Identification of Oxidized Amino Acid Residues in the Vicinity of the Mn(4)CaO(5) Cluster of Photosystem II: Implications for the Identification of Oxygen Channels within the Photosystem

As a light-driven water-plastoquinone oxidoreductase, Photosystem II produces molecular oxygen as an enzymatic product. Additionally, under a variety of stress conditions, reactive oxygen species are produced at or near the active site for oxygen evolution. In this study, Fourier-transform ion cyclotron resonance mass spectrometry was used to identify oxidized amino acid residues located in several core Photosystem II proteins (D1, D2, CP43, and CP47) isolated from spinach Photosystem II membranes. While the majority of these oxidized residues (81%) are located on the oxygenated solvent-exposed surface of the complex, several residues on the CP43 protein ((354)E, (355)T, (356)M, and (357)R) which are in close proximity (<15 ?) to the Mn(4)CaO(5) active site are also modified. These residues appear to be associated with putative oxygen/reactive oxygen species exit channel(s) in the photosystem. These results are discussed within the context of a number of computational studies which have identified putative oxygen channels within the photosystem.     

X-ray spectroscopy of the Mn4Ca cluster in the water-oxidation complex of Photosystem II

The water-oxidation complex of Photosystem II (PS II) contains a heteronuclear cluster of 4 Mn atoms and a Ca atom. Ligands to the metal cluster involve bridging O atoms, and O and N atoms from amino acid side-chains of the D1 polypeptide of PS II, with likely additional contributions from water and CP43. Although moderate resolution X-ray diffraction-based structures of PS II have been reported recently, and the location of the Mn₄Ca cluster has been identified, the structures are not resolved at the atomic level. X-ray absorption (XAS), emission (XES), resonant inelastic X-ray scattering (RIXS) and extended X-ray absorption fine structure (EXAFS) provide independent and potentially highly accurate sources of structural and oxidation-state information. When combined with polarized X-ray studies of oriented membranes or single-crystals of PS II, a more detailed picture of the cluster and its disposition in PS II is obtained.     

Mimicking the electron donor side of Photosystem II in artificial photosynthesis

This review focuses on our recent efforts in synthetic ruthenium–tyrosine–manganese chemistry mimicking the donor side reactions of Photosystem II. Tyrosine and tryptophan residues were linked to ruthenium photosensitizers, which resulted in model complexes for proton-coupled electron transfer from amino acids. A new mechanistic model was proposed and used to design complexes in which the mechanism could be switched between concerted and step-wise proton-coupled electron transfer. Moreover, a manganese dimer linked to a ruthenium complex could be oxidized in three successive steps, from Mn₂^II,II to Mn₂^III,IV by the photo-oxidized ruthenium sensitizer. This was possible thanks to a charge compensating ligand exchange in the manganese complex. Detailed studies of the ligand exchange suggested that at high water concentrations, each oxidation step is coupled to a proton-release of water-derived ligands, analogous to the oxidation steps of the manganese cluster of Photosystem II.     

Structurally conserved channels in cyanobacterial and plant photosystem II

In the cyanobacterial photosystem II (PSII), the O4-water chain in the D1 and CP43 proteins, a chain of water molecules that are directly H-bonded to O4 of the Mn₄Ca cluster, is linked with a channel that connects the protein bulk surface along with a membrane-extrinsic protein subunit, PsbU (O4-PsbU channel). The cyanobacterial PSII structure also shows that the O1 site of the Mn₄Ca cluster has a chain of H-bonded water molecules, which is linked with the channel that proceeds toward the bulk surface via PsbU and PsbV (O1-PsbU/V channel). Membrane-extrinsic protein subunits PsbU and PsbV in cyanobacterial PSII are replaced with PsbP and PsbQ in plant PSII. However, these four proteins have no structural similarity. It remains unknown whether the corresponding channels also exist in plant PSII, because water molecules are not identified in the plant PSII cryo-electron microscopy (cryo-EM) structure. Using the cyanobacterial and plant PSII structures, we analyzed the channels that proceed from the Mn₄Ca cluster. The cyanobacterial O4-PsbU and O1-PsbU/V channels were structurally conserved as the channel that proceeds along PsbP toward the protein bulk surface in the plant PSII (O4-PsbP and O1-PsbP channels, respectively). Calculated protonation states indicated that in contrast to the original geometry of the plant cryo-EM structure, protonated PsbP-Lys166 may form a salt-bridge with ionized D1-Glu329 and protonated PsbP-Lys173 may form a salt-bridge with ionized PsbQ-Asp28 near the O1-PsbP channel. The existence of these channels might explain the molecular mechanism of how PsbP can interact with the Mn₄Ca cluster.     

