Takahiro Henmi

Turnover of the aggregates and cross-linked products of the D1 protein generated by acceptor-side photoinhibition of photosystem II

It is known that the reaction-center binding protein D1 in photosystem (PS) II is degraded significantly during photoinhibition. The D1 protein also cross-links covalently or aggregates non-covalently with the nearby polypeptides in PS II complexes by illumination. In the present study, we detected the adducts between the D1 protein and the other reaction-center binding protein D2 (D1/D2), the alpha-subunit of cyt b(559) (D1/cyt b(559)), and the antenna chlorophyll-binding protein CP43 (D1/CP43) by SDS/urea-polyacrylamide gel electrophoresis and Western blotting with specific antibodies. The adducts were observed by weak and strong illumination (light intensity: 50-5000 microE m(-2) s(-1)) of PS II membranes, thylakoids and intact chloroplasts from spinach, under aerobic conditions. These results indicate that the cross-linking or aggregation of the D1 protein is a general phenomenon which occurs in vivo as well as in vitro with photodamaged D1 proteins. We found that the formation of the D1/D2, D1/cyt b(559) and D1/CP43 adducts is differently dependent on the light intensity; the D1/D2 heterodimers and D1/cyt b(559) were formed even by illumination with weak light, whereas generation of the D1/CP43 aggregates required strong illumination. We also detected that these D1 adducts were efficiently removed by the addition of stromal components, which may contain proteases, molecular chaperones and the associated proteins. By two-dimensional SDS/urea-polyacrylamide gel electrophoresis, we found that several stromal proteins, including a 15-kDa protein are effective in removing the D1/CP43 aggregates, and that their activity is resistant to SDS.

Towards structural elucidation of eukaryotic photosystem II: Purification, crystallization and preliminary X-ray diffraction analysis of photosystem II from a red alga

Crystal structure of photosystem II (PSII) has been reported from prokaryotic cyanobacteria but not from any eukaryotes. In the present study, we improved the purification procedure of PSII dimers from an acidophilic, thermophilic red alga Cyanidium caldarium, and crystallized them in two forms under different crystallization conditions. One had a space group of P222(1) with unit cell constants of a=146.8 A, b=176.9 A, and c=353.7 A, and the other one had a space group of P2(1)2(1)2(1) with unit cell constants of a=209.2 A, b=237.5 A, and c=299.8 A. The unit cell constants of both crystals and the space group of the first-type crystals are different from those of cyanobacterial crystals, which may reflect the structural differences between the red algal and cyanobacterial PSII, as the former contains a fourth extrinsic protein of 20 kDa. X-ray diffraction data were collected and processed to a 3.8 A resolution with the first type crystal. For the second type crystal, a post-crystallization treatment of dehydration was employed to improve the resolution, resulting in a diffraction data of 3.5 A resolution. Analysis of this type of crystal revealed that there are 2 PSII dimers in each asymmetric unit, giving rise to 16 PSII monomers in each unit cell, which contrasts to 4 dimers per unit cell in cyanobacterial crystals. The molecular packing of PSII within the unit cell was constructed with the molecular replacement method and compared with that of the cyanobacterial crystals.

Characterization of the stromal protease(s) degrading the cross-linked products of the D1 protein generated by photoinhibition of photosystem II

When photosystem (PS) II-enriched membranes are exposed to strong light, cross-linking of the intrinsic D1 protein with the surrounding polypeptides and degradation of the D1 protein take place. The cross-linking of the D1 protein with the alpha-subunit of cytochrome b(559) is suggested to be an early event of photoinduced damage to the D1 protein (Barbato et al., FEBS Lett. 309 (1992) 165-169). The relationship between the cross-linking and the degradation of the D1 protein, however, is not yet clear. In the present study, we show that the addition of stromal extract from chloroplasts degrades the 41 kDa cross-linked product of D1/cytochrome b(559) alpha-subunit and enhances the degradation of the D1 protein. Incubation of the preilluminated PS II-enriched membranes with the stromal extract at 25 degrees C causes the degradation of the cross-linked product by more than 70%. The activity of the stromal extract showed a pH optimum at 8.0, and was enhanced by the addition of ATP or GTP. Consistent with the nucleotide effect, this stromal activity was eliminated by the preincubation of the stromal extract with apyrase, which hydrolyzes nucleotides. Also, the stromal activity was nearly fully inhibited by a serine-type protease inhibitor, 3,4-dichloroisocoumarin, which suggests participation of a serine-type protease(s).

X-ray crystallographic and biochemical characterizations of a mutant photosystem II complex from Thermosynechococcus vulcanus with the psbTc gene inactivated by an insertion mutation.

The characterization of a PsbTc-truncated mutant photosystem II complex is described.

Crystallization and Crystal Structure Analysis of a Mutant Photosystem II Complex Lacking PsbI from Thermosynechococcus vulcanus

Psbi Is One Of The Low Molecular Mass, Intrinsic Membrane Protein Of Photosystem Ii (Psii) Tightly Associated With The Isolated D1/D2 Reaction Center Complex. The Location Of Psbi Has Been Identified To Be In A Peripheral Area Of Psii Dimer Surrounded By D1 And Cp43 In The Crystal Structure Of A Thermophilic Cyanobacterium At 3 aResolution (Loll Et Al. 2005). In Order To Study The Role Of Psbi From A Structural Point Of View, We Purified Psii Dimer Complexes From A Psbi-Deletion Mutant Of Thermosynechococcus Vulcanus, Crystallized, And Analyzed The Crystal Structure Of The Psbi-Deleted Psii. Our Results Confirmed The Position Of Psbi In The Current Structure, And Showed That The Isolated Mutant Psii Had A Maximum Decrease Of 30% In Oxygen Evolution Compared With That Of Wild-Type Psii. The Amount Of Psii Monomer Was Found To Increase Significantly In Thylakoids Of The Psbideletion Mutant Than That Of Wild-Type, In Agreement With The Previously Reported Results That Psbi Is Required For Maintaining The Stability Of Psii Dimer In Vivo.

Purification And Crystallization Of Photosystem Ii Dimer Complex From A Red Alga Cyanidium Caldarium

The central part of photosystem II (PSII) is highly conserved from prokaryotic cyanobacteria to eukaryotes; however, there are some apparent differences in the extrinsic proteins involved in oxygen evolution among different organisms. So far, the crystal structure of PSII from cyanobacteria has been reported, whereas no reports have been published on the structure of any eukaryotic PSII. Red alga is one of the eukaryotic algae closely related to cyanobacteria, but its PSII differs from that of cyanobacteria in that the former contains a 20 kDa protein, a unique, fourth extrinsic protein. In order to elucidate the structure of red algal PSII and its differences with cyanobacterial PSII, we purified and crystallized PSII from an acidophilic, thermophilic red alga Cyanidium caldarium. In this study, the previously published procedure for purification of PSII from the red alga was improved, yielding a highly purified PSII dimer preparation with high oxygen-evolving activities comparable with that of thermophilic cyanobacterial PSII. We obtained three-dimensional crystals of the red algal PS II under several conditions, and characterized the crystals by X-ray diffraction. The results showed that the red algal PSII crystals had a space group of P2221, which is different from that of cyanobacterial crystals. The unit cell parameters of red algal PSII were also different from those of cyanobacterial PSII, with a and c axes longer and b axis shorter than those of cyanobacterial PSII.