Structural organization of proteins on the oxidizing side of photosystem II. Two molecules of the 33-kDa manganese-stabilizing proteins per reaction center.

The 33-kDa manganese-stabilizing protein stabilizes the manganese cluster in the oxygen-evolving complex. There has been, however, a considerable amount of controversy concerning the stoichiometry of this photosystem II (PS II) component. In this paper, we have verified the extinction coefficient of the manganese-stabilizing protein by amino acid analysis, determined the manganese content of oxygen-evolving photosystem II membranes and reaction center complex using inductively coupled plasma spectrometry, and determined immunologically the amount of the manganese-stabilizing protein associated with photosystem II. Oxygen-evolving photosystem II membranes and reaction center complex preparations contained 258 +/- 11 and 67 +/- 3 chlorophyll, respectively, per tetranuclear manganese cluster. Immunoquantification of the manganese-stabilizing protein using mouse polyclonal antibodies on "Western blots" demonstrated the presence of 2.1 +/- 0.2 and 2.0 +/- 0.3 molecules of the manganese-stabilizing protein/tetranuclear manganese cluster in oxygen-evolving PS II membranes and highly purified PS II reaction center complex, respectively. Since the manganese-stabilizing protein co-migrated with the D2 protein in our electrophoretic system, accurate immunoquantification required the inclusion of CaCl2-washed PS II membrane proteins or reaction center complex proteins in the manganese-stabilizing protein standards to compensate for the possible masking effect of the D2 protein on the binding of the manganese-stabilizing protein to Immobilon-P membranes. Failure to include these additional protein components in the manganese-stabilizing protein standards leads to a marked underestimation of the amount of the manganese-stabilizing protein associated with these photosystem II preparations.     

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.     

Interaction of CPa-1 with the manganese-stabilizing protein of photosystem II: identification of domains cross-linked by 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide.

The structural organization of photosystem II proteins has been investigated by use of the zero-length protein cross-linking reagent 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide and monoclonal and polyclonal antibody reagents. Photosystem II membranes were treated with 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide which cross-links amino groups to carboxyl groups which are in van der Waals contact. This treatment did not affect the oxygen evolution rates of these membranes and increased the retention of oxygen evolution after CaCl2 washing. Analysis of the proteins cross-linked by this treatment indicated that two cross-linked species with apparent molecular masses of 95 and 110 kDa were formed which cross-reacted with antibodies against both the 33-kDa manganese-stabilizing protein and the chlorophyll protein CPa-1. Cleavage of the 110-kDa cross-linked species with cyanogen bromide followed by N-terminal sequence analysis was used to identify the peptide fragments of CPa-1 and the manganese-stabilizing protein which were cross-linked. Two cyanogen bromide fragments were identified with apparent molecular masses of 50 and 25 kDa. N-Terminal sequence analysis of the 50-kDa cyanogen bromide fragment indicates that this consists of the C-terminal 16.7-kDa fragment of CPa-1 and the intact manganese-stabilizing protein. This strongly suggests that the manganese-stabilizing protein is cross-linked to the large extrinsic loop domain of CPa-1. N-Terminal analysis of the 25-kDa cyanogen bromide fragment indicates that this consists of the C-terminal 16.7-kDa peptide of CPa-1 and the N-terminal 8-kDa peptide of the manganese-stabilizing protein.(ABSTRACT TRUNCATED AT 250 WORDS)     

Carboxylate groups on the manganese-stabilizing protein are required for its efficient binding to photosystem II

