Yorinao Inoue

Mn-preserving extraction of 33-, 24- and 16-kDa proteins from O2-evolving PS II particles by divalent salt-washing

Divalent salt‐washing of O2‐evolving PS II particles caused total liberation of 33‐, 24‐ and 16‐kDa proteins, but the resulting PS II particles retained almost all amounts of Mn present in initial particles. The retained Mn was EPR‐silent when the particles were kept in high concentrations of divalent salt. By divalent salt‐washing, the activity of diphenylcarbazide (DPC) photooxidation was not affected at all, neither suppressed nor enhanced, while O2 evolution was totally inactivated. These results indicate that Mn can be kept associated with PS II particles even after liberation of the 33‐kDa protein, and suggest that the 33‐kDa protein is probably not responsible for binding Mn onto membranes, but is possibly responsible for maintaining the function of Mn atoms in the O2‐evolving center.

Ca2+-dependent restoration of O2-evolving activity in CaCl2-washed PS II particles depleted of 33, 24 and 16 kDa proteins

CaCl2‐washing of O2‐evolving PS II particles liberated 33, 24 and 16 kDa proteins concomitant with inactivation of O2 evolution, whereas almost all Mn remained associated with membranes [FEBS Lett. 164 (1983) 252–260], and the lost O2 evolution was significantly restored when 33 kDa protein rebound to the washed membranes [FEBS Lett. 166 (1984) 381–384]. Half of the Mn atoms retained in CaCl2‐washed particles were unstably associated with the membrane, being gradually released during incubation in the absence of Ca2+, whereas in the presence of Ca2+ the release of Mn was suppressed concomitant with partial reactivation of O2 evolution. These results were interpreted as indicating that Ca2+ as well as 33 kDa protein maintains the conformation around the Mn‐binding sites as required for O2 evolution.

Effect of trypsin on PS-II particles. Correlation between Hill-activity, Mn-abundance and peptide pattern

The effect of trypsin treatment on Photosystem-II particles has been investigated by measurements of oxygen evolution, 2,6-dichlorophenolindophenol (DCIP)-reduction and Mn-abundance and by analyzing the peptide pattern. The following results were obtained. (1) Trypsin modifies both the acceptor and donor side of PS II, but striking differences are observed for the pH dependence: whereas the acceptor side is severely attacked between pH 5.5 and 9.0, the destruction of the donor side (oxygen-evolving capacity) by trypsin becomes significant only at pH values higher than 7.25. (2) The pH-dependence of the susceptibility of oxygen evolution to trypsin closely resembles that observed in inside-out thylakoids (Renger, G., Volker, M. and Weiss, W. (1984) Biochim. Biophys. Acta 766, 582–591). (3) The effect of trypsin on the functional integrity of water oxidation cannot be due to an attack on the surface exposed 16 kDa, 24 kDa and 33 kDa polypeptides, because they are digested rapidly even at pH 6.5, where the oxygen-evolving capacity remains almost unaffected. (4) Trypsination of PS-II particles as well as of the isolated 33 kDa protein leads to a 15 kDa fragment. In trypsinized PS-II particles this fragment remains membrane-bound. The amount of the 15 kDa fragment and Mn content are correlated with the oxygen-evolving capcity. These results indicate pH-dependent structural modifications at the donor side of System II which make target proteins accessible to trypsin. The 33 kDa protein is inferred to play a regulatory role in photosynthetic oxygen evolution and this function is realized by only a part of the protein, i.e., the 15 kDa fragment, that remains resistant to mild trypsination.

A new photosystem II reaction center component (4.8 kDa protein) encoded by chloroplast genome

The photosystem II reaction center complex, so‐called D1‐D2‐cytochrome b‐559 complex, isolated from higher plants contains a new component of about 4.8 kDa [(1988) Plant Cell Physiol. 29, 1233–1239]. The partial amino acid sequence of this component from spinach was determined after release of N‐terminal blockage. The determined sequence matched an open reading frame (ORF36) of the chloroplast genome from tobacco and liverwort, which is located downstream from the psbK gene and forms an operon with psbK. The predicted product consists of 36 amino acid residues and has a single membrane‐spanning segment. High homology between the tobacco and liverwort genes, and its presence in the reaction center complex suggest an important role for this component in the photosystem II complex. Since this gene corresponds to a part of the formerly designated psbI gene, we propose to revise the definition of psbI as the gene encoding the 4.8 kDa reaction center component.

Discrete extraction of the Ca atom functional for O2 evolution in higher plant photosystem II by a simple low pH treatment

Treatment of spinach PS II membranes with a citrate solution at pH 3.0 totally inactivated O2 evolution concomitant with a 50% decrease in Ca abundance. Notably, neither the abundance of Mn and extrinsic proteins nor the activity of DPC photooxidation was at all affected by the treatment. The treated membranes evolved O2 at a high rate in the presence of exogenous Ca2+, but the activity was sensitive to EDTA. However, when the treated membranes were incubated with Ca2+ for a few tens of minutes, the O2‐evolving activity became EDTA‐resistant, suggesting a firm re‐ligation of Ca2+ to the Ca‐binding site. It was indicated that spinach PS II contains two Ca atoms per reaction center, and that the low pH citrate treatment selectively removes one of the two Ca atoms that is specifically functional for O2 evolution, even in the presence of all three extrinsic proteins.

