Regeneration of the high-affinity manganese-binding site in the reaction center of an oxygen-evolution deficient mutant of Scenedesmus by protease action

The O₂-evolution deficient mutant (LF-1) of Scenedesmus obliquus inserts an unprocessed D1 protein into the thylakoid membrane and binds less than half the wild type (WT) level of Mn. LF-1 photosystem II (PS II) membrane fragments lack that part of the high-affinity Mn²⁺-binding site found in WT membranes which may be associated with histidine residues on the D1 protein (Seibert et al. 1989 Biochim Biophys Acta 974: 185–191). Hsu et al. (1987 Biochim Biophys Acta 890: 89–96) purport that the high-affinity site (characterized by competitive inhibition of DPC-supported DCIP photoreduction by M concentrations of Mn²⁺) in Mn-extracted PS II membranes is also the binding site for Mn functional in O₂ evolution. Proteases (papain, subtilisin, and carboxypeptidase A) can be used to regenerate the high-affinity Mn²⁺-binding site in LF-1 PS II membranes but not in thylakoids. Experiments with the histidine modifier, DEPC, suggest that the regenerated high-affinity Mn²⁺-binding sites produced by either subtilisin or carboxypeptidase A treatments were the same sites observed in WT membranes. However, none of the protease treatments produced LF-1 PS II membranes that could be photoactivated. Reassessment of the processing studies of Taylor et al. (1988 FEBS Lett 237: 229–233) lead us to believe that their procedure also does not result in substantial photoactivation of LF-1 PS II membranes. We conclude that (1) the unprocessed carboxyl end of the D1 protein in LF-1 is located on the lumenal side of the PS II membrane, (2) the unprocessed fragment physically obstructs or perturbs that part of the high-affinity Mn²⁺-binding site undetectable in LF-1, and (3) the D1 protein must be processed at the time of insertion into the membrane for normal O₂-evolution function to result.Abbreviations Chl chlorophyll - DCBQ 2,6-dichloro-1,4-benzoquinone - DCIP 2,6-dichlorophenol indophenol - DEPC diethylpryocarbonate - DPC 1,5-diphenylcarbazide - HEPES 4-(2-hydroxyethyl)-1-piperazine-ethanesulfonic acid - LDS-PAGE lithium dodecylsulfate polyacrylamide gel electrophoresis - LF-1 a low-fluorescent mutant of Scenedesmus obliquus - MES 4-morpholineethanesulfonic acid - PS II photosystem II - PMSF phenylmethylsulfonyl fluoride - RC photosystem II reaction center - Tris tris(hydroxymethyl)aminomethane - WT wild type Operated by the Midwest Research Institute for the U.S. Department of Energy under contract DE-AC-02-83CH10093.     

Specific 125I labeling of D1 (herbicide-binding protein): An indication that D1 functions on both the donor and acceptor sides of photosystem II

When ¹²⁵I⁻ was given as an artificial electron donor to non-O₂-evolving thylakoids of spinach, a 29 kDa polypeptide was specifically tagged by ¹²⁵I due to its photooxidation by PS II [(1985) Plant Cell Physiol. 26, 1093–1100]. We examined precisely the ¹²⁵I-labeling pattern in comparison with azido[¹⁴C]atrazine photoaffinity labeling of D1 and immunoblotting with anti-D1 and anti-D2, and found that D1 (herbicidebinding protein) of PS II reaction center complex is specifically tagged by ¹²⁵I in three different species of higher plants (spinach, pea and wheat) and a thermophilic cyanobacterium (Synechococcus vulcanus). It was suggested that D1 bears the photooxidation site or has a domain very close to the photooxidation site on the donor side of PS II, in addition to the well established binding site for Q_b and herbicides on the acceptor side of PS II.     