The effects of the modification of carboxylate groups on the manganese-stabilizing protein of photosystem II were investigated. Carboxylate groups (including possibly the C-terminus) on the manganese-stabilizing protein were modified with glycine methyl ester in a reaction facilitated by 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide. The manganese-stabilizing protein that was modified while associated with NaCl-washed photosystem II membranes contained 1-2 modified carboxylates, whereas the protein that was modified while free in solution contained 4 modified carboxylates. Both types of modified protein could reconstitute oxygen evolution at high manganese-stabilizing protein to photosystem II reaction center ratios. However, the protein that had been modified in solution exhibited a dramatically altered binding affinity for photosystem II. No such alteration in binding affinity was observed for the protein that had been modified while associated with the photosystem. Mapping of the sites of modification was carried out by trypsin and Staphylococcus V8 protease digestion of the modified proteins and analysis by matrix-assisted laser desorption/ionization mass spectrometry. These studies indicated that the domains (157)D-(168)D and (212)E-(247)Q (C-terminus) are labeled only when the manganese-stabilizing protein is modified in solution. Modified carboxylates in these domains are responsible for the altered binding affinity of this protein for the photosystem.     

Isolation and characterization of oxygen-evolving thylakoid membranes and photosystem II particles from a marine diatom Chaetoceros gracilis

Thylakoid membranes retaining high oxygen-evolving activity (about 250 micromol O(2)/mg Chl/h) were prepared from a marine centric diatom, Chaetoceros gracilis, after disruption of the cells by freeze-thawing. We also succeeded in purification of Photosystem II (PSII) particles by differential centrifugation of the thylakoid membranes after treatment with 1% Triton X-100. The diatom PSII particles showed an oxygen-evolving activity of 850 and 1045 micromol O(2)/mg Chl/h in the absence and presence of CaCl(2), respectively. The PSII particles contained fucoxanthin chlorophyll a/c-binding proteins in addition to main intrinsic proteins of CP47, CP43, D2, D1, cytochrome b559, and the antenna size was estimated to be 229 Chl a per 2 molecules of pheophytin. Five extrinsic proteins were stoichiometrically released from the diatom PSII particles by alkaline Tris-treatment. Among these five extrinsic proteins, four proteins were red algal-type extrinsic proteins, namely, PsbO, PsbQ', PsbV and PsbU, whereas the other one was a novel, hypothetical protein. This is the first report on isolation and characterization of diatom PSII particles that are highly active in oxygen evolution and retain the full set of extrinsic proteins including an unknown protein.     

Construction and characterization of cyanobacterial mutants lacking the manganese-stabilizing polypeptide of photosystem II.

Mutants of the cyanobacterium Synechocystis sp. Pasteur Culture Collection (PCC) 6803 that specifically lack the extrinsic 33-kDa manganese-stabilizing polypeptide of the photosystem II oxygen-evolving complex have been constructed by two independent methods. Cartridge mutagenesis was used to insertionally inactivate the psbO gene of one mutant and completely delete the psbO gene of the other mutant. These mutants have no detectable manganese-stabilizing polypeptide, but they do accumulate steady-state levels of the intrinsic photosystem II polypeptides D1, D2, and CP-43 that are comparable to wild-type, as determined by immunoblot analysis. Measurement of the evolution of the relative quantum yields of chlorophyll fluorescence following actinic flash excitation indicates that though the concentration of reaction centers in mutant cells is comparable to that of wild-type cells, approximately 40% of these centers harbor a fluorescence-quenching species other than P680+. The mutants are capable of photoautotrophic growth at a slower rate than that of wild-type. Under conditions of Ca2+ depletion where wild-type growth is unaffected, the mutants are unable to grow at all. The manganese-stabilizing protein, therefore, enhances the binding of Ca2+ or protects the reaction center at low Ca2+ concentrations. The mutant evolve oxygen at approximately 70% of the wild-type rate, but are completely photoinactivated by high light intensities. Our results indicate that the manganese-stabilizing polypeptide is not absolutely required for photosystem II assembly or function in cyanobacteria, but its absence does lead to an enhanced sensitivity to photoinhibition.     

Interaction of CPa-1 with the manganese-stabilizing protein of photosystem II: identification of domains on CPa-1 which are shielded from N-hydroxysuccinimide biotinylation by the manganese-stabilizing protein.

