Partial degradation of the extrinsic 23-kDa protein of the Photosystem II complex of spinach

Chymotrypsin eliminated nine amino acid residues at the amino-terminal side of the extrinsic 23-kDa protein of the oxygen-evolving Photosystem II complex of spinach. The resultant 22-kDa fragment was able to bind to the Photosystem II complex but with lowered binding affinity. However, once the 22-kDa fragment bound to the complex, it retained most functions of the 23-kDa protein; the fragment provided a binding site for the extrinsic 18-kDa protein, preserved a tight trap for Ca²⁺ in the complex, and shifted the optimum Cl⁻ concentration for oxygen evolution from 30 to 10 mM, although it was less effective in sustaining oxygen evolution at Cl⁻ concentrations below 10 mM. These observations suggest that the elimination of nine amino acid residues at the amino-terminal region of the 23-kDa protein does not significantly alter the conformation of the protein, except for partial modification of its binding site and its interaction with Cl⁻.     

Inhibition of the oxygen-evolving complex of photosystem II and depletion of extrinsic polypeptides by nickel

The toxic effect of Ni²⁺ on photosynthetic electron transport was studied in a photosystem II submembrane fraction. It was shown that Ni²⁺ strongly inhibits oxygen evolution in the millimolar range of concentration. The inhibition was insensitive to NaCl but significantly decreased in the presence of CaCl₂. Maximal chlorophyll fluorescence, together with variable fluorescence, maximal quantum yield of photosystem II, and flash-induced fluorescence decays were all significantly declined by Ni²⁺. Further, the extrinsic polypeptides of 16 and 24 kDa associated with the oxygen-evolving complex of photosystem II were depleted following Ni²⁺ treatment. It was deduced that interaction of Ni²⁺ with these polypeptides caused a conformational change that induced their release together with Ca²⁺ from the oxygen-evolving complex of photosystem II with consequent inhibition of the electron transport activity.     

The extrinsic proteins of Photosystem II

Years of genetic, biochemical, and structural work have provided a number of insights into the oxygen evolving complex (OEC) of Photosystem II (PSII) for a variety of photosynthetic organisms. However, questions still remain about the functions and interactions among the various subunits that make up the OEC. After a brief introduction to the individual subunits Psb27, PsbP, PsbQ, PsbR, PsbU, and PsbV, a current picture of the OEC as a whole in cyanobacteria, red algae, green algae, and higher plants will be presented. Additionally, the role that these proteins play in the dynamic life cycle of PSII will be discussed.     

Requirements for different combinations of the extrinsic proteins in specific cyanobacterial Photosystem II mutants

The crystallographic data available for Photosystem II (PS II) in cyanobacteria has now provided complete structures for loop E from CP43 and CP47 as well as the extrinsic subunits PsbO, PsbU and PsbV. Protein interactions between these subunits are essential for stable water splitting and there is evidence that the binding of PsbU facilitates optimal energy transfer from the phycobilisome. Interactions between PsbO and CP47 may also play a role in dimer stabilization while loop E of CP43 contributes directly to the water-splitting reaction. Recent evidence also suggests that homologs of PsbP and PsbQ play key roles in cyanobacterial PS II, and under nutrient-deficient conditions PsbQ appears essential for photoautotrophic growth.     

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

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

The structure and function of the 33 kDa extrinsic protein of Photosystem II: A critical assessment

In this review the structure and function of the 33 kDa protein of Photosystem II is examined. Significant controversies exist concerning the solution secondary structure of the protein, the location of its binding site(s) within Photosystem II, the amino acid residues of the 33 kDa protein required for binding and its stoichiometry within the photosystem. The studies which examine these topics are considered from a critical perspective. A hypothetical model of the folding of the 33 kDa extrinsic protein which is supported by site-specific labeling studies and site-directed mutagenesis experiments is presented. Additionally, the function of the protein within the photosystem is unclear. We present a hypothesis that the 33 kDa protein is involved in maintaining the chloride associated with photosynthetic oxygen evolution in close proximity to the oxygen-evolving site.     

