Isao Enami

Is the primary cause of thermal inactivation of oxygen evolution in spinach PS II membranes release of the extrinsic 33 kDa protein or of Mn

Abstract Incubation of PS II membranes at 45–50°C for several min resulted in strong inactivation of oxygen evolution, concomitant with release of Mn and the extrinsic proteins of 33, 23 and 17 kDa. No correlation was found between loss of the activity and release of the 33 kDa protein or Mn. However, involvement of the protein release in the mechanism of heat-inactivation was suggested by stabilization of the activity against heat-treatment by immobilization of the 33 kDa protein with a water-soluble carbodiimide. Furthermore, a linear correlation was found between extents of heat-inactivation and amounts of the 33 kDa protein released in the presence of 50 mM CaCl2, which greatly accelerated inactivation of oxygen evolution, release of the 33 kDa protein and aggregation of PS II membranes at high temperatures. Evidence was obtained indicating that the 33 kDa protein released at high temperatures rebinds to its functional site when temperature is lowered but CaCl2 suppresses rebinding of the protein by promoting intensive aggregation of the membranes. Thus, the activity survived in the presence of CaCl2 is proportional to the amounts of the protein remained attached to the membranes during heat-treatment. By contrast, release of Mn was not affected by addition of CaCl2 so that enhanced inactivation of oxygen evolution was not accompanied by corresponding increase in the amount of Mn released. It is concluded, therefore, that the primary cause of heat-inactivation of oxygen evolution is dissociation of the 33 kDa protein but not that of Mn.

Plugging a Molecular Wire into Photosystem I: Reconstitution of the Photoelectric Conversion System on a Gold Electrode

Plug and play: Photoinduced electron transfer occurs from photoexcited P700 in photosystem I (PSI) to a gold surface (see picture). A novel molecular connector system is used, in which an artificial molecular wire, which is assembled on the gold surface, was plugged into PSI by reconstitution. Analysis of the photoelectron transfer kinetics proved both the output of electrons from PSI and the effectiveness of the molecular wire.

Structures and functions of the extrinsic proteins of photosystem II from different species

This minireview presents a summary of information available on the variety and binding properties of extrinsic proteins that form the oxygen-evolving complex of photosystem II (PSII) of cyanobacteria, red alga, diatom, green alga, euglena, and higher plants. In addition, the structure and function of extrinsic PsbO, PsbV, and PsbU proteins are summarized based on the crystal structure of thermophilic cyanobacterial PSII together with biochemical and genetic studies from various organisms.

Isolation and characterization of a Photosystem II complex from the red alga Cyanidium caldarium: association of cytochrome c-550 and a 12 kDa protein with the complex.

A Photosystem II (PS II) complex was purified from an acidophilic as well as a thermophilic red alga, Cyanidium caldarium. The purified PS II complex was essentially devoid of phycobiliproteins and other contaminating components, and showed a high oxygen-evolving activity of 2375 mumol O2/mg Chl per h using phenyl-p-benzoquinone as the electron acceptor. The expression of this high activity did not require addition of exogenous Ca2+, although EDTA reduced the activity by 40%. This effect of EDTA can be reversed not only by Ca2+ but also by Mg2+; a similar Mg2+ effect has been observed in purified cyanobacterial PS II but not in higher plant PS II. Immunoblotting analysis indicated the presence of major intrinsic polypeptides commonly found in PS II from cyanobacteria and higher plants as well as the extrinsic 33 kDa protein. Antibodies against the extrinsic 23 and 17 kDa proteins of higher plant PS II, however, did not crossreact with any polypeptides in the purified PS II, indicating the absence of these proteins in the red alga. In contrast, two other extrinsic proteins of 17 and 12 kDa were present in the red algal PS II; they were released by 1 M Tris or Urea/NaCl treatment but not by 1 M NaCl. The 17 kDa polypeptide was identified to be cytochrome c-550 from heme-staining, immunoblot analysis and N-terminal amino acid sequencing, and the 12 kDa protein was found to be homologous to the 12 kDa extrinsic protein of cyanobacterial PS II from its N-terminal sequence. These results indicate that PS II from the red alga is closely related to PS II from cyanobacteria rather than to that from higher plants, and that the replacement of PS II extrinsic cytochrome c-550 and the 12 kDa protein by the extrinsic 23 and 17 kDa proteins occurred during evolution from red algae to green algae and higher plants.