The structural organization of photosystem II proteins has been investigated by use of the amino group-labeling reagent N-hydroxysuccinimidobiotin (NHS-biotin) and calcium chloride-washed photosystem II membranes. We have previously shown that the presence of the extrinsic, manganese-stabilizing protein on photosystem II membranes prevents the modification of lysyl residues located on the chlorophyll protein CPa-1 (CP-47) by NHS-biotin [Bricker, T. M., Odom, W. R., & Queirolo, C. B. (1988) FEBS Lett. 231, 111-117]. Upon removal of the manganese-stabilizing protein by calcium chloride-washing, CPa-1 can be specifically modified by treatment with NHS-biotin. Preparative quantities of biotinylated CPa-1 were subjected to chemical cleavage with cyanogen bromide. Two major biotinylated peptides were identified with apparent molecular masses of 11.8 and 15.7 kDa. N-terminal sequence analysis of these peptides indicated that the 11.8-kDa peptide was 232G-330M and that the 15.7-kDa peptide was 360P-508V. The 15.7-kDa CNBr peptide was subjected to limited tryptic digestion. The two smallest tryptic fragments identified migrated at apparent molecular masses of 9.1 (nonbiotinylated) and 7.5 kDa (biotinylated). N-terminal sequence analysis and examination of the predicted amino acid sequences of these peptides suggest that the 9.1-kDa fragment was 422R-508V and that the 7.5-kDa fragment was 360P-421A. These results strongly suggest that two NHS-biotinylated domains, 304K-321K and 389K-419K, become exposed on CPa-1 when the manganese-stabilizing protein is removed by CaCl2 treatment. Both of these domains lie in the large extrinsic loop E of CPa-1.     

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.     

The manganese-stabilizing protein is required for photosystem II assembly/stability and photoautotrophy in higher plants

Interfering RNA was used to suppress the expression of two genes that encode the manganese-stabilizing protein of photosystem II in Arabidopsis thaliana, MSP-1 (encoded by psbO-1, At5g66570), and MSP-2 (encoded by psbO-2, At3g50820). A phenotypic series of transgenic plants was recovered that expressed high, intermediate, and low amounts of these two manganese-stabilizing proteins. Chlorophyll fluorescence induction and decay analyses were performed. Decreasing amounts of expressed protein led to the progressive loss of variable fluorescence and a marked decrease in the fluorescence quantum yield (F(v)/F(m)) in both the absence and the presence of dichloromethylurea. This result indicated that the amount of functional photosystem II reaction centers was compromised in the plants that exhibited intermediate and low amounts of the manganese-stabilizing proteins. An analysis of the decay of the variable fluorescence in the presence of dichlorophenyldimethylurea indicated that charge recombination between Q ((A-)) and the S(2) state of the oxygen-evolving complex was seriously retarded in the plants that expressed low amounts of the manganese stabilizing proteins. This may have indicated a stabilization of the S(2) state in the absence of the extrinsic component. Immunological analysis of the photosystem II protein complement indicated that significant losses of the CP47, CP43, and D1 proteins occurred upon the loss of the manganese-stabilizing proteins. This indicated that these extrinsic proteins were required for photosystem II core assembly/stability. Additionally, although the quantity of the 24-kDa extrinsic protein was only modestly affected by the loss of the manganese-stabilizing proteins, the 17-kDa extrinsic protein dramatically decreased. The control proteins ribulose bisphosphate carboxylase and cytochrome f were not affected by the loss of the manganese-stabilizing proteins; the photosystem I PsaB protein, however, was significantly reduced in the low expressing transgenic plants. Finally, it was determined that the transgenic plants that expressed low amounts of the manganese-stabilizing proteins could not grow photoautotrophically.     