On the role of the N-terminus of the extrinsic 33 kDa protein of Photosystem II

The role of the N-terminus of the extrinsic 33 kDa protein of Photosystem II has been investigated by means of site-directed mutagenesis and cross-linking. Replacement of Asp-9 resulted in a dramatic increase in proteolytic sensitivity leading to the degradation of the protein forming a 31 kDa fragment with an undefined N-terminus. This fragment was unable to restore oxygen evolution. However, the variants of the 33 kDa protein which remained intact could reconstitute oxygen evolution as effectively as the wild-type protein. Cross-linking experiments with a water-soluble carbodiimide revealed that mutagenesis of residue D9 led to the disruption of an intramolecular salt bridge. Therefore we suggest that the N-terminus of the 33 kDa protein is necessary for maintaining the binding ability of the protein to Photosystem II but might not be involved in binding itself.     

Quantitative analysis of membrane components in a highly active O2-evolving photosystem II preparation from spinach chloroplasts

Stoichiometry of membrane components associated with Photosystem II was determined in a highly active O₂-evolving Photosystem II preparation isolated from spinach chloroplasts by the treatment with digitonin and Triton X-100. From the analysis with sodium dodecyl sulfate polyacrylamide gel electrophoresis and Triton X-114 phase partitioning, the preparation was shown to contain the reaction center protein (43 kDa), the light-harvesting chlorophyll-protein complex (the main band, 27 kDa), the herbicide-binding protein (32 kDa) and cytochrome b-559 (10 kDa) as hydrophobic proteins, and three proteins (33, 24 and 18 kDa) which probably constitute the O₂-evolution enzyme complex as hydrophilic proteins. These proteins were associated stoichiometrically with the Photosystem II reaction center: one Photosystem II reaction center, approx. 200 chlorophyll, one high-potential form of cytochrome b-559, one low-potential form of cytochrome b-559, one 33 kDa protein, one (to two) 24 kDa protein and one (to two) 18 kDa protein. Measurement of fluorescence induction showed the presence of three electron equivalents in the electron acceptor pool on the reducing side of Photosystem II in our preparation. Three molecules of plastoquinone A were detected per 200 chlorophyll molecules with high-performance liquid chromatography. The Photosystem II preparation contained four managanese atoms per 200 chlorophyll molecules.     

Molecular mechanism of action of Pb and Zn on water oxidizing complex of photosystem II

Pb²⁺ and Zn²⁺ inhibition of photosystem II (PSII) activity was reported to be mediated via displacement of native inorganic cofactors (Cl⁻, Ca²⁺ and Mn²⁺) from the oxygen evolving complex, OEC [Rashid and Popovic (1990) FEBS Lett. 271, 181–184; Rashid et al. (1991) Photosynth. Res. 30, 123–130]. Since the binding sites of these cofactors are protected by a shield of three extrinsic polypeptides (17, 23 and 33 kDa), we investigated whether these metal ions affect the extrinsic polypeptide shield of OEC. By immunoblotting with antibodies recognizing the 23 and 33 kDa polypeptides, we showed that both the metal ions significantly dissociated the 23 kDa (+17 kDa) polypeptide, and partially dissociated the 33 kDa. Ca²⁺, one of the important inorganic cofactors of oxygen evolution, strongly prevented the dissociating action of Pb²⁺ but did not prevent the action of Zn²⁺. The probable molecular mechanism of action of Pb²⁺ and Zn²⁺ on PSII OEC is discussed.     

Kinetics of the oxygen-evolving complex in salt-washed photosystem II preparations

