Requirement of Manganese for Electron Donation of Hydrogen Peroxide in Photosystem II Reaction Center Complex

Mn²⁺ was required for the electron donating reaction from H₂O₂,but not for that from diphenylcarbazide (DPC), in the PS IIreaction center complex which was prepared from spinach chloroplastsby Triton X-100 extraction. The reaction center complex showeda high activity of 2,6-dichloroindophenol (DCIP) photoreductionin the presence of DPC, but a low activity with H₂O₂. The H₂O₂-supportedDCIP photoreduction was suppressed by EDTA and enhanced by asmall amount of Mn²⁺. Ca²⁺ and Mg²⁺ could not replace Mn²⁺.The activation by Mn²⁺ and its binding showed two binding sitesof Mn²⁺ in the reaction center complex, with high (1.5?10⁷ M^–1)and low (1 ? 10⁶ M^–1) binding constants. (Received November 8, 1986; Accepted April 10, 1987)     

Inhibition of the Hill Reaction by Tris and Restoration by Electron Donation to Photosystem II

Experiments in which chloroplasts were washed with tris and tricine buffers at different pH's indicated that the non-protonated (uncharged) form of tris was inhibitory to the Hill reaction while the protonated form of tris and the zwitterionic forms of tricine were non-inhibitory. Buffers analogous to tris and tricine gave similar results. Photoreduction of NADP could be restored to the inhibited chloroplasts by adding the reduced forms of p-hydroquinone, p-aminophenol, p-phenylenediamine, benzidine, semicarbazide, and dihydroxydiphenyl, all of which donated electrons to photosystem II. Photoreduction of ferricyanide was shown with those donor systems (benzidine and semicarbazide) which did not react chemically with ferricyanide. Photophosphorylation was also restored with all of the electron donors except semicarbazide.     

Electron donation in Photosystem II

The electron transfer resulting from illumination and dark storage of PS II has been studied using EPR signals from several electron carriers. The recombination of D⁺ (Signal II) and Q⁻_A formed by illumination occurred during dark storage at 77 K and was used to deplete reaction centres of D⁺. The donor D was then shown to be oxidized in the dark by the S₂ state of the oxygen-evolving complex. A slow change which occurred during dark storage of PS II samples was detected using the power saturation characteristics of D. We interpret this effect on D to be an indirect result of a rearrangement of the manganese complex during long-term dark adaptation. A role for D in the stability, protection and perhaps initial manganese binding of the oxygen-evolving complex is suggested.     

Electron microscopy in structural studies of Photosystem II

Various techniques of electron microscopy (EM) such as ultrathin sectioning, freeze-fracturing, freeze-etching, negative staining and (cryo-)electron crystallography of two-dimensional crystals have been employed, since now, to obtain much of the structural information of the Photosystem II (PS II) pigment–protein complex at both low and high resolution. This review summarizes information about the structure of this membrane complex as well as its arrangement and interactions with the antenna proteins in thylakoid membranes of higher plants and cyanobacteria obtained by means of EM. Results on subunit organization, with the emphasis on the proteins of the oxygen-evolving complex (OEC), are compared with the data obtained by X-ray crystallography of cyanobacterial PS II. This revised version was published online in August 2006 with corrections to the Cover Date.     

Mechanism of Electron Flow through the QB Site in Photosystem II. 1. Kinetics of the Reduction of Electron Acceptors at the QB and Plastoquinone Sites in Photosystem II Particles from the Cyanobacterium Synechococcus vulcanus