Presence in Photosystem II Core Complexes of a 34-Kilodalton Polypeptide Required for Water Photolysis

Photosystem II (PSII) reaction center core complexes have been isolated and characterized from wild type (WT) Scenedesmus obliquus and from its LF-1 mutant. LF-1 thylakoids are blocked on the oxidizing side of PSII and have a reduced Mn content. Visible absorption and low temperature fluorescence spectra of both core complexes are identical and resemble those reported for spinach (Satoh, Butler 1978 Plant Physiol 61: 373-379). Lithium dodecyl sulfate-polycrylamide gel electrophoresis reveals that a protein alteration, originally observed in thylakoid membranes (Metz, Wong, Bishop 1980 FEBS Lett 114: 61-66), is retained in the PSII core particles. That is, a 34-kilodalton (kD) polypeptide, present in the WT core complex, is missing in the mutant, and the core complex of the mutant contains a 36-kD protein not present in the WT. The 34-kD intrinsic protein is also observed in O₂-evolving PSII preparations and PSII core complexes from spinach. It is distinct from the 33-kD extrinsic protein first reported by T. Kuwabara and N. Murata (1979 Biochim Biophys Acta 581: 228-236). We suggest that the 34-kD protein is a site of Mn binding in the PSII membrane.     

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.     

Evidence for two different herbicide-binding proteins at the reducing side of Photosystem II

The binding behaviour at the thylakoid membrane of the radioactively labelled phenolic inhibitors 2-iodo-4-nitro-6-[2′,3′-³H]isobutylphenol and 3,5-diiodo-4-hydroxy[U-¹⁴C]benzonitrile (ioxynil) has been studied. As judged from displacement experiments with other herbicides, phenolic herbicides and herbicides as represented best by 3-(3,4-dichlorophenyl)-1,1-dimethylurea have different binding sites at the reducing side of Photosystem II. The binding parameters of phenolic herbicides are not, or only slightly, affected by trypsin treatment of chloroplasts. In chloroplasts, besides free pigments, lipids, and the light-harvesting chlorophyll $\text{a}\text{b}$ protein complex, a protein of molecular weight 41 000 is radioactively labelled by the photoaffinity label 4-nitro-2-azido-6-[2′,3′-³H]isobutylphenol. The amount of radioactivity bound to the 41 kDa protein is diminished if chloroplasts are incubated with 3-(3,4-dichlorophenyl)-1,1-dimethylurea prior to addition of the photoaffinity label, but not if the 2,4-dinitrophenyl ether of 2-iodo-4-nitrothymol is used instead. These two compounds are characteristic representatives of inhibitiors acting at the reducing or the oxidizing site of plastoquinone, respectively. Based on these results, a model for two different herbicide-binding proteins at the reducing side of Photosystem II is presented.     

Identification of the phosphorylation site of an 8.3 kDa protein from photosystem II of spinach

The four principal phosphoproteins of PS II cores (8.3, 32, 34 and 44 kDa) give rise to distinct tryptic phosphopeptides which have been purified by affinity chromatography on Fe³⁺-chelating Sepharose and reverse-phase HPLC. The tryptic phosphopeptide derived from the 8.3 kDa protein has the sequence NH₂-Ala-Thr-Gln-Thr-Val-Glu-Ser-Ser-Ser-Arg. It corresponds to the N-terminus of the chloroplast psbH gene product, except for the loss of the initiating N-formylmethionine. The peptide is phosphorylated on the first threonyl residue. Differences between the phosphorylation sites of the 8.3 kDa protein and LHC II are consistent with the hypothesis that thylakoids contain two distinct redox-controlled protein kinases differing in substrate specificity.     

Herbicide and plastoquinone-binding proteins of Photosystem II reaction center complexes from the thermophilic cyanobacterium,Synechococcus sp.