Copper effect on the protein composition of photosystem II

We provide data from in vitro experiments on the polypeptide composition, photosynthetic electron transport and oxygen evolution activity of intact photosystem II (PSII) preparations under Cu(II) toxicity conditions. Low Cu(II) concentrations (Cu(II) per PSII reaction centre unit≤230) that caused around 50% inhibition of variable chlorophyll a fluorescence and oxygen evolution activity did not affect the polypeptide composition of PSII. However, the extrinsic proteins of 33, 24 and 17 kDa of the oxygen-evolving complex of PSII were removed when samples were treated with 300 μ M CuCl₂ (Cu(II) per PSII reaction centre unit=1 400). The LHCII antenna complex and D1 protein of the reaction centre of PSII were not affected even at these Cu(II) concentrations. The results indicated that the initial inhibition of the PSII electron transport and oxygen-evolving activity induced by the presence of toxic Cu(II) concentrations occurred before the damage of the oxygen-evolving complex. Indeed, more than 50% inhibition could be achieved in conditions where its protein composition and integrity was apparently preserved.     

Light-dependent degradation of the D1 protein in photosystem II is accelerated after inhibition of the water splitting reaction

Strong illumination of oxygen-evolving organisms inhibits the electron transport through photosystem II (photoinhibition). In addition the illumination leads to a rapid turnover of the D1 protein in the reaction center of photosystem II. In this study the light-dependent degradation of the D1 reaction center protein and the light-dependent inhibition of electron-transport reactions have been studied in thylakoid membranes in which the oxygen evolution has been reversibly inhibited by Cl- depletion. The results show that Cl(-)-depleted thylakoid membranes are very vulnerable to damage induced by illumination. Both the D1 protein and the inhibition of the oxygen evolution are 15-20 times more sensitive to illumination than in control thylakoid membranes. The presence, during the illumination, of the herbicide 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) prevented both the light-dependent degradation of the D1 protein and the inhibition of the electron transport. The protection exerted by DCMU is seen only in Cl(-)-depleted thylakoid membranes. These observations lead to the proposal that continuous illumination of Cl(-)-depleted thylakoid membranes generates anomalously long-lived, highly oxidizing radicals on the oxidizing side of photosystem II, which are responsible for the light-induced protein damage and inhibition. The presence of DCMU during the illumination prevents the formation of these radicals, which explains the protective effects of the herbicide. It is also observed that in Cl(-)-depleted thylakoid membranes, oxygen evolution (measured after the readdition of Cl-) is inhibited before electron transfer from diphenylcarbazide to dichlorophenolindophenol.(ABSTRACT TRUNCATED AT 250 WORDS)     

Preparation and Characterization of Heat-Stable and Very Active Oxygen-Evolving Photosystem II Particles from the Thermophilic Cyanobacterium, Synechococcus elongatu

Very active and heat-stable oxygen-evolving photosystem II particleswere isolated from the thermophilic cyanobacterium Synechococcuselongatus by treatment of thylakoid membranes with a non-ionicdetergent, sucrose monolaurate (SML). The particles were analyzedin a comparison with photosystem II particles prepared withß-octylglucoside (OG). The two preparations had similarpolypeptide compositions, which were caracterized by high levelsof polypeptides from phycobilisomes. The ratio of chlorophylla to QA was 45 and there were four Mn atoms and one tightlybound Ca²⁺ ion per QA in the particles prepared with SML. Thepreparations were thermophilic, showing substantial rates ofoxygen evolution at temperatures up to 60°C. The maximumrates attained at 45°C were as high as 6.0 mmoles O₂ mg^–1Chl h^–1. PS II particles prepared with OG were similarlythermostable but were less active in oxygen evolution at alltemperatures examined. Kinetic analysis of flash-induced absorptiontransients revealed that about 22% and 28% of photosystem IIreaction centers were not associated with the functional QBsite in the SML- and OG-particles, respectively. When correctedfor the inactive reaction centers, the maximum rates of oxygenevolution by SML- and OG-particles were 7.7 and 7.0 mmoles O₂mg^–1 Chl h^–1, which correspond to half times of1.9 and 2.1 ms for the first-order electron transfer, respectively.Comparison of these half times with those of the S-state transitionand the release of oxygen indicates that the overall photosystemII electron transport is limited by the reduction of added electronacceptors and not by release of oxygen. ³On leave from National Chemical Laboratory for Industry, Higashi1-1, Tsukuba, Ibaraki 305     