Various derivatives of benzoquinone (BQ) were found to be reducedat two sites [the Q_B and plastoquinone (PQ) sites] in photosystemII particles from Synechococcus vulcanus, and the relationshipbetween the structures of BQs and the kinetics of such reductionat each site were studied. Affinities of BQs for both the Q_B and the PQ site and the maximumturnover rates at the two sites were estimated by computer simulationof the dependence on the concentration of the BQ of the rateof oxygen evolution. Affinities of BQs for the Q_B sites determinedby this method agreed well with those determined from competitionbetween BQs and DCMU for the Q_B site. All methyl-substituted BQs had low affinities for the Q_B site,and tetramethyl-p-benzoquinone had almost no affinity. An increasein the number of chlorine atoms in the quinone ring increasedthe affinity, and the position of such substitutions had a greateffect on the affinity when two positions on the ring were occupiedby two chlorine atoms or methyl groups. The affinities of BQs for the PQ site were almost the same forall BQs tested in this experiment but the maximum turnover ratesat this site varied greatly from one derivative to another. The results are consistent with the hypothesis that the bindingof the PQ molecule to the Q_B site is attributable not to itsquinone ring but to its isoprenoid chain and, moreover, thatthe electron transfer through the Q_B site occurs not by replacementof the PQ molecule but by donation of electrons to another PQmolecule. (Received September 20, 1994; Accepted February 27, 1995)     

Models for Proton-Coupled Electron Transfer in Photosystem II

The coupling of proton and electron transfers is a key part of the chemistry of photosynthesis. The oxidative side of photosystem II (PS II) in particular seems to involve a number of proton-coupled electron transfer (PCET) steps in the S-state transitions. This mini-review presents an overview of recent studies of PCET model systems in the authors’ laboratory. PCET is defined as a chemical reaction involving concerted transfer of one electron and one proton. These are thus distinguished from stepwise pathways involving initial electron transfer (ET) or initial proton transfer (PT). Hydrogen atom transfer (HAT) reactions are one class of PCET, in which H⁺ and e ⁻ are transferred from one reagent to another: AH+B→A+BH, roughly along the same path. Rate constants for many HAT reactions are found to be well predicted by the thermochemistry of hydrogen transfer and by Marcus Theory. This includes organic HAT reactions and reactions of iron-tris(α-diimine) and manganese-(μ-oxo) complexes. In PS II, HAT has been proposed as the mechanism by which the tyrosine Z radical (Y_Z) oxidizes the manganese cluster (the oxygen evolving complex, OEC). Another class of PCET reactions involves transfer of H⁺ and e ⁻ in different directions, for instance when the proton and electron acceptors are different reagents, as in AH–B+C⁺→A–HB⁺+C. The oxidation of Y_Z by the chlorophyll P680 + has been suggested to occur by this mechanism. Models for this process – the oxidation of phenols with a pendent base – are described. The oxidation of the OEC by Y_Z could also occur by this second class of PCET reactions, involving an Mn–O–H fragment of the OEC. Initial attempts to model such a process using ruthenium-aquo complexes are described. An erratum to this article can be found at     

Hydrogen Peroxide Inhibits Photosynthetic Electron Transport in Cells of Cyanobacteria

The effect of H₂O₂ on photosynthetic O₂ evolution and photosynthetic electron transfer in cells of cyanobacteria Anabaena variabilis and Anacystis nidulans was studied. The following experiments were performed: 1) directly testing the effect of exogenous H₂O₂; 2) testing the effect of intracellular H₂O₂ generated with the use of methyl viologen (MV); 3) testing the effect of inhibiting intracellular H₂O₂ decomposition by salicylic acid (SA) and 3-amino-1,2,4-triazole (AT). H₂O₂ inhibited photosynthetic O₂ evolution and light-induced reduction of p-benzoquinone (BQ) + ferricyanide (FeCy) in the Hill reaction. The I₅₀ value for H₂O₂ was 0.75 mM. Photosynthetic electron transfer in the cells treated with H₂O₂ was not maintained by H₂O₂, NH₂OH, 1,5-diphenylcarbazide, tetraphenylboron, or butylated hydroxytoluene added as artificial electron donors for Photosystem (PS) II. The H₂O CO₂, H₂O MV (involving PSII and PSI) and H₂O BQ + FeCy (chiefly dependent on PSII) electron transfer reactions were inhibited upon incubation of the cells with MV, SA, or AT. The N,N,N",N"-tetramethyl-p-phenylenediamine MV (chiefly dependent on PSI) electron transfer was inhibited by SA and AT but was resistant to MV. The results show that H₂O₂ inhibits photosynthetic electron transfer. It is unlikely that H₂O₂ could be a physiological electron donor in oxygenic photosynthesis.     