The Sll0606 Protein Is Required for Photosystem II Assembly/Stability in the Cyanobacterium Synechocystis sp. PCC 6803

An insertional transposon mutation in the sll0606 gene was found to lead to a loss of photoautotrophy but not photoheterotrophy in the cyanobacterium Synechocystis sp. PCC 6803. Complementation analysis of this mutant (Tsll0606) indicated that an intact sll0606 gene could fully restore photoautotrophic growth. Gene organization in the vicinity of sll0606 indicates that it is not contained in an operon. No electron transport activity was detected in Tsll0606 using water as an electron donor and 2,6-dichlorobenzoquinone as an electron acceptor, indicating that Photosystem II (PS II) was defective. Electron transport activity using dichlorophenol indolephenol plus ascorbate as an electron donor to methyl viologen, however, was the same as observed in the control strain. This indicated that electron flow through Photosystem I was normal. Fluorescence induction and decay parameters verified that Photosystem II was highly compromised. The quantum yield for energy trapping by Photosystem II (F_V/F_M) in the mutant was less than 10% of that observed in the control strain. The small variable fluorescence yield observed after a single saturating flash exhibited aberrant Q_A⁻ reoxidation kinetics that were insensitive to dichloromethylurea. Immunological analysis indicated that whereas the D2 and CP47 proteins were modestly affected, the D1 and CP43 components were dramatically reduced. Analysis of two-dimensional blue native/lithium dodecyl sulfate-polyacrylamide gels indicated that no intact PS II monomer or dimers were observed in the mutant. The CP43-less PS II monomer did accumulate to detectable levels. Our results indicate that the Sll0606 protein is required for the assembly/stability of a functionally competent Photosystem II.     

The 28 kDa apoprotein of CP 26 in PS II binds copper

Photosystem II (PS II) particles isolated from spinach in the presence of 10 M CuSO₄ contained 1.2 copper/300 Chl that was resistant to EDTA. When CuSO₄ was not added during the isolation, PS II particles contained variable amounts of copper resistant to EDTA (0.1–1.1 copper/300 Chl). No correlation was found between copper content and oxygen evolving capacity of the PS II particles. To identify the copper binding protein, we developed a fractionation procedure which included solubilisation of PS II particles followed by precipitation with polyethylene glycol. A 22-fold purification of copper with respect to protein was achieved for a 28 kDa protein. Partial amino acid sequence of a 13 kDa fragment, obtained after V8 (endo Glu-C) protease treatment, showed identity with CP 26 over a 14 amino acid stretch. EPR measurements on the purified protein suggest oxygen and/or nitrogen as ligands for copper but tend to exclude sulfur. We conclude that the 28 kDa apoprotein of CP 26 from spinach binds one copper per molecule of CP 26. A possible function for this copper protein in the xanthophyll cycle is discussed.Abbreviations CP 26 and CP 29 chlorophyll a/b protein complex 26 and 29 - LHC II light-harvesting chlorophyll a/b protein complex of Photosystem II - SB14 sulfobetaine 14 A preliminary report of these results was presented at the IX Int. Congress on Photosynthesis, Nagoya, Japan, 1992.     

Sequence variation at the oxygen-evolving centre of photosystem II: a new class of ‘rogue’ cyanobacterial D1 proteins

Photosystem II is the oxygen-evolving enzyme of photosynthesis. It is a membrane-bound protein-pigment complex. The oxygen is produced at the oxygen-evolving centre (OEC), a Mn₄CaO₅ metallocluster, which is largely ligated by amino acids of the D1 protein. The OEC-ligating residues are invariant between most cyanobacteria and higher plants. In this study, a new class of cyanobacterial D1 proteins has been identified in which the OEC metal-ligating residues are very different to the consensus. This new class of ‘rogue’ D1 proteins is associated with diazotrophic cyanobacteria. Their function, activity and origins are discussed.     