The kinetics of flash-induced electron transport were investigated in oxygen-evolving Photosystem II preparations, depleted of the 23 and 17 kDa polypeptides by washing with 2 M NaCl. After dark-adaptation and addition of the electron acceptor 2,5-dichloro-p-benzoquinone, in such preparations approx. 75% of the reaction centers still exhibited a period 4 oscillation in the absorbance changes of the oxygen-evolving complex at 350 nm. In comparison to the control preparations, three main effects of NaCl-washing could be observed: the half-time of the oxygen-evolving reaction was slowed down to about 5 ms, the misses and double hits parameters of the period 4 oscillation had changed, and the two-electron gating mechanism of the acceptor side could not be detected anymore. EPR-measurements on the oxidized secondary donor Z⁺ confirmed the slower kinetics of the oxygen-releasing reaction. These phenomena could not be restored by readdition of the released polypeptides nor by the addition of CaCl₂, and are ascribed to deleterious action of the highly concentrated NaCl. Otherwise, the functional coupling of Photosystem II and the oxygen-evolving complex was intact in the majority of the reaction centers. Repetitive flash measurements, however, revealed P⁺Q⁻ recombination and a slow Z⁺ decay in a considerable fraction of the centers. The flash-number dependency of the recombination indicated that this reaction only appeared after prolonged illumination, and disappeared again after the addition of 20 mM CaCl₂. These results are interpreted as a light-induced release of strongly bound Ca²⁺ in the salt-washed preparations, resulting in uncoupling of the oxygen-evolving system and the Photosystem II reaction center, which can be reversed by the addition of a relatively high concentration of Ca²⁺.     

D1 protein of photosystem II: The light sensor in chloroplasts

Light, controls the “blueprint” for chloroplast development, but at high intensities is toxic to the chloroplast. Excessive light intensities inhibit primarily photosystem II electron transport. This results in generation of toxic singlet oxygen due to impairment of electron transport on the acceptor side between pheophytin and Q_B -the secondary electron acceptor. High light stress also impairs electron transport on the donor side of photosystem II generating highly oxidizing species Z⁺ and P680⁺. A conformationsl change in the photosystem II reaction centre protein Dl affecting its Q_B-binding site is involved in turning the damaged protein into a substrate for proteolysis. The evidence indicates that the degradation of D1 is an enzymatic process and the protease that degrades D1 protein has been shown to be a serine protease Although there is evidence to indicate that the chlorophyll a-protein complex CP43 acts as a serine-type protease degrading Dl, the observed degradation of Dl protein in photosystem II reaction centre particlesin vitro argues against the involvement of CP43 in Dl degradation. Besides the degradation during high light stress of Dl, and to a lesser extent D2-the other reaction centre protein, CP43 and CP29 have also been shown to undergo degradation. In an oxygenic environment, Dl is cleaved from its N-and C-termini and the disassembly of the photosystem II complex involves simultaneous release of manganese and three extrinsic proteins involved in oxygen evolution. It is known that protein with PEST sequences are subject to degradation; D1 protein contains a PEST sequence adjacent to the site of cleavage on the outer side of thylakoid membrane between helices IV and V. The molecular processes of “triggering” of Dl for proteolytic degradation are not clearly understood. The changes in structural organization of photosystem II due to generation of oxy-radicals and other highly oxidizing species have also not been resolved. Whether CP43 or a component of the photosystem II reaction centre itself (Dl. D2 or cy1 b559 subunits), which may be responsible for degradation of Dl, is also subject to light modification to become an active protease, is also not known. The identity of proteases degrading Dl, LHCII and CP43 and C29 remains to be established     

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

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

Precursor of the nuclear-encoded extrinsic 30 kDa protein in photosystem II of Euglena gracilis Z is not a polyprotein

Polyprotein-type precursors have been reported for the nuclear-encoded proteins such as the small subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) and the apoproteins of light-harvesting chlorophyll-protein (LHC) in Euglena. We report here that the precursor of the extrinsic 30 kDa protein of photosystem II (PS II) encoded by nuclear DNA is not a polyprotein. The precursor was identified as a 45 kDa protein by immunoprecipitation of in vitro translation products of mRNA and by a pulse-chase experiment. It is probable that the structure of the precursor of the nuclear-encoded protein in Euglena chloroplast is closely related to the feature of assembly, as well as of transport, of the protein in chloroplast.     

Structural predictions on the 33 kDa extrinsic protein associated to the oxygen evolving complex of photosynthetic organisms

Modern computational methods for protein structure prediction have been used to study the structure of the 33 kDa extrinsic membrane protein, associated to the oxygen evolving complex of photosynthetic organisms. A multiple alignment of 14 sequences of this protein from cyanobacteria, algae and plants is presented. The alignment allows the identification of fully conserved residues and the recognition of one deletion and one insertion present in the plant sequences but not in cyanobacteria. A tree of similarity, deduced from pair-wise comparison and cluster analysis of the sequences, is also presented. The alignment and the consensus sequence derived are used for prediction the secondary structure of the protein. This prediction indicates that it is a mainly-beta protein (25–38% of -strands) with no more than 4% of -helix. Fold recognition by threading is applied to obtain a topological 2D model of the protein. In this model the secondary structure elements are located, including several highly conserved loops. Some of these conserved loops are suggested to be important for the binding of the 33 kDa protein to Photosystem II and for the stability of the manganese cluster. These structural predictions are in good agreement with experimental data reported by several authors.     