Electron transfer in the water-oxidizing complex of Photosystem II

An overview is presented of secondary electron transfer at the electron donor side of Photosystem II, at which ultimately two water molecules are oxidized to molecular oxygen, and the central role of manganese in catalyzing this process is discussed. A powerful technique for the analysis of manganese redox changes in the water-oxidizing mechanism is the measurement of ultraviolet absorbance changes, induced by single-turnover light flashes on dark-adapted PS II preparations. Various interpretations of these ultraviolet absorbance changes have been proposed. Here it is shown that these changes are due to a single spectral component, which presumably is caused by the oxidation of Mn(III) to Mn(IV), and which oscillates with a sequence +1, +1, +1, –3 during the so-called S₀ S₁ S₂ S₃ S₀ redox transitions of the oxygen-evolving complex. This interpretation seems to be consistent with the results obtained with other techniques, such as those on the multiline EPR signal, the intervalence Mn(III)-Mn(IV) transition in the infrared, and EXAFS studies. The dark distribution of the S states and its modification by high pH and by the addition of low concentrations of certain water analogues are discussed. Finally, the patterns of proton release and of electrochromic absorbance changes, possibly reflecting the change of charge in the oxygen-evolving system, are discussed. It is concluded that nonstoichiometric patterns must be considered, and that the net electrical charge of the system probably is the highest in state S₂ and the lowest in state S₁.     

Two Plastoquinone A Molecules Are Required for Photosystem II Activity: Analysis in Hexane-Extracted Photosystem II Particles

Prenylquinones were extracted with hexane from lyophilized oxygen-evolvingphotosystem II particles prepared from spinach chloroplasts.Determination by high performance liquid chromatography showedthat two molecules of plastoquinone A remained per reactioncenter after the extraction, in contrast to the presence ofthree to four plastoquinone A molecules before the extraction.Electron transfer from water to phenyl-p-quinone was not inhibitedby the extraction. Measurement of EPR signal II and microsecondchlorophyll fluorescence kinetics showed that hexane did notextract quinones which were acting as the secondary electrondonor (Z) and the primary electron acceptor (Q_A) in photosystemII. These results, as well as the effect of quinone extractionon oxygen evolution, indicate that two molecules of plastoquinoneA acting as Z and Q_A are essential for the activity of photosystemII. An artificial donor phenyl-p-quinone probably accepts electronfrom Q_A at the same site as the intrinsic secondary electronacceptor (Q_B). Q_A and Z seem to be surrounded by special microenvironmentswhich differ from that of bulk quinones, and are resistant tohexane treatment. (Received November 27, 1984; Accepted April 30, 1985)     

Photosystem II Core Phosphorylation Heterogeneity, Differential Herbicide Binding, and Regulation of Electron Transfer in Photosystem II Preparations from Spinach

The effect of photosystem II core phosphorylation on the secondary quinone acceptor of photosystem II (Q_B) domain environment was analyzed by comparative herbicide-binding studies with photosystem II preparations from spinach (Spinacia oleracea L.). It was found that phosphorylation reduces the binding affinity for most photosynthetic herbicides. The binding of synthetic quinones and of the electron acceptor 2,6-dichlorophenolindophenol is also reduced by photosystem II phosphorylation. Four photosystem II core populations isolated from membranes showed different extents of phosphorylation as well as different degrees of affinity for photosynthetic herbicides. These findings support the idea that heterogeneity of photosystem II observed in vivo could be, in part, due to phosphorylation.     