The mechanism of the degradation of psaB gene product, one of the photosynthetic reaction center subunits of photosystem I, upon photoinhibition

The psaB gene product (PsaB protein), one of the reaction center subunits of Photosystem I (PS I), was specifically degraded by light illumination of spinach thylakoid membranes. The degradation of the protein yielded N-terminal fragments of molecular mass 51 kDa and 45 kDa. The formation of the 51 kDa fragment was i) partially suppressed by the addition of phenylmethylsulfonyl fluoride or 3,4-dichloroisocoumarin, which are inhibitors of serine proteases, and ii) enhanced in the presence of hydrogen peroxide during photoinhibitory treatment, but iii) not detected following hydrogen peroxide treatment in the dark. These results suggest that the hydroxyl radical produced at the reduced iron-sulfur centers in PS I triggers the conformational change of the PS I complex, which allows access of a serine-type protease to PsaB. This results in the formation of the 51 kDa N-terminal fragment, presumably by cleavage on the loop exposed to the stromal side, between putative helices 8 and 9. On the other hand, the formation of the 45 kDa fragment, which was enhanced in the presence of methyl viologen but did not accompany the photoinhibition of PS I, was not affected by the addition of hydrogen peroxide or protease inhibitors. Another fragment of 18 kDa was identified as a C-terminal counterpart of the 45 kDa fragment. N-terminal sequence analysis of the 18 kDa fragment revealed that the cleavage occurred between Ala500 and Val501 on the loop exposed to the lumenal side, between putative helices 7 and 8 of the PsaB protein.

Isolation and characterization of Photosystem II complexes which lack light-harvesting chlorophyll a/b proteins but retain three extrinsic proteins related to oxygen evolution from spinach

Oxygen-evolving Photosystem II (PS II) complexes, which were largely deprived of major light-harvesting chlorophyll a/b proteins (LHC II) but still associated with the 33 kDa, 23 kDa and 17 kDa extrinsic proteins related to oxygen evolution, were isolated from spinach oxygen-evolving PS II membranes with a non-ionic detergent, n-heptyl thioglucoside. A minor antenna chlorophyll-protein (CP 29) was present but in reducted amounts. The complexes contained all the constituent subunits of PS II reaction center core complexes, the 47 kDa and 43 kDa chlorophyll-carrying proteins, the D1 and D2 proteins and cytochrome b-559. In addition, three hydrophobic proteins of 29 kDa (CP 29 apoprotein), 20 kDa and 10 kDa were present. The antenna size was 80 chlorophyll a per QA, or 76 chlorophyll a per 4 Mn, and the complexes contained about 1 Ca2+ per PS II. With phenyl- or dichloro-p-benzoquinone as electron acceptor, the complexes showed high rates of oxygen evolution in the absence of exogenously added Ca2+. The activity became, however, strongly Ca2+-dependent when the 23 kDa and 17 kDa proteins, but not the bound Ca2+, had been removed with 1.5 M NaCl. The Ca2+ requirement disappeared on reconstitution of the complexes with the two proteins. The complexes were compared with other oxygen-evolving preparations having different polypeptide compositions and functions of several subunit proteins and Ca2+ in PS II electron transport are discussed.

Total immobilization of the extrinsic 33 kDa protein in spinach Photosystem II membrane preparations. Protein stoichiometry and stabilization of oxygen evolution

(1) Treatment of oxygen-evolving Photosystem II membrane fragments (PS II membranes) with a zero-length crosslinker, l-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) led to immobilization of all the extrinsic 33 kDa protein molecules without any significant effects on the oxygen-evolving activity and oscillation patterns of flash-induced oxygen evolution and thermoluminescence B band. (2) With increasing concentration of EDC, the chlorophyll-binding 47 kDa protein decreased in parallel with the 33 kDa protein, yielding a crosslinked product consisting of one each of the two proteins. The results, which indicate that the two proteins are present in equimolar amounts in PS II membranes, are consistent with the stoichiometry of one copy of the 33 kDa protein per PS II unit. (3) The total immobilization of the 33 kDa protein stabilized 40 to 60% of the oxygen-evolving activity against urea/NaCl−, CaCl2− and MgCl2-wash, which otherwise solubilize the three extrinsic proteins and strongly inactivate oxygen evolution. The result implies that extraction of the extrinsic proteins may not be the sole cause of the inactivation of oxygen evolution by these treatments. (4) The crosslinking of the 33 kDa protein with EDC had no protecting effect against Tris-, NH2OH- and pH 9.0-treatments. However, the stability of oxygen evolution at alkaline pH levels was slightly but significantly increased by treatment of PS II membranes with dithiobis(suc-cinimidylpropionate), which specifically modifies amino groups.