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.     

Inhibition of photosystem II photochemistry by Cr is caused by the alteration of both D1 protein and oxygen evolving complex

The effect of chromium (Cr) on photosystem II (PSII) electron transport and the change of proteins content within PSII complex were investigated. When Lemna gibba was exposed to Cr during 96 h, growth inhibition was found to be associated with an alteration of the PSII electron transport at both PSII oxidizing and reducing sides. Investigation of fluorescence yields at transients K, J, I, and P suggested for Cr inhibitory effect to be located at the oxygen-evolving complex and Q_A reduction. Those Cr-inhibitory effects were related to the change of the turnover of PSII D1 protein and the alteration of 24 and 33 kDa proteins of the oxygen-evolving complex. The inhibition of the PSII electron transport and the formation of reactive oxygen species induced by Cr were highly correlated with the decrease in the content of D1 protein and the amount of 24 and 33 kDa proteins. Therefore, functional alteration of PSII activity by Cr was closely related with the structural change within PSII complex.     

The carboxyl-terminal processing of precursor D1 protein of the photosystem II reaction center

The D1 protein, a key subunit of photosystem II reaction center, is synthesized as a precursor form with a carboxyl-terminal extension, in oxygenic photosynthetic organisms with some exceptions. This part of the protein is removed by the action of an endopeptidase, and the proteolytic processing is indispensable for the manifestation of oxygen-evolving activity in photosynthesis. The carboxyl-terminus of mature D1 protein, which appears upon the cleavage, has recently been demonstrated to be a ligand for a manganese atom in the Mn₄Ca-cluster, which is responsible for the water oxidation chemistry in photosystem II, based on the isotope-edited Fourier transform infrared spectroscopy and the X-ray crystallography. On the other hand, the structure of a peptidase involved in the cleavage of precursor D1 protein has been resolved at a higher resolution, and the enzyme–substrate interactions have extensively been analyzed both in vivo and in vitro. The present article briefly summarizes the history of research and the present state of our knowledge on the carboxyl-terminal processing of precursor D1 protein in the photosystem II reaction center.     

The structure and formation of host-parasite pit connections between the red algal alloparasiteHarveyella mirabilis and its red algal hostOdonthalia floccosa

Summary Harveyella mirabilis is a colourless red algal alloparasite which grows on and within its photosynthetic hostOdonthalia floccosa. Cells ofHarveyella establish secondary pit connections (PCs) with other parasite cells and with cells of the host. Small, uninucleate conjunctor cells are produced by parasite cells and remain connected to them by PCs. Conjunctor cells may fuse with either an adjacent host or parasite cell, with the parasite-conjunctor cell PC becoming either a host-parasite or parasite-parasite secondary PC. Occasionally the conjunctor cell does not fuse with an adjacent cell (either host or parasite) and degenerates. The secondary pit plug which forms between a parasite cell and its conjunctor cell always develops with two structurally distinct surfaces characteristic of a host-parasite pit plug. Only if the conjunctor cell fuses with another parasite cell will the structure of the pit plug be altered to that of a parasite-parasite pit plug. Fungal hyphae also invade the region of infection, andHarveyella cells respond by producing nonfunctional conjunctor cells that grow towards adjacent hyphae. Evidence suggests that secondary PCs may be induced to form mechanically, by the physical presence of another cell, rather than in direct response to a message received from an adjacent cell. The mechanism of secondary PC formation described here is similar to that reported for the closely related alloparasiteHolmsella and may be common to a number of red algal parasitic associations. Helen Margaret Quirk, B. Sc. (Hons), M. Sc. (1953–1982), student, research assistant and friend, died after a long illness on October 24, 1982.     

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.