Electron donation to Photosystem II in reaction center preparations

The room-temperature EPR characteristics of Photosystem II reaction center preparations from spinach, pokeweed and Chlamydomonas reinhardii have been investigated. In all preparations a light-induced increase in EPR Signal II, which arises from the oxidized form of a donor to P-680⁺, is observed. Spin quantitation, with potassium nitrosodisulfonate as a spin standard, demonstrates that the Signal II species, Z^?, is present in approx. 60% of the reaction centers. In response to a flash, the increase in Signal II spin concentration is complete within the 98 μs response time of our instrument. The decay of Z^? is dependent on the composition of the particle suspension medium and is accelerated by addition of either reducing agents or lipophilic anions in a process which is first order in these reagents. Comparison of these results with optical data reported previously (Diner, B.A. and Bowes, J.M. (1981) in Proceedings of the 5th International Congress on Photosynthesis (Akoyunoglou, G., ed.), Vol. 3, pp. 875–883, Balaban, Philadelphia), supports the identification of Z with the P-680⁺ donor, D₁. From the polypeptide composition of the particles used in this study, we conclude that Z is an integral component of the reaction center and use this conclusion to construct a model for the organization of Photosystem II.     

Three-Dimensional Electron Microscopy of the Photosystem II Core Complex

A three-dimensional image of the spinach photosystem II core complex composed of CP47, D1, D2, cytochromeb-559, andpsbI gene product was reconstructed at 20-Å resolution from the two-dimensional crystals negatively stained with phosphotungstate. Confirming the previous proposal, the crystal had ap22₁2₁symmetry. One PSII core complex was measured to be 80 × 80 Å in the membrane plane and 88 Å normal to it. The mass distribution was asymmetric about the lipid bilayer, consistent with predictions from the amino acid sequences. The lumenal mass consisted of three domains forming a characteristic triangular platform with another domain on top of it. Three stromal domains were smaller and linearly arranged. Due to strong stain exclusion in the hydrophobic core part of the lipid bilayer, the transmembrane region appeared to be imaged with a reversed contrast. Inverting the contrast resulted in a reasonable density distribution for that part. Thus, though the information on the transmembrane region is limited, the domain structure of the PSII core complex was revealed and allowed us to propose a model for the arrangement of subunits in the PSII core complex.     

Influence of Hyperthyroidism on Superoxide Radical and Hydrogen Peroxide Production by Rat Liver Submitochondrial Particles

Administration of daily doses of 0.1 mg of 3, 5, 3'-triiodothyronine (T₃)/kg body weight for 3 consecutive days to fed rats elicited a calorigenic response in the animals, in concomitance with a 36% increase in the rate of O₂ consumption by the liver. In these conditions, liver submitochondrial particles (SMP) from T₃-treated rats exhibited marked increases in the rate of superoxide radical generation, both in the presence of NADH (142%) or succinate (152%). Furthermore, liver SMP from hyperthyroid animals released hydrogen peroxide at higher rates than those of euthyroid rats, either under basal conditions or in the succinate-supported process, both in the absence and presence of antimycin-A. It is concluded that the hyperthyroid state in the rat leads to a drastic enhancement in the capacity of liver mitochondria to produce active oxygen species, which correlates with the elevated respiratory rate observed in the intact organ.     

Photophosphorylation Associated with Photosystem II: II. Effects of Electron Donors, Catalyst Oxidation, and Electron Transport Inhibitors on Photosystem II Cyclic Photophosphorylation

Incubation of KCN-Hg-NH₂OH-inhibited spinach (Spinacia oleracea L.) chloroplasts with p-phenylenediamine for 10 minutes in the dark prior to illumination produced rates of photosystem II cyclic photophosphorylation up to 2-fold greater than the rates obtained without incubation. Partial oxidation of p-phenylenediaine with ferricyanide produced a similar stimulation of ATP synthesis; addition of dithiothreitol suppressed the stimulation observed with incubation. Addition of ferricyanide in amounts sufficient to oxidize completely p-phenylenediamine failed to inhibit completely photosystem II cyclic activity. This is due at least in part to the fact that the ferrocyanide produced by oxidation of p-phenylenediamine is itself a catalyst of photosystem II cyclic photophosphorylation. N,N,N′N′-Tetramethyl-p-phenylenediamine catalyzes photosystem II cyclic photophosphorylation at rates approaching those observed with p-phenylenediamine. The activities of both proton/electron and electron donor catalysts of the photosystem II cycle are inhibited by dibromothyoquinone and antimycin A. These findings are interpreted to indicate that photosystem II cyclic photophosphorylation requires the operation of endogenous membrane-bound electron carriers for optimal coupling of ATP synthesis to electron transport.     