Photoaffinity labeling of Synechococcus Photosystem (PS) II preparations with radioactive azido-derivatives of three herbicides and of plastoquinone was carried out to identify herbicide and plastoquinone-binding proteins. [¹⁴C]Azido-atrazine and [¹⁴C]azido-monuron specifically labeled the 28 kDa polypeptide of the PS II reaction center complex, which is sensitive to 3-(3′,4′-dichlorophenyl)-1,1-dimethylurea (DCMU). No specific labeling of this polypeptide with azido-atrazine was found in CP2-b (PS II reaction center lacking the 40 kDa subunit) which is insensitive to DCMU. [³H]Azido-dinoseb reacted with the 28 kDa polypeptide and the 47 kDa chlorophyll-carrying protein. The labeling with [³H]azido-plastoquinone resulted in the incorporation of the radioactivity exclusively into the 47 kDa polypeptide. It is concluded that the 28 kDa polypeptide is the herbicide-binding protein of the cyanobacterium and that the 47 kDa polypeptide has a binding site for plastoquinone and for phenol-type herbicides.     

Polypeptide composition of a Photosystem II core complex. Presence of a herbicide-binding protein

The polypeptide composition of a Photosystem II (PS II) core complex from higher plant chloroplasts has been characterized by subjecting the isolated complex to sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Two polypeptides in the 40–50 kDa size class, attributed to the chlorophyll a-binding apoproteins of PS II, were resolved when the urea concentration in the SDS-polyacrylamide gel electrophoresis was greater than 1 M. The two chlorophyll a-binding proteins were dissimilar in their primary structure based upon their different hydrolysis products on SDS-polyacrylamide gel electrophoresis following papain treatment. The core complex contained three additional polypeptides. Two polypeptides in the 30–34 kDa size class were resolved when the urea concentration in the gel system was increased to greater than 4 M. One of the polypeptides in this size class was identified as the herbicide-binding protein from azido[¹⁴C]atrazine labeling studies. The herbicide-binding protein displayed an anomalous electrophoretic migration behavior in SDS-polyacrylamide gel electrophoresis in the presence or absence of urea; its apparent molecular weight decreased when the urea concentration increased. The fifth protein component of the core complex was attributed to cytochrome b-559 which was found to consist of the ascorbate- and dithionite-reducible forms in the samples prior to SDS solubilization.     

Autotrophic cells of the <Emphasis Type="Italic">Synechocystis psbH</Emphasis> deletion mutant are deficient in synthesis of CP47 and accumulate inactive PS II core complexes

Cells of the psbH deletion mutant IC7 of the cyanobacterium Synechocystis PCC 6803 grown in the absence of glucose contain strongly reduced levels of chlorophyll when compared with cells grown in the presence of glucose, or compared with wild-type (WT) cells. Low-temperature fluorescence emission spectra revealed decreased content of both active PS II (Photosystem II) and PS I (Photosystem I) complexes. Analysis of thylakoid membrane complexes of IC7 by native electrophoresis showed a similar set of chlorophyll–proteins, namely a PS II core complex and trimeric and monomeric PS II complexes, as in WT. However, in contrast to WT, the ³⁵S-methionine protein labeling pattern of the mutant exhibited no preferential labeling of the D1 protein in the PS II core complexes, and the labeled D1 and D2 proteins accumulated predominantly in the PS II reaction center lacking CP47. The results show that in autotrophically grown cells of the psbH deletion mutant, selective D1 turnover is inhibited and synthesis of CP47 becomes a limiting step in the PS II assembly.     

Photoaffinity labeling and biochemical characterization of binding proteins for pheromones,juvenile hormones,and peptides

Radiolabeled photoaffinity analogs can be used to purify and characterize proteins involved in pheromone perception, juvenile hormone (JH) action, and neuropeptide reception. Several photoaffinity analogs and purification strategies are described for each of these physiological targets. First, a diazoacetate photoaffinity label is used to selectively modify the pheromone binding protein of the gypsy moth, Lymantria dispar. Reverse-phase HPLC is then employed to fractionate the male antennal proteins. Second, a tandem procedure involving preparative isoelectric focusing (IEF) and ion-exchange (IEX) HPLC is employed for the purification of the Manduca sexta hemolymph juvenile hormone binding protein (JHBP), which has now been cloned and sequenced. A separate application of this strategy for the purification of the 29 kDa JH I/methoprene receptor proteins from epidermal nuclei of M. sexta larvae is outlined. A new photolabel, farnesyl diazoketone, has been employed for the characterization of crustacean hemolymph methyl farnesoate binding proteins. Third, the development of neuropeptide photoaffinity labels is described for two systems: (1) the red pigment concentrating hormone (RPCH) of shrimp and (2) the allatostatins isolated from the brain of the cockroach Diploptera punctata.     