Specific loss of the extrinsic 18 KDa protein from Photosystem II upon heating to 47 °C causes inactivation of oxygen evolution likely due to Ca release from the Mn-complex

Exposure of Photosystem II (PS II) membrane particles from spinach to a temperature of 47 °C caused the rapid release of the 18 kDa protein in parallel to inactivation of oxygen evolution. Previously, it has been suggested that the first heat-jump response involves rapid Ca release from the Mn complex of O₂-evolution, followed by the slower release of (2 + 2) Mn^II ions [Pospisil P et al. (2003) Biophys J 84: 1370–1386]. Here, the predicted biphasic Mn^II release to the bulk was verified by atomic absorption spectroscopy (AAS). Analysis of laser flash-induced delayed fluorescence transients suggests that the loss of the essential Ca ion from the Mn₄Ca complex in the dark is due to the loss of the 18 kDa protein. The S₂-state multiline EPR signal of the Mn complex was still generated in heat-treated PS II presumably lacking Ca, but retaining four Mn ions.Dedicated to Professor Norio Murata on the occasion of his retirement     

Oxidized Amino Acid Residues in the Vicinity of QA and PheoD1 of the Photosystem II Reaction Center: Putative Generation Sites of Reducing-Side Reactive Oxygen Species

Under a variety of stress conditions, Photosystem II produces reactive oxygen species on both the reducing and oxidizing sides of the photosystem. A number of different sites including the Mn₄O₅Ca cluster, P₆₈₀, Pheo_D1, Q_A, Q_B and cytochrome b₅₅₉ have been hypothesized to produce reactive oxygen species in the photosystem. In this communication using Fourier-transform ion cyclotron resonance mass spectrometry we have identified several residues on the D1 and D2 proteins from spinach which are oxidatively modified and in close proximity to Q_A (D1 residues ²³⁹F, ²⁴¹Q, ²⁴²E and the D2 residues ²³⁸P, ²³⁹T, ²⁴²E and ²⁴⁷M) and Pheo_D1 (D1 residues ¹³⁰E, ¹³³L and ¹³⁵F). These residues may be associated with reactive oxygen species exit pathways located on the reducing side of the photosystem, and their modification may indicate that both Q_A and Pheo_D1 are sources of reactive oxygen species on the reducing side of Photosystem II.     

Fourier transform infrared and mass spectrometry analyses of a site-directed mutant of D1-Asp170 as a ligand to the water-oxidizing Mn4CaO5 cluster in photosystem II

The Mn₄CaO₅ cluster, the catalytic center of water oxidation in photosystem II (PSII), is coordinated by six carboxylate and one imidazole ligands. The roles of these ligands in the water oxidation mechanism remain largely unknown. In this study, we constructed a D1-D170H mutant, in which the Asp ligand bridging Mn and Ca ions was replaced with His, in the cyanobacterium Synechocystis sp. PCC 6803, and analyzed isolated PSII core complexes using Fourier transform infrared (FTIR) difference spectroscopy and mass spectrometry (MS). The S₂-minus-S₁ FTIR difference spectrum of the PSII complexes of the D1-D170H mutant showed features virtually identical to those of the wild-type PSII. MS analysis further showed that ~70% of D1 proteins from the PSII complexes of D1-D170H possessed the wild-type amino acid sequence, although only the mutated sequence was detected in genomic DNA in the same batch of cells for PSII preparations. In contrast, a D1-S169A mutant as a control showed a modified FTIR spectrum and only a mutated D1 protein. It is thus concluded that the FTIR spectrum of the D1-D170H mutant actually reflects that of wild-type PSII, whereas the Mn₄CaO₅ cluster is not formed in PSII with D1-D170H mutation. Although the mechanism of production of the wild-type D1 protein in the D1-D170H mutant is unknown at present, a caution is necessary in the analysis of site-directed mutants of crucial residues in the D1 protein, and mutation has to be confirmed not only at the DNA level but also at the amino acid level.     