Manganese binding to the 23 kDa extrinsic protein of Photosystem II

The recombinant form of the extrinsic 23 kDa protein (psbP) of Photosystem II (PSII) was studied with respect to its capability to bind Mn. The stoichiometry was determined to be one manganese bound per protein. A very high binding constant, K(A)=10(-17) M(-1), was determined by dialysis of the Mn containing protein against increasing EDTA concentration. High Field EPR spectroscopy was used to distinguish between specific symmetrically ligated Mn(II) from those non-specifically Mn(II) attached to the protein surface. Upon Mn binding PsbP exhibited fluorescence emission with maxima at 415 and 435 nm when tryptophan residues were excited. The yield of this blue fluorescence was variable from sample to sample. It was likely that different conformational states of the protein were responsible for this variability. The importance of Mn binding to PsbP in the context of photoactivation of PSII is discussed.     

Manganese Peroxidase-Dependent Oxidation of Glyoxylic and Oxalic Acids Synthesized by Ceriporiopsis subvermispora Produces Extracellular Hydrogen Peroxide

The ligninolytic system of the basidiomycete Ceriporiopsis subvermispora is composed of manganese peroxidase (MnP) and laccase. In this work, the source of extracellular hydrogen peroxide required for MnP activity was investigated. Our attention was focused on the possibility that hydrogen peroxide might be generated by MnP itself through the oxidation of organic acids secreted by the fungus. Both oxalate and glyoxylate were found in the extracellular fluid of C. subvermispora cultures grown in chemically defined media, where MnP is also secreted. The in vivo oxidation of oxalate was measured; ¹⁴CO₂ evolution was monitored after addition of exogenous [¹⁴C]oxalate to cultures at constant specific activity. In standard cultures, evolution of CO₂ from oxalate was maximal at day 6, although the MnP titers were highest at day 12, the oxalate concentration was maximal (2.5 mM) at day 10, and the glyoxylate concentration was maximal (0.24 mM) at day 5. However, in cultures containing low nitrogen levels, in which the pH is more stable, a better correlation between MnP titers and mineralization of oxalate was observed. Both MnP activity and oxidation of [¹⁴C]oxalate were negligible in cultures lacking Mn(II). In vitro assays confirmed that Mn(II)-dependent oxidation of [¹⁴C]oxalate by MnP occurs and that this reaction is stimulated by glyoxylate at the concentrations found in cultures. In addition, both organic acids supported phenol red oxidation by MnP without added hydrogen peroxide, and glyoxylate was more reactive than oxalate in this reaction. Based on these results, a model is proposed for the extracellular production of hydrogen peroxide by C. subvermispora.     

Characteristics of the Photosystem II reaction centre. II. Electron donors

The properties of Photosystem II electron donation were investigated by EPR spectrometry at cryogenic temperatures. Using preparations from mutants which lacked Photosystem I, the main electron donor through the Photosystem II reaction centre to the quinone-iron acceptor was shown to be the component termed Signal II. A radical of 10 G line width observed as an electron donor at cryogenic temperatures under some conditions probably arises through modification of the normal pathway of electron donation. High-potential cytochrome b-559 was not observed on the main pathway of electron donation. Two types of PS II centres with identical EPR components but different electron-transport kinetics were identified, together with anomalies between preparations in the amount of Signal II compared to the quinone-iron acceptor. Results of experiments using cells from mutants of Scenedesmus obliquus confirm the involvement of the Signal II component, manganese and high-potential cytochrome b-559 in the physiological process leading to oxygen evolution.