Site-directed mutagenesis of glutamate residues in the large extrinsic loop of the photosystem II protein CP 43 affects oxygen-evolving activity and PS II assembly

The psbC gene encodes the intrinsic chlorophyll protein CP 43, a component of photosystem II in higher plants, green algae, and cyanobacteria. Oligonucleotide-directed mutagenesis was used to introduce mutations into the portion of psbC that encodes the large extrinsic loop E of CP 43 in the cyanobacterium Synechocystis 6803. Three mutations, E293Q, E339Q, and E352Q, each produced a strain with impaired photosystem II activity. The E293Q mutant strain grew photoautotrophically at rates comparable to the control strain. Immunological analyses of several PS II components indicated that this mutant accumulated normal quantities of PS II proteins. However, this mutant evolved oxygen to only 56% of control rates at saturating light intensities. Measurements of total variable fluorescence yield indicated that this mutant assembled approximately 60% of the fully functional PS II centers found in the control strain. The E339Q mutant grew photoautotrophically at a severely reduced rate. Both immunological analysis and variable fluorescence yield experiments indicated that E339Q assembled a normal complement of PS II centers. However, this mutant was capable of evolving oxygen to only 20% of control rates. Variable fluorescence yield experiments demonstrated that this mutant was inefficient at using water as an electron donor. Both E293Q and E339Q strains exhibited an increased (approximately 2-fold) sensitivity to photoinactivation. The E352Q mutant was the most severely affected. This mutant failed to grow photoautotrophically and exhibited essentially no capacity for oxygen evolution. Measurements of total variable fluorescence yield indicated that this mutant assembled no functional PS II centers. Immunological analysis of isolated thylakoid membranes from E352Q revealed a complete absence of CP 43 and reduced levels of both the D1 and manganese-stabilizing proteins. These results suggest that the mutations E293Q and E339Q each produce a defect associated with the oxygen-evolving complex of photosystem II. The E352Q mutation appears to affect the stability of the PS II complex. This is the first report showing that alteration of negatively charged residues in the CP 43 large extrinsic loop results in mutations affecting PS II assembly/function.     

Gluconeogenesis predominates in periportal regions of the liver lobule

Rates of gluconeogenesis from lactate were calculated in periportal and pericentral regions of the liver lobule in perfused rat livers from increases in O2 uptake due to lactate. When lactate (0.1-2.0 mM) was infused into livers from fasted rats perfused in either anterograde or the retrograde direction, a good correlation (r = 0.97) between rates of glucose production and extra O2 uptake by the liver was observed as expected. Rates of oxygen uptake were determined subsequently in periportal and pericentral regions of the liver lobule by placing miniature oxygen electrodes on the liver surface and measuring the local change in oxygen concentration when the flow was stopped. Basal rates of oxygen uptake of 142 +/- 11 and 60 +/- 4 mumol X g-1 X h-1 were calculated for periportal and pericentral regions, respectively. Infusion of 2 mM lactate increased oxygen uptake by 71 mumol X g-1 X h-1 in periportal regions and by 29 mumol X g-1 X h-1 in pericentral areas of the liver lobule. Since the stoichiometry between glucose production and extra oxygen uptake is well-established, rates of glucose production in periportal and pericentral regions of the liver lobule were calculated from local changes in rates of oxygen uptake for the first time. Maximal rates of glucose production from lactate (2 mM) were 60 +/- 7 and 25 +/- 4 mumol X g-1 X h-1 in periportal and pericentral zones of the liver lobule, respectively. The lactate concentrations required for half-maximal glucose synthesis were similar (0.4-0.5 mM) in both regions of the liver lobule in the presence or absence of epinephrine (0.1 microM). In the presence of epinephrine, maximal rates of glucose production from lactate were 79 +/- 5 and 59 +/- 3 mumol X g-1 X h-1 in periportal and pericentral regions, respectively. Thus, gluconeogenesis from lactate predominates in periportal areas of the liver lobule during perfusion in the anterograde direction; however, the stimulation by added epinephrine was greatest in pericentral areas. Differences in local rates of glucose synthesis may be due to ATP availability, as a good correlation between basal rates of O2 uptake and rates of gluconeogenesis were observed in both regions of the liver lobule in the presence and absence of epinephrine. In marked contrast, when livers were perfused in the retrograde direction, glucose production was 28 +/- 5 mumol X g-1 X h-1 in periportal areas and 74 +/- 6 mumol X g-1 X h-1 in pericentral regions.(ABSTRACT TRUNCATED AT 400 WORDS)     