EPR signal II in cyanobacterial Photosystem II reaction-center complexes with and without the 40 kDa chlorophyll-binding subunit

The steady-state amplitude and flash-induced kinetics of EPR signal II in two Photosystem II (PS II) reaction center protein complexes from Synechococcus were measured to probe the organization of species involved in the PS II electron-transfer chain. A PS II reaction center complex (E-1) which has 47, 40, 31, 28 and 9 kDa subunits shows both fast decaying (signal II_f) and slowly decaying (signal II_s+u) EPR components. The amplitude of signal II_f, which represents Z (the donor to P-680), is about 1 spin per 30 Chl. This corresponds to one spin per reaction center in this preparation. Signal II_s+u, the slowly decaying component of signal II, reflects D, a donor to PS II on a side chain from the path of water oxidation in higher plants and algae. Signal II_s+u is present in the E-1 preparation in a ratio of about 1 spin per 40 Chl. Flash-induced signal II_f in E-1 shows biexponential decay with half-times of 20 ms and 300 ms. In a PS II reaction center complex (CP2b) which has 47, 31, 28 and 9 kDa subunits, but no 40 kDa subunit, an appreciable amount of signal II_f is observed (about 1 per 50 Chl). Less than 1 spin per 400 Chl of signal II_s+u is visible in this sample. The kinetics of Z⁺ reduction (signal II_f) in CP2b is similar to that seen in E-1 preparations, indicating that CP2b contains all of the molecules necessary for primary charge separation and secondary electron donation from Z.     

Photoinhibition and Recovery of Photosynthesis in Canker-susceptible and Resistant Needles of Cypress (Cupressus sempervirens L.)

The aim of the present investigation was to test the hypothesis that the cypress canker caused by a fungus (Seiridium cardinale) infection induced effects on photosynthesis which could be related to photoinhibition and the process of recovery in susceptible and resistant needles. Photoinhibition of photosynthesis and recovery was studied in canker‐infected susceptible and resistant needles of cypress (Cupressus sempervirens L.) under controlled conditions (irradiation of detached needles to approximately 1900 μmol/m²/s). The degree of photoinhibition was determined by means of the ratio of variable to maximum chlorophyll (Chl) fluorescence (F_v/F_m) and electron transport measurements. The potential efficiency of photosystem (PS) II, F_v/F_m declined, and F_o increased significantly in canker‐susceptible needles, while F_o did not change in resistant needles. In isolated thylakoids, high light (HL) decreased the rate of whole chain and PS II activity markedly more in susceptible than in resistant needles. A smaller reduction of PS I activity was noticed only in susceptible needles. Upon subsequent dark incubation, fast recovery was noticed in both needle types and reached maximum rates of PS II efficiency similar to those noticed in non‐photoinhibited needles. The artificial exogenous electron donors such as diphenyl carbazide (DPC), NH₂OH and Mn²⁺ failed to restore the HL induced loss of PS II activity in susceptible needles, while DPC and NH₂OH significantly restored it in resistant needles. The results suggest that HL inactivates the donor side of PS II in resistant and the acceptor side of PS II in susceptible needles. The results on the quantification of the PS II reaction centre protein D1 and 33 kDa protein of water‐splitting complex following HL exposure showed pronounced differences between susceptible and resistant needles. The marked loss of PS II activity in HL‐irradiated needles was due to the marked loss of D1 protein in susceptible and 33 kDa protein in resistant needles, respectively.     

