h(-1) even in the absence of Cl- and Ca2+ ions. (2) NaCl-treatment removed not only PsbP and PsbQ but also a part of PsbO from Euglena PSII, indicating that PsbO binds to Euglena PSII more loosely than those of other organisms. Treatments by urea/NaCl, alkaline Tris or CaCl2 completely removed the three extrinsic proteins from Euglena PSII. (3) Each of the Euglena extrinsic proteins bound directly to PSII independent of the other extrinsic proteins, which is similar to the binding properties of the extrinsic proteins in a green alga, Chlamydomonas reinhardtii. (4) One of the significant features of Euglena PSII is that the oxygen evolution was not enhanced by Ca2+. When CaCl2-treated Euglena PSII was reconstituted with PsbO, the oxygen-evolving activity was stimulated by the addition of NaCl, but no further stimulation was observed by CaCl2. (5) Oxygen evolution of Euglena PSII reconstituted with PsbO from C. reinhardtii or spinach instead of that from Euglena also showed no enhancement by Ca2+, whereas a significant enhancement of oxygen evolution was observed by Ca2+ when the green algal or higher plant PSII was reconstituted with Euglena PsbO instead of their own PsbO. These results indicate that the PSII intrinsic proteins instead of the extrinsic PsbO protein, are responsible for the stimulation of oxygen evolution by Ca2+. Sequence comparison of major PSII intrinsic proteins revealed that PsbI of Euglena PSII is remarkably different from other organisms in that Euglena PsbI possesses extra 16-17 residues exposed to the luminal side. This may be related to the loss of enhancement of oxygen evolution by Ca2+ ion.     

Nitrogen ligation to the manganese cluster of Photosystem II in the absence of the extrinsic proteins and as a function of pH

Three extrinsic proteins (PsbO, PsbP and PsbQ), with apparent molecular weights of 33, 23 and 17 kDa, bind to the lumenal side of Photosystem II (PS II) and stabilize the manganese, calcium and chloride cofactors of the oxygen evolving complex (OEC). The effect of these proteins on the structure of the tetramanganese cluster, especially their possible involvement in manganese ligation, is investigated in this study by measuring the reported histidine-manganese coupling [Tang et al. (1994) Proc Natl Acad Sci USA 91: 704–708] of PS II membranes depleted of none, two or three of these proteins using ESEEM (electron spin echo envelope modulation) spectroscopy. The results show that neither of the three proteins influence the histidine ligation of manganese. From this, the conserved histidine of the 23 kDa protein can be ruled out as a manganese ligand. Whereas the 33 and 17 kDa proteins lack conserved histidines, the existence of a 33 kDa protein-derived carboxylate ligand has been posited; our results show no evidence for a change of the manganese co-ordination upon removal of this protein. Studies of the pH-dependence of the histidine–manganese coupling show that the histidine ligation is present in PS II centers showing the S₂ multiline EPR signal in the pH-range 4.2–9.5. This revised version was published online in June 2006 with corrections to the Cover Date.     

Cross-reconstitution of various extrinsic proteins and photosystem II complexes from cyanobacteria, red algae and higher plants

Photosystem II (PSII) contains different extrinsic proteins required for oxygen evolution among different organisms. Cyanobacterial PSII contains the 33 kDa, 12 kDa proteins and cytochrome (cyt) c-550; red algal PSII contains a 20 kDa protein in addition to the three homologous cyanobacterial proteins; whereas higher plant PSII contains the 33 kDa, 23 kDa and 17 kDa proteins. In order to understand the binding and functional properties of these proteins, we performed cross-reconstitution experiments with combinations of PSII and extrinsic proteins from three different sources: higher plant (spinach), red alga (Cyanidium caldarium) and cyanobacterium (Synechococcus vulcanus). Among all of the extrinsic proteins, the 33 kDa protein is common to all of the organisms and is totally exchangeable in binding to PSII from any of the three organisms. Oxygen evolution of higher plant and red algal PSII was restored to a more or less similar level by binding of any one of the three 33 kDa proteins, whereas oxygen evolution of cyanobacterial PSII was restored to a larger extent with its own 33 kDa protein than with the 33 kDa protein from other sources. In addition to the 33 kDa protein, the red algal 20 kDa, 12 kDa proteins and cyt c-550 were able to bind to cyanobacterial and higher plant PSII, leading to a partial restoration of oxygen evolution in both organisms. The cyanobacterial 12 kDa protein and cyt c-550 partially bound to the red algal PSII, but this binding did not restore oxygen evolution. The higher plant 23 kDa and 17 kDa proteins bound to the cyanobacterial and red algal PSII only through non-specific interactions. Thus, only the red algal extrinsic proteins are partially functional in both the cyanobacterial and higher plant PSII, which implies a possible intermediate position of the red algal PSII during its evolution from cyanobacteria to higher plants.     

The action of mercury on the binding of the extrinsic polypeptides associated with the water oxidizing complex of photosystem II

Mercury (Hg²⁺), a sulfhydryl group reactant, wasused to probe structure-function relationships in photosystem II (PSII). In the present work, we investigated the impact of mercury on the polypeptide composition of PSII submembrane preparations. Electrophoretic analysis revealed that the incubation of the membranes in the presence of mercury produces the depletion of a polypeptide of molecular weight of 33 kDa. This polypeptide corresponds to the extrinsic protein EP33 of the oxygen evolving complex removed following urea treatment. However, the two closely related extrinsic polypeptides of 16 and 23 kDa, usually removed concomitantly after urea treatment, remained unaffected after the mercury treatment. These data demonstrated the existence of an intrinsic binding site for EP23. The molecular mode of action of mercury in the oxygen evolving complex of PSII is discussed.