Effects of trypsin and calcium chloride on signal IIs in oxygen-evolving PS II preparations

Photosystem II oxygen-evolving preparations exhibited a reversible loss of signal IIs hyperfine structure when treated with 1.0 M CaCl2. A progressive irreversible loss of hyperfine structure was observed upon trypsin treatment of these preparations. These treatments appear to alter the environment of the radical responsible for signal IIs. Gel electrophoresis of trypsin-treated photosystem II preparations indicates that three polypeptides (45, 32-34, and 26 kDa) are altered with the same kinetics as observed for the trypsin-induced loss of signal IIs. Two of these polypeptides (45 and 32-34 kDa) are core components of photosystem II.     

Carboxylate groups on the manganese-stabilizing protein are required for efficient binding of the 24 kDa extrinsic protein to photosystem II

The effects of the modification of carboxylate groups on the manganese-stabilizing protein on the binding of the 24 kDa extrinsic protein to Photosystem II were investigated. Carboxylate groups on the manganese-stabilizing protein were modified with glycine methyl ester in a reaction facilitated by 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide. The manganese-stabilizing protein which was modified while associated with NaCl-washed membranes could bind to calcium chloride-washed PS II membranes and reconstitute oxygen evolution in a manner similar to that observed for unmodified manganese-stabilizing protein (Frankel, L.K, Cruz, J. C. and Bricker, T. M. (1999) Biochemistry 38, 14271-14278). However, PS II membranes reconstituted with this modified protein were defective in their ability to bind the extrinsic 24 kDa protein of Photosystem II. Mapping of the sites of modification was carried out by trypsin and Staphylococcus V8 protease digestion of the modified protein and analysis by MALDI mass spectrometry. These studies indicated that the domains (1)E-(71)D, (97)D-(144)D, and (180)D-(187)E are labeled when the manganese-stabilizing protein is bound to NaCl-washed Photosystem II membranes. We hypothesize that modified carboxylates, possibly residues (1)E, (32)E, (139)E, and/or (187)E, in these domains are responsible for the altered binding affinity of the 24 kDa protein observed.     

Photophosphorylation Associated with Photosystem II: I. Photosystem II Cyclic Photophosphorylation Catalyzed by p-Phenylenediamine

Incubation of spinach chloroplast membranes for 90 minutes in the presence of 50 mm KCN and 100 mum HgCl(2) produces an inhibition of photosystem I activity which is stable to washing and to storage of the chloroplasts at -70 C. Subsequent exposure of these preparations to NH(2)OH and ethylenediaminetetraacetic acid destroys O(2) evolution and flow of electrons from water to oxidized p-phenylenediamine, but two types of phosphorylating cyclic electron flow can still be observed. In the presence of 3-(3,4-dichlorophenyl)-1,1'-dimethylurea, phenazinemethosulfate catalyzes ATP synthesis at a rate 60% that observed in uninhibited chloroplasts. C-Substituted p-phenylenediamines will also support low rates of photosystem I-catalyzed cyclic photophosphorylation, but p-phenylenediamine is completely inactive. When photosystem II is not inhibited, p-phenylenediamine will catalyze ATP synthesis at rates up to 90 mumol/hr.mg chlorophyll. This reaction is unaffected by anaerobiosis, and an action spectrum for ATP synthesis shows a peak at 640 nm. These results are interpreted as evidence for the existence of photosystem II-dependent cyclic photophosphorylation in these chloroplast preparations.     