COOH-terminal processing of polypeptide D1 of the photosystem II reaction center of Scenedesmus obliquus is necessary for the assembly of the oxygen-evolving complex

Mutant LF-1 of the green alga Scenedesmus obliquus has been described by Metz and co-workers (Metz, J. G., Pakrasi, H., Seibert, M., and Arntzen, C. J. (1986) FEBS Lett. 205, 269-274) to be inactive for light-driven oxygen evolution, despite a functional Photo-system II reaction center. A polypeptide, D1, implicated in the ligation of the primary photoreactants of photosystem II, was shown to migrate with an apparent higher molecular mass on LDS-PAGE in the mutant than in the wild-type (WT) strain. We show here that polypeptide D1 is synthesized in a precursor form in Scenedesmus WT. Following synthesis and insertion into the thylakoid membrane, a 1.5-2-kDa oligopeptide is clipped off with a half-time of 1-2 min, yielding the mature 34-kDa form of the polypeptide. No processing of polypeptide D1 from mutant LF-1 was observed to take place. We show here that polypeptide D1 of LF-1 displays an identical proteolytic fingerprint pattern to the precursor D1 polypeptide of the wild-type strain. These both have molecular masses about 1.5-2 kDa higher than that of the mature WT polypeptide. A polyclonal antibody elicited by a synthetic oligopeptide (14-mer), predicted from the psbA gene nucleotide sequence to be homologous to the COOH terminus of the precursor D1 of spinach, cross-reacts only with D1 of mutant LF-1 and not with mature D1 of spinach, Chlamydomonas, or of Scenedesmus WT. This observation demonstrates that the greater molecular mass of polypeptide D1 from mutant LF-1 and of Scenedesmus WT precursor D1 is derived from a COOH-terminal extension. We conclude that the LF-1 mutant lacks the appropriate nuclear-encoded protease which processes polypeptide D1 at its COOH terminus from the precursor to the mature form. Such processing would appear to be a necessary step toward the stable incorporation of manganese into the oxygen-evolving site.     

Theoretical study of the binding site and mode of action for photosystem II herbicides

Several features of a proteinaceous binding site and a molecular mode of action are proposed for photosystem II (PS II) herbicides based upon a variety of experimental and theoretical evidence. Experimental studies have established that PS II herbicides bind non-covalently to a 32 kdalton protein in the PS II complex and inhibit electron transfer between the first quinone (Q) and the second quinone (B) on the reducing side of PS II. The herbicides each contain hydrophobic components as well as a flat polar component with a dipole moment in the range of 3–5 Debyes. The primary function of the hydrophobic components is to increase the lipid solubility of the entire herbicide molecule; the secondary function of the hydrophobic components is to fit the hydrophobic surface of the herbicide binding site. It is proposed that the flat polar component binds electrostatically at a highly polar protein site, probably a protein salt bridge or the terminus of a protein alpha helix. Further, it is proposed that the PS II herbicides shift the equilibrium Q^?Bz?QB^? to the left (i) by reducing the magnitude of an anion-stabilizing electric field across the B-binding site, or (ii) by inhibiting the conformational relaxation or protonation of the PS II protein in response to reduction of B to B^?, or (iii) by displacing the quinone head of B from its binding site. Ab initio molecular quantum mechanical calculations have been carried out to investigate the electrostatic interactions in specific herbicide-binding site models.     

