Noriaki Tamura

Photoactivation of the water-oxidizing complex in photosystem II membranes depleted of Mn, Ca and extrinsic proteins. II: Studies on the functions of Ca2+

The effects of Ca 2+ addition to PS II membranes depleted of Ca, Mn and the 17/23 kDa proteins (NH 2 OH-TMF-2) on photoactivation of the water-oxidizing Mn-complex were studied. Lightincubation of NH 2 OH-TMF-2 with MnCl 2 but no Ca 2+ resulted in an increase of EDTA non-extractable membrane-bound Mn abundance; however, the resulting Mn-complex was inactive in O 2 evolution. Post-addition and incubation of Ca 2+ with these membranes in darkness conferred catalytic water-oxidizing activity to the membranes and caused release of one or two membrane-bound Mn atoms per reaction center. Among divalent cations, the hierarchy of effectiveness for conferring activity was Ca 2+ > Sr 2+ ≫ Ba 2+ ,Mg 2+ , a result similar to the cation specificity for reconstitution of water-oxidizing activity of PS II membranes depleted of Ca 2+ and 17/23 kDa proteins (Ghanotakis, D.F., Babcock, G.T. and Yocum, C.F. (1984) FEBS Lett. 167, 127–130). Whereas NH 2 OH-TMF-2 photoactivated in Mn 2+ /Ca 2+ exhibited a typical period-four oscillation of thermoluminescence B-band, membranes photoactivated in Mn 2+ only showed a high yield of B-band with an upshifted peak temperature aftera single flash but no subsequent oscillatory behavior. Post-incubation in darkness with divalent cations (effectiviry of Ca 2+ > Sr 2+ ≫ Ba 2+ ,Mg 2+ ) permitted the subsequent period-four oscillation of the B-band. These results indicate: (1) photoligation of Mn 2+ into the Mn-complex does not require Ca 2+ addition to NH 2 OH-TMF-2; (2) the Mn-complex formed during photoactivation in the absence of Ca 2+ are inactive in water oxidation, a consequence of an inhibition of the S 2 → S 3 transition; and (3) inactive complexes are converted to catalytically active complexes by dark incubation with Ca 2+ . In analyses for the thermoluminescence D-band (S 2 Q − A with NH 2 OH-TMF-2 photoactivated in Mn 2+ only, the normal D-band and an additional emission band with a high peak temperature of 45 °C were observed. Dark incubation with Ca 2+ before illumination to populate S 2 Q − A charge pair diminished the 45 °C emission without a proportionate increase of the normal D-band.

Assignment of histidine residues in Dl protein as possible ligands for functional manganese in photosynthetic water-oxidizing complex

By use of a chemical modifier, the ligand amino-acid residue(s) for manganese ligation in the photosynthetic water-oxidation center was investigated. The following was found. (1) Treatment with diethyl pyrocarbonate (DEPC), a histidine modifier, caused a loss of photoactivation capability of NH2OH-treated PS II membranes (devoid of Mn). (2) DEPC-induced loss of photoactivation capability showed pH dependence with a pKaof 6.9 and was reversed by subsequent treatment with NH2OH, both indicative of specific involvement of histidine residue in the modification. (3) DEPC modification was protected by the presence of Mn photoligated in the center, and DEPC-modified centers drastically lost the Mn binding affinity accompanied by a decrease in rate of generation of the unstable intermediate state involved in the multi-quantum process of photoactivation. (4) [14C]DEPC treatment radiolabelled several PS II proteins, but the labelling intensities of D1 and a 60 kDa band due to D1/D2 heterodimer were specifically suppressed among others by the presence of photoligated Mn. Based on this, it was inferred that D1 protein contains a ligand histidine residue(s) specifically interacting with Mn functional in water oxidation.

Lack of photoactivation capacity in Scenedesmus obliquus LF-1 results from loss of half the high-affinity manganese-binding site: Relationship to the unprocessed D1 protein