Crystal Structure of Monomeric Photosystem II from Thermosynechococcus elongatus at 3.6-? Resolution

The membrane-embedded photosystem II core complex (PSIIcc) uses light energy to oxidize water in photosynthesis. Information about the spatial structure of PSIIcc obtained from x-ray crystallography was so far derived from homodimeric PSIIcc of thermophilic cyanobacteria. Here, we report the first crystallization and structural analysis of the monomeric form of PSIIcc with high oxygen evolution capacity, isolated from Thermosynechococcus elongatus. The crystals belong to the space group C222₁, contain one monomer per asymmetric unit, and diffract to a resolution of 3.6 Å. The x-ray diffraction pattern of the PSIIcc-monomer crystals exhibit less anisotropy (dependence of resolution on crystal orientation) compared with crystals of dimeric PSIIcc, and the packing of the molecules within the unit cell is different. In the monomer, 19 protein subunits, 35 chlorophylls, two pheophytins, the non-heme iron, the primary plastoquinone Q_A, two heme groups, 11 β-carotenes, 22 lipids, seven detergent molecules, and the Mn₄Ca cluster of the water oxidizing complex could be assigned analogous to the dimer. Based on the new structural information, the roles of lipids and protein subunits in dimer formation of PSIIcc are discussed. Due to the lack of non-crystallographic symmetry and the orientation of the membrane normal of PSIIcc perpendicular (∼87°) to the crystallographic b-axis, further information about the structure of the Mn₄Ca cluster is expected to become available from orientation-dependent spectroscopy on this new crystal form.     

Some properties of Photosystem II preparations from the cyanobacterium Synechococcus sp. — presence of an l-amino acid oxidase in Photosystem II complexes from Synechococcus sp.

A highly active O₂-evolving Photosystem II complex has been purified from the cyanobacterium Synechococcus sp., and this complex has been compared with the Photosystem II complex previously isolated from this cyanobacterium (Ohno, T., Satoh, K. and Katoh, S. (1986) Biochim. Biophys. Acta 852, 1–8). Further treatment of the O₂-evolving complex with the detergent sodium taurodesoxycholate resulted in a complex which consisted mainly of the 47 and 40 kDa peptides and which had lost the O₂-evolving activity, but which could still reduce 2,6-dichlorophenolindophenol with 1,5-diphenylcarbazide. Previously, we have shown that a flavoprotein of 49 kDa which has an l-amino acid oxidase activity under certain conditions, is a component of highly active Photosystem II preparations from the cyanobacterium Anacystis nidulans (Pistorius, E.K. and Gau, A.E. (1986) FEBS Lett. 206, 243–248). Based on immunological studies with the antiserum raised against the l-amino acid oxidase protein from A. nidulans, we show that a protein which cross-reacts with this antiserum is present in the highly purified Photosystem II preparations from Synechococcus sp. Moreover, an l-amino acid oxidase activity could also be detected in Photosystem II preparations from Synechococcus sp. The enzyme preferentially oxidizes basic l-amino acids as l-arginine, l-ornithine, 2,3-diamino propionic acid and l-citrulline. In contrast to the enzyme from A. nidulansl-lysine is not oxidized. The here shown presence of an l-amino acid oxidase protein in Photosystem II preparations from Synechococcus sp. is an additional support of our hypothesis that a flavoprotein is a functional component of the water-oxidizing enzyme complex.     

Mn2+ reduces Yz + in manganese-depleted Photosystem II preparations

Manganese in the oxygen-evolving complex is a physiological electron donor to Photosystem II. PS II depleted of manganese may oxidize exogenous reductants including benzidine and Mn²⁺. Using flash photolysis with electron spin resonance detection, we examined the room-temperature reaction kinetics of these reductants with Y_z ⁺, the tyrosine radical formed in PS II membranes under illumination. Kinetics were measured with membranes that did or did not contain the 33 kDa extrinsic polypeptide of PS II, whose presence had no effect on the reaction kinetics with either reductant. The rate of Y_z ⁺ reduction by benzidine was a linear function of benzidine concentration. The rate of Y_z ⁺ reduction by Mn²⁺ at pH 6 increased linearly at low Mn²⁺ concentrations and reached a maximum at the Mn²⁺ concentrations equal to several times the reaction center concentration. The rate was inhibited by K⁺, Ca²⁺ and Mg²⁺. These data are described by a model in which negative charge on the membrane causes a local increase in the cation concentration. The rate of Y_z ⁺ reduction at pH 7.5 was biphasic with a fast 400 s phase that suggests binding of Mn²⁺ near Y_z ⁺ at a site that may be one of the native manganese binding sites.Abbreviations PS II Photosystem II - Y_D tyrosine residue in Photosystem II that gives rise to the stable Signal II EPR spectrum - Y_z tyrosine residue in Photosystem II that mediates electron transfer between the reaction center chlorophyll and the site of water oxidation - ESR electron spin resonance - DPC diphenylcarbazide - DCIP dichlorophenolindophenol     