Crosslinking between the 33 kDa extrinsic protein and the 47 kDa chlorophyll‐carrying protein of the PS II reaction center core complex

Using highly purified oxygen‐evolving photosystem II complexes from spinach, the 33 kDa extrinsic protein was found to crosslink with the 47 kDa chlorophyll‐carrying protein with a cleavable bifunctional crosslinking reagent, dithiobis(succinimidylpropionate).

Nearest neighbor relationships among constituent proteins of oxygen-evolving Photosystem II membranes: binding and function of the extrinsic 33 kDa protein

Nearest neighbor relationships among constituent proteins of oxygen-evolving Photosystem II (PS II) membrane preparations from spinach were investigated by means of crosslinking with a cleavable bifunctional crosslinker, dithiobis(succinimidylpropionate) (DSP). (1) Diagonal gel electrophoresis revealed crosslinking between two extrinsic proteins of 17 and 23 kDa, between the extrinsic 33 kDa protein and the 47 kDa chlorophyll-carrying protein and between the 26 and 27 kDa apoproteins of light-harvesting chlorophyll a/b protein. In addition, a product which involved a protein of 29 kDa was detected. (2) Amounts of the extrinsic proteins crosslinked were determined by washing DSP-treated membranes with high concentrations of urea and NaCl, or CaCl 2 . Neither of the two extrinsic proteins of 17 and 23 kDa was crosslinked with intrinsic membrane proteins, whereas 15 to 20% of the 33 kDa protein was immobilized by treatment with 0.1% DSP. Oxygen evolution became resistant to the urea/NaCl-wash proportionally to the amount of the 33 kDa protein crosslinked, indicating that the 33 kDa protein covalently bound to the 47 kDa protein is still fully functional. (3) The crosslinking of the 33 kDa protein accompanied by parallel increases in the amount of Mn remained unextracted, and the rate of oxygen evolution survived after 30-min treatment of PS IImembranes at pH 9.0. Thus the 33 kDa protein has a protective effect on the Mn cluster at the alkaline pH. In contrast, Mn was mostly extracted with a high concentration of Tris, irrespective of the crosslinking of the 33 kDa protein.

Distribution of the extrinsic proteins as a potential marker for the evolution of photosynthetic oxygen-evolving photosystem II

Distribution of photosystem II (PSII) extrinsic proteins was examined using antibodies raised against various extrinsic proteins from different sources. The results showed that a glaucophyte (Cyanophora paradoxa) having the most primitive plastids contained the cyanobacterial‐type extrinsic proteins (PsbO, PsbV, PsbU), and the primitive red algae (Cyanidium caldarium) contained the red algal‐type extrinsic proteins (PsO, PsbQ′, PsbV, PsbU), whereas a prasinophyte (Pyraminonas parkeae), which is one of the most primitive green algae, contained the green algal‐type ones (PsbO, PsbP, PsbQ). These suggest that the extrinsic proteins had been diverged into cyanobacterial‐, red algal‐ and green algal‐types during early phases of evolution after a primary endosymbiosis. This study also showed that a haptophyte, diatoms and brown algae, which resulted from red algal secondary endosymbiosis, contained the red algal‐type, whereas Euglena gracilis resulted from green algal secondary endosymbiosis contained the green algal‐type extrinsic proteins, suggesting that the red algal‐ and green algal‐type extrinsic proteins have been retained unchanged in the different lines of organisms following the secondary endosymbiosis. Based on these immunological analyses, together with the current genome data, the evolution of photosynthetic oxygen‐evolving PSII was discussed from a view of distribution of the extrinsic proteins, and a new model for the evolution of the PSII extrinsic proteins was proposed.