The high-affinity binding site for Mn 2+ is characterized by a decrease in 1,5-diphenylcarbazide (DPC) to 2,6-dichlorophenolindophenol (DCIP) electron transport with NH 2 OH-treated spinach Photosystem II (PS II) membrane fragments when micromolar amounts of Mn 2+ are present in the assay. This site is purported to be the binding site for Mn, functional in O 2 evolution (Hsu, B.-D., Lee, J.-Y. and Pan, R.-L. (1987) Biochim. Biophys. Acta 890, 89–96). We have examined this site in PS II-enriched membranes from Scenedesmus obliquus wild-type (WT) and LF-1 mutant cells. LF-1 inserts an unprocessed D1 protein into the photosynthetic membrane, binds approx. 40% of the functional Mn as WT, and does not evolve O 2 (Metz, J.G., Pakrasi, H.B., Seibert, M. and Arntzen, C.J. (1986) FEBS Lett. 205, 269–274). The dissociation constant for added high-affinity Mn 2+ is about 0.3–0.4 μM in wheat, WT, and LF-1 PS II. However, the relative amount of available high-affinity Mn 2+ -binding site is about half as much in LF-1 PS II membranes compared to wheat, spinach, and WT PS II membranes. Despite the fact that LF-1 PS II can photoligate Mn, LF-1 cannot be photoactivateid as can NH 2 OH-treated WT PS II. LF-1 subjected to photoactivating conditions does not reach S 2 as determined by thermoluminescence. This work indicates that the Hsu et al. high-affinity Mn 2+ site is actually at least two sites, one of which is missing in LF-1, and that successful photoactivation potential requires the presence of all high-affinity Mn 2+ site. The fact that the full complement of high-affinity Mn 2+ -binding site is observed in isolated spinach PS II reaction center (D1/D2/cytochrome b -559) complex demonstrates that other PS II core proteins do not affect the high-affinity site. Histidine chemical modifier experiments show that one component of the high-affinity site is probably associated with histidine(s) and that this component is missing in LF-1. We conclude that histidine(s) on the Dl protein provides ligand(s) for part of the Mn required for O 2 -evolution function and that the balance of the Mn is bound by other amino acids on the proteins composing the PS II reaction center.

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.

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).

Photoactivation and Photoinhibition of the O 2 -Evolving Complex in Dark-Grown Spruce Seedlings

Coniferous seedlings form highly-developed chloroplasts even during dark germination (1,2). The chloroplasts, however, cannot evolve oxygen since the assembly of the tetra-Mn cluster does not occur. The water-oxidizing complex in PS II membranes isolated from dark-grown spruce cotyledons can be assembled in vitro (3), as reported with NH2OH-treated PS II membranes from wheat leaves (4). This indicates that the light-induced assembly of water-oxidizing complex (photoactivation) in the dark-grown spruce cotyledons does not require de novo protein synthesis.

Functional Role of Fibrillin5 in Acclimation to Photooxidative Stress

The functional role of a lipid-associated soluble protein, fibrillin5 (FBN5), was determined with the Arabidopsis thaliana homozygous fbn5-knockout mutant line (SALK_064597) that carries a T-DNA insertion within the FBN5 gene. The fbn5 mutant remained alive, displaying a slow growth and a severe dwarf phenotype. The mutant grown even under growth light conditions at 80 µmol m-2 s-1 showed a drastic decrease in electron transfer activities around PSII, with little change in electron transfer activities around PSI, a phenomenon which was exaggerated under high light stress. The accumulation of plastoquinone-9 (PQ-9) was suppressed in the mutant, and >90% of the PQ-9 pool was reduced under growth light conditions. Non-photochemical quenching (NPQ) in the mutant functioned less efficiently, resulting from little contribution by energy-dependent quenching (qE). The ultrastructure of thylakoids in the mutant revealed that their grana were unstacked and transformed into loose and disordered structures. Light-harvesting complex (LHC)-containing large photosystem complexes and photosystem core complexes in the mutant were less abundant than those in wild-type plants. These results suggest that the lack of FBN5 causes a decrease in PQ-9 and imbalance of the redox state of PQ-9, resulting in misconducting both short-term and long-term control of the input of light energy to photosynthetic reaction centers. Furthermore, in the fbn5 mutant, the expression of genes involved in jasmonic acid biosynthesis was suppressed to ≤10% of that in the wild type under both growth-light and high-light conditions, suggesting that FBN5 functions as a transmitter of 1O2 in the stroma.

Reassembly of the Photosynthetic Water-Oxidizing Complex on the Thylakoid Membranes

The manganese cluster catalyzing the photosynthetic water-oxidation is ligated to the PS2 reaction center proteins, the D1 and D2 proteins (1). Under illumination, vigorous photoreactions occur on the PS2 reaction center, resulting in photodamage and the subsequent repair of the PS2 proteins, particularly the D1 protein (2, 3). Thus, the manganese cluster may be disassembled and (re-)assembled on the thylakoids, in concern with recycling of the PS2 reaction centers. However, it remains open where and how the Mn cluster is assembled in vivo.