Purification of highly active oxygen-evolving photosystem II from Chlamydomonas reinhardtii

A method is described for the isolation and purification of active oxygen-evolving photosystem II (PS II) membranes from the green alga Chlamydomonas reinhardtii. The isolation procedure is a modification of methods evolved for spinach (Berthold et al. 1981). The purity and integrity of the PS II preparations have been assesssed on the bases of the polypeptide pattern in SDS-PAGE, the rate of oxygen evolution, the EPR multiline signal of the S₂ state, the room temperature chlorophyll a fluorescence yield, the 77 K emission spectra, and the P700 EPR signal at 300 K. These data show that the PS II characteristics are increased by a factor of two in PS II preparations as compared to thylakoid samples, and the PS I concentration is reduced by approximately a factor ten compared to that in thylakoids.Abbreviations BSA bovine serum albumin - Chl chlorophyll - DCBQ 2,6-dichloro-p-benzoquinone - DCMU (diuron) 3-(3,4-dichlorophenyl)-1,1-dimethylurea - DMQ 2,5-dimethyl-p-benzoquinone - EDTA ethylenediamine tetraacetic acid - EPR electron paramagnetic resonance - Hepes N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid - MES 2-[N-Morpholino]ethanesulfonic acid - OEE oxygen evolving enhancer - PS II photosystem II - SDS-PAGE sodium dedocyl sulfate polyacrylamide gel electrophoresis     

Influence of the 33 kDa manganese-stabilizing protein on the structure and substrate accessibility of the oxygen-evolving complex of photosystem II

The 33 kDa manganese-stabilizing extrinsic protein binds to the lumenal side of photosystem II (PS II) close to the Mn(4)Ca cluster of the oxygen-evolving complex, where it limits access of small molecules to the metal site. Our previous finding that the removal of this protein did not alter the magnetic coupling regime within the manganese cluster, measured by electron spin-echo envelope modulation [Gregor, W., and Britt, R. D. (2000) Photosynth. Res. 65, 175-185], prompted us to examine whether this accessibility control is also true for substrate water, using the same pulsed EPR technique. Comparing the deuteron modulation of the S(2)-state multiline signal of PS II membranes, equilibrated with deuterated water (D(2)O) after removal or retention of the 33 kDa protein, we observed no change in the number and the distance of deuterons magnetically coupled to manganese, indicating that the number and distance of water molecules bound to the manganese cluster are independent of bound 33 kDa protein in the S(1) state, in which the sample was poised prior to cryogenic illumination. A simple modulation depth analysis revealed a distance of 2.5-2.6 A between the closest deuteron and manganese. These results are in agreement with our refined X-ray absorption analysis. The manganese K-edge positions, reflecting their oxidation states, and the extended X-ray absorption fine structure amplitudes and distances between the manganese ions and their oxygen and nitrogen ligands (1.8, 2.7, and 3.3-3.4 A) were independent of bound 33 kDa protein.     

Epitope mapping of the monoclonal antibody FAC2 on the apoprotein of Cpa-1 in photosystem II

Using a combination of cyanogen bromide cleavage and endoproteinase digestion we have shown that the putative epitope for the monoclonal antibody FAC2 lies in the region 360Pro(-391)Ser on the apoprotein of CPa-1. This region lies entirely within the large extrinsic loop of this protein. We have shown previously that the epitope of FAC2 becomes exposed in oxygen-evolving membranes upon treatment with alkaline Tris which releases all four of the manganese associated with the oxygen-evolving site of photosystem II. The epitope is not exposed, however, after CaCl(2) treatment and exposure to low concentrations of chloride, conditions which lead to the release of two of the four manganeses associated with the oxygen-evolving site. These results suggest that, upon release of the chloride-insensitive manganese from photosystem II membranes, a conformational change occurs which leads to the exposure of 360Pro(-391)Ser on CPa-1 to the monoclonal antibody FAC2.