Energy transfer for low temperature fluorescence in PS II mutant thylakoids

The Chl-protein complexes of three maize (Zea mays L.) mutants and one barley (Hordeum vulgare L.) mutant were analyzed using low temperature Chl fluorescence emissions spectroscopy and LDS-polyacrylamide gel electrophoresis. The maize mutants hcf-3, hcf-19, and hcf-114 all exhibited a high Chl fluorescence (hcf) phenotype indicating a disruption of the energy transfer within the photosynthetic apparatus. The mutations in each of these maize mutants affects Photosystem II. The barley mutant analyzed was the well characterized Chl b-less mutant chlorina-f2, which did not exhibit the hcf phenotype. Chlorina-f2 was used because no complete Chl b-less mutant of maize is available. Analysis of hcf-3, hcf-19, and hcf-114 revealed that in the absence of CP43, LHC II can still transfer excitation energy to CP47. These results suggest that in mutant membranes LHC II can interact with CP47 as well as CP43. This functional interaction of LHC II with CP47 may only occur in the absence of CP43, however, it is possible that LHC II is positioned in the thylakoid membranes in a manner which allows association with both CP43 and CP47.Abbreviations hcf high chlorophyll fluorescence - LDS lithium dodecyl sulfate - LHC II light-harvesting complex of Photosystem II - LHC I light-harvesting complex of Photosystem I - CPIa chlorophyll-protein complex consisting of LHC I and the PS I core complex - CPI chlorophyll-protein complex consisting of the PS I core complex - CP47 47 kDa chlorophyll-protein of the Photosystem II core - CP43 43 kDa chlorophyll-protein of the Photosystem II core - CP29 29 kDa chlorophyll-protein of Photosystem II - CP26 26 kDa chlorophyll-protein of Photosystem II - CP24 24 kDa chlorophyll-protein of Photosystem II - fp free pigments     

Presence of a flavoprotein in O2-evolving Photosystem II preparations from the cyanobacterium Anacystis nidulans

A highly active O₂-evolving Photosystem II complex which was greatly depleted of phycobiliproteins was isolated from the cyanobacterium Anacystis nidulans. This complex contained the flavoprotein with l-amino acid oxidase activity which we have previously shown to be present in thylakoid preparations of this cyanobacterium (Pistorius, E.K. and Voss, H. (1982) Eur. J. Biochem. 126, 203–209). One of the most prominent polypeptides in this O₂-evolving Photosystem II complex had a molecular weight of 49 kDa. This polypeptide co-chromatographed on SDS-polyacrylamide gels with the purified l-amino acid oxidase which consists of two subunits of 49 kDa. The antagonistic effect of CaCl₂ on the two examined reactions could also be demonstrated with this O₂-evolving Photosystem II complex: CaCl₂ stimulated photosynthetic O₂ evolution, but inhibited the l-amino acid oxidase activity. Both reactions were inhibited by o-phenanthroline. These results further support a functional relationship between the flavoprotein with l-amino acid oxidase activity and Photosystem II activities in A. nidulans. However, we only found 1 mol FAD per 350–650 mol chlorophyll, although 1 gatom Mn per 5–10 mol chlorophyll was present. When we assume a photosynthetic unit of about 40 chlorophylls, then in most preparations the FAD values were more than a factor of 10 too low. Results which we obtained with the purified l-amino acid oxidase showed that the FAD values were in most enzyme samples lower than the theoretically expected value of 2 mol FAD per mol enzyme. Moreover, in some cases the absorption spectrum of the enzyme showed substantial deviations from the spectrum of oxidized FAD. These experiments indicated that the flavin in the enzyme could partly exist in a form which was different from ‘authentic oxidized FAD’. We do not yet know the chemical nature of this ‘modified flavin’.     