Identification of a chloroplast membrane polypeptide associated with the oxidizing side of photosystem II by the use of select low-fluorescent mutants of Scenedesmus

Comparison of photochemical activities and variable fluorescence yield characteristics of whole cells and isolated chloroplast particles of low-fluorescent, photosystem II mutants of $\text{Scenedesmus}$$\text{obliquus}$ to those of the wild-type showed that several strains were affected primarily on the oxidizing side of photosystem II. In strains LF-1, LF-3, and LF-5 analysis of the manganese content of isolated chloroplast membranes showed a predominant shift in the $\text{Mn}\text{400}$ Chl from the wild-type value (4.3) to values near 1.5; this difference was also associated with a near total loss of cytochrome b-559 (high potential). Examination of chloroplast membrane polypeptides by gel electrophoresis revealed a decrease only in the mobility of one band in all three mutants; the apparent molecular weight was shifted from 34 kilodalton in the wild-type to 36 kilodalton in the mutants. Evidence is presented suggesting that the 34 kilodalton polypeptide of the wild-type is probably associated with the manganese requiring portion of the water-splitting apparatus of photosystem II.     

Depletion of Photosystem II-extrinsic proteins. I. Effects on O2- and N2-flash yields and steady-state O2 evolution

Wheat O₂-evolving Photosystem II (PS II) membranes having a PS II unit of approx. 200 chlorophylls (Chl), approx. 4 Mn/200 Chl, less than 1 P-700/3000 Chl and an electron-acceptor pool of approx. 2.5 equiv./PS II were analyzed and compared with wheat PS II membranes depleted (at least 90%) of the 17 and 23 kDa proteins by NaCl extraction during Triton X-100 isolation of membranes. Extraction of these proteins caused approx. 50% decrease in O₂ evolution in any light regime and an increase of approx. 2 equiv./PS II of the electron-acceptor pool, but affected neither Mn abundance, photoreduction of DCIP by tetraphenylboron, or N₂ yield (from NH₂OH) from a single flash. Mass spectrometric analyses of O₂ flash yields in the presence of potassium ferricyanide showed that both chloroplasts and the unextracted PS II membranes yielded oscillations compatible with S₀/S₁/S₂/S₃ of 25:75:0:0 and α (0.1) and β (0.05). Depletion of 17 and 23 kDa proteins resulted in a two-fold increase in α, approx. 25–40% disconnection of the S state complex from the PS II trap complex but with no change in β. Preincubation of control or extracted PS II membranes with potassium ferricyanide permitted a significant double-hit on the first flash. In the absence of an added electron acceptor, N₂ flash yields were more sustained with 17 and 23 kDa depleted than with 17 and 23 kDa sufficient PS II membranes. In contrast, no significant O₂ flash yields were observed with extracted PS II preparations under these conditions (control PS II membranes showed a predictable O₂ pattern before damping after only 5–6 flashes). These results suggest that extraction of the 17 and 23 kDa proteins results in an increase of pool size on the PS II acceptor side (seen as unmasking ‘Component C’). ‘Component C’ can mediate electron transfer from Q⁻ to Z⁺ (S₂).     

The roles of the extrinsic subunits in Photosystem II as revealed by EPR

The effects of selective removal of extrinsic proteins on donor side electron transport in oxygen-evolving PS II particles were examined by monitoring the decay time of the EPR signal from the oxidized secondary donor, Z⁺, and the amplitude of the multiline manganese EPR signal. Removal of the 16 and 24 kDa proteins by washing with 1 M NaCl inhibits oxygen evolution, but rapid electron transfer to Z⁺ still occurs as evidenced by the near absence of Signal II_f. The absence of a multiline EPR signal shows that NaCl washing induces a modification of the oxygen-evolving complex which prevents the formation of the S₂ state. This modification is different from the one induced by chloride depletion of PS II particles, since in these a large multiline EPR signal is found. After removal of the 33 kDa protein with 1 M MgCl₂, Signal II_f is generated after a light flash. Readdition of the 33 kDa component to the depleted membranes accelerates the reduction of Z⁺. Added calcium ions show a similar effect. These findings suggest that partial advancement through the oxygen-evolving cycle can occur in the absence of the 16 and 24 kDa proteins. The 33 kDa protein, on the other hand, may be necessary for such reactions to take place.     