Protein composition of the photosystem II core complex in genetically engineered mutants of the cyanobacterium Synechocystis sp. PCC 6803

The presence of four photosystem II proteins, CP47, CP43, D1 and D2, was monitored in mutants of Synechocystis sp. PCC 6803 that have modified or inactivated genes for CP47, CP43, or D2. It was observed that: (1) thylakoids from mutants without a functional gene encoding CP47 are also depleted in D1 and D2; (2) inactivation of the gene for CP43 leads to decreased but significant levels of CP47, D1 and D2; (3) deletion of part of both genes encoding D2, together with deletion of part of the CP43-encoding gene causes a complete loss of CP47 and D1; (4) thylakoids from a site-directed mutant in which the His-214 residue of D2 has been replaced by asparagine do not contain detectable photosystem II core proteins. However, in another site-directed mutant, in which His-197 has been replaced by tyrosine, some CP47 as well as breakdown products of CP43, but no D1 and D2, can be detected. These data could indicate a central function of CP47 and D2 in stable assembly of the photosystem II complex. CP43, however, is somewhat less critical for formation of the core complex, although CP43 is required for a physiologically functional photosystem II unit. A possible model for the assembly of the photosystem II core complex is proposed.

EPR evidence for a modified S-state transition in chloride-depleted Photosystem II

Abstract The role of chloride on the S-state transition in spinach Photosystem II (PS II) particles was investigated by EPR spectroscopy at low temperature and the following results were obtained. (1) After excitation by continuous light at 200 K, chloride-depleted particles did not show the EPR multiline signal associated with the S2 state, but only showed the broad signal at g = 4.1. The S2 multiline signal was completely restored upon chloride repletion. (2) In the absence of chloride the S2 multiline signal was not induced by a single flash excitation at 0°C. However, upon addition of chloride after the flash the signal was developed in darkness. (3) The amplitude of the multiline S2 signal thus developed upon chloride addition after flash illumination did not show oscillations dependent upon flash number. These results indicate that the O2-evolving complex in chloride-depleted PS II membranes is able to store at least one oxidizing equivalent, a modified S2 state, which does not give rise to the multiline signal. Addition of chloride converts this oxidizing equivalent to the normal S2 state which gives rise to the multiline signal. The modified S2 state is more stable than the normal S2 state, showing decay kinetics about 20-times slower than those of the normal S2 state, and the formation of higher S states is blocked.

X-ray Detection of the Period-Four Cycling of the Manganese Cluster in Photosynthetic Water Oxidizing Enzyme.

X-ray absorption near-edge structure spectra of the manganese (Mn) cluster in physiologically native intermediate states of photosynthetic water oxidation induced by short laser flash were measured with a compact heat-insulated chamber equipped with an x-ray detector near the sample surface. The half-height energy of the Mn Kedge showed a period-four oscillation dependent on cycling of the Joliot-Koks oxygen clock. The flash number-dependent shift in the Mn K-edge suggests that the Mn cluster is oxidized by one electron upon the S0-to-S1, S1-to-S2, and S2-to-S3 transitions and then reduced upon the S3-to-S0 transition that releases molecular oxygen.

Crystal structure of a repair enzyme of oxidatively damaged DNA. MutM (FPG), from an extreme thermophile, Thermus thermophilus HB8

The MutM [formamidopyrimidine DNA glycosylase (Fpg)] protein is a trifunctional DNA base excision repair enzyme that removes a wide range of oxidatively damaged bases (N‐glycosylase activity) and cleaves both the 3′‐ and 5′‐phosphodiester bonds of the resulting apurinic/apyrimidinic site (AP lyase activity). The crystal structure of MutM from an extreme thermophile, Thermus thermophilus HB8, was determined at 1.9 Å resolution with multiwavelength anomalous diffraction phasing using the intrinsic Zn2+ ion of the zinc finger. MutM is composed of two distinct and novel domains connected by a flexible hinge. There is a large, electrostatically positive cleft lined by highly conserved residues between the domains. On the basis of the three‐dimensional structure and taking account of previous biochemical experiments, we propose a DNA‐binding mode and reaction mechanism for MutM. The locations of the putative catalytic residues and the two DNA‐binding motifs (the zinc finger and the helix–two‐turns–helix motifs) suggest that the oxidized base is flipped out from double‐stranded DNA in the binding mode and excised by a catalytic mechanism similar to that of bifunctional base excision repair enzymes.

Simple and discrete isolation of an O2-evolving PS II reaction center complex retaining Mn and the extrinsic 33 kDa protein

Oxygen evolution PS II Reaction center 33 kDa protein Octylglucopyranoside Photosynthesis