EPR spectroscopy of the manganese cluster of photosystem II

Electron paramagnetic resonance (EPR) spectroscopy is a valuable tool for understanding the oxidation state and chemical environment of the Mn₄Ca cluster of photosystem II. Since the discovery of the multiline signal from the S₂ state, EPR spectroscopy has continued to reveal details about the catalytic center of oxygen evolution. At present EPR signals from nearly all of the S-states of the Mn₄Ca cluster, as well as from modified and intermediate states, have been observed. This review article describes the various EPR signals obtained from the Mn₄Ca cluster, including the metalloradical signals due to interaction of the cluster with a nearby organic radical.     

Subunit composition of CP43-less photosystem II complexes of Synechocystis sp. PCC 6803: implications for the assembly and repair of photosystem II

Photosystem II (PSII) mutants are useful experimental tools to trap potential intermediates involved in the assembly of the oxygen-evolving PSII complex. Here, we focus on the subunit composition of the RC47 assembly complex that accumulates in a psbC null mutant of the cyanobacterium Synechocystis sp. PCC 6803 unable to make the CP43 apopolypeptide. By using native gel electrophoresis, we showed that RC47 is heterogeneous and mainly found as a monomer of 220 kDa. RC47 complexes co-purify with small Cab-like proteins (ScpC and/or ScpD) and with Psb28 and its homologue Psb28-2. Analysis of isolated His-tagged RC47 indicated the presence of D1, D2, the CP47 apopolypeptide, plus nine of the 13 low-molecular-mass (LMM) subunits found in the PSII holoenzyme, including PsbL, PsbM and PsbT, which lie at the interface between the two momomers in the dimeric holoenzyme. Not detected were the LMM subunits (PsbK, PsbZ, Psb30 and PsbJ) located in the vicinity of CP43 in the holoenzyme. The photochemical activity of isolated RC47-His complexes, including the rate of reduction of P680⁺, was similar to that of PSII complexes lacking the Mn₄CaO₅ cluster. The implications of our results for the assembly and repair of PSII in vivo are discussed.     

The sensitivity of Photosystem II to damage by UV-B radiation depends on the oxidation state of the water-splitting complex

The water-oxidizing complex of Photosystem II is an important target of ultraviolet-B (280-320 nm) radiation, but the mechanistic background of the UV-B induced damage is not well understood. Here we studied the UV-B sensitivity of Photosystem II in different oxidation states, called S-states of the water-oxidizing complex. Photosystem II centers of isolated spinach thylakoids were synchronized to different distributions of the S₀, S₁, S₂ and S₃ states by using packages of visible light flashes and were exposed to UV-B flashes from an excimer laser (λ = 308 nm). The loss of oxygen evolving activity showed that the extent of UV-B damage is S-state-dependent. Analysis of the data obtained from different synchronizing flash protocols indicated that the UV-sensitivity of Photosystem II is significantly higher in the S₃ and S₂ states than in the S₁ and S₀ states. The data are discussed in terms of a model where UV-B-induced inhibition of water oxidation is caused either by direct absorption within the catalytic manganese cluster or by damaging intermediates of the water oxidation process.     

A role of the C-terminal extension of the photosystem II D1 protein in sensitivity of the cyanobacterium Synechocystis PCC 6803 to photoinhibition.

The D1 protein, a key protein subunit of Photosystem II complex (PSII), is synthesised as a precursor (pD1) with a carboxyl-terminal extension. In the cyanobacterium Synechocystis sp. PCC 6803, this extension consists of 16 amino acid residues and it is cleaved by a specific protease in two putative steps with the final cleavage after the residue Ala344. In order to define the importance of the extension for the functioning of PSII, we constructed and characterized several site-directed mutants of Synechocystis that differ in the length and amino acid sequence of this extension. The mutant lacking the entire C-terminal extension exhibited slightly increased sensitivity to photoinhibition. Analysis of the PSII assembly in the mutant by the blue-native electrophoresis in combination with radioactive labelling revealed an increased level of the unassembled D1 protein in this strain. Replacement of the amino acid residue Asn359 by His or Asp also led to the higher vulnerability to photoinhibition of both mutants. In the Asn359His mutant, this vulnerability was accompanied by an increased level of the PSII core lacking CP43 indicating limitation of the repair cycle in the CP43 reassembly step.