A photoaffinity label for the siderophore-mediated iron transport system in Neurospora crassa

A photoaffinity derivative of the coprogen class of siderophores, p-azidobenzoylcoprogen B, has been synthetized. In the dark it is recognized and taken up by the iron transport system in Neurospora crassa (arg-5, ota, aga) in a concentration-dependent manner (K_m = 6 μM, V_max = 0.2 nmol·min⁻¹·mg⁻¹). It is also a competitive inhibitor of coprogen uptake, K_I ≈ 5 μM. Photolyssis of cells with near-ultraviolet light during transport in the presence of the photoaffinity label results in approx. 50% irreversible inhibition of both coprogen and ferrichrysin uptake. Uptake of p-azidobenzoylcoprogen B itself is also inhibited upon illumination. It is proposed that this affinity label be used in isolation of the iron receptor protein(s) in N. crassa.     

Photosynthetic water oxidation: The protein framework

Approximately 20 protein subunits are associated with the PS II complex, not counting subunits of peripheral light-harvesting antenna complexes. However, it is not yet established which proteins specifically are involved in the water-oxidation process. Much evidence supports the concept that the D1/D2 reaction center heterodimer not only plays a central role in the primary photochemistry of Photosystem II, but also is involved in electron donation to P680 and in ligation of the manganese cluster. This evidence includes (a) the primary donor to P680 has been shown to be a redox-active tyrosyl residue (Tyr161) in the D1 protein, and (b) site-directed mutagenesis and computer-assisted modeling of the reaction center heterodimer have suggested several sites with a possible function in manganese ligation. These include Asp170, Gln165 and Gln189 of the D1 protein and Glu69 of the D2 protein as well as the C-terminal portion of the mature D1 protein. Also, hydrophilic loops of the chlorophyll-binding protein CP43 that are exposed at the inner thylakoid surface could be essential for the water-splitting process.In photosynthetic eukaryotes, three lumenal extrinsic proteins, PS II-O (33 kDa), PS II-P (23 kDa) and PS II-Q (16 kDa), influence the properties of the manganese cluster without being involved in the actual catalysis of water oxidation. The extrinsic proteins together may have multiple binding sites to the integral portion of PS II, which could be provided by the D1/D2 heterodimer and CP47. A major role for the PS II-O protein is to stabilize the manganese cluster. Most experimental evidence favors a connection of the PS II-P protein with binding of the Cl^- and Ca²⁺ ions required for the water oxidation, while the PS II-Q protein seems to be associated only with the Cl^- requirement. The two latter proteins are not present in PS II of prokaryotic organisms, where their functions may be replaced by a 10–12 kDa subunit and a newly discovered low-potential cytochrome c-550.Abbreviations PS II Photosystem II - PCC Pasteur Culture Collection     

PsbX maintains efficient electron transport in Photosystem II and reduces susceptibility to high light in Synechocystis sp. PCC 6803

PsbX is a 4.1 kDa intrinsic Photosystem II (PS II) protein, found together with the low-molecular-weight proteins, PsbY and PsbJ, in proximity to cytochrome b₅₅₉. The function of PsbX is not yet fully characterized but PsbX may play a role in the exchange of the secondary plastoquinone electron acceptor Q_B with the quinone pool in the thylakoid membrane. To study the role of PsbX, we have constructed a PsbX-lacking strain of Synechocystis sp. PCC 6803. Our studies indicate that the absence of PsbX causes sensitivity to high light and impairs electron transport within PS II. In addition to a change in the Q_B-binding pocket, PsbX-lacking cells exhibited sensitivity to sodium formate, suggesting altered binding of the bicarbonate ligand to the non-heme iron between the sequential plastoquinone electron acceptors Q_A and Q_B. Experiments using ³⁵S-methionine revealed high-light-treated PsbX-lacking cells restore PS II activity during recovery under low light by an increase in the turnover of PS II-associated core proteins. These labeling experiments indicate the recovery after exposure to high light requires both selective removal and replacement of the D1 protein and de novo PS II assembly.