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We isolated and sequenced a cDNA clone encoding a minor chlorophyll a/b-binding protein, CP26, which is associated with the light-harvesting complex II of Chlamydomonas reinhardtii. Protein sequences of internal peptide fragments from purified CP26 were determined and used to identify a cDNA clone. The 1.1 kb lhcb5gene codes for a polypeptide of 289 amino acids with a predicted molecular weight of 30 713. The lhcb5gene product could reconstitute with chlorophylls and xanthophylls to form a green band on a gel. Although the expression of many lhcbgenes are strictly regulated by light, the lhcb5gene was only loosely regulated. We propose that a plant acclimatizes itself to the light environment by quantitatively and qualitatively modulating the light-harvesting complex. Characterization of the primary structure and the implications of its unique expression are discussed.

One of the photosystem II reaction center proteins, D1, is encoded by the psbAgene and is synthesized as a precursor form with a carboxyl-terminal extension that is subsequently cleaved between Ala-344 and Ser-345. We have generated three psbAtransformants of the green alga Chlamydomonas reinhardtiiin which Ala-344 or Ser-345 have been substituted with Pro or Glu (A344P, S345E, and S345P) to understand the effects of the amino acid substitutions on the processing of the precursor D1. S345E grew photoautotrophically and showed PSII activity like the wild type. However, A344P and S345P were unable to grow photoautotrophically and were significantly photosensitive. A344P was deficient in the processing of precursor D1 and in oxygen-evolving activity, but assembled photosystem II complex capable of charge separation. In contrast, both precursor and mature forms of D1 accumulated in S345P cells from the logarithmic phase and the cells evolved oxygen at 18% of wild-type level. However, S345P cells from the stationary phase contained mostly the mature D1 and showed a twofold increase in oxygen-evolving activity. The rate of processing of the accumulated pD1 was estimated to be about 100 times slower than in the wild type. It is therefore concluded that the functional oxygen-evolving complex is assembled when the precursor D1 is processed, albeit at a very low rate. These results suggest the functional significance of the amino acid residues at the processing site of the precursor D1.

The S2state of the oxygen-evolving Mn-cluster of Photosystem II (PS II) is known to have different forms that exhibit the g=2 multiline and g= 4.1 EPR signals. These two spin forms are interconvertible at > 200 K and the relative amplitudes of the two signals are dependent on the species of cryoprotectant and alcohol contained in the medium. Also, it was recently found that the mutiline form can be converted to the g= 4.1 form by absorption of near-infrared light by the Mn-cluster itself at around 150 K [Boussac et al. (1996) Biochemistry 35: 6984–6989]. We have used light-induced Fourier transform infrared (FTIR) difference spectroscopy to study the structural difference in these two S2forms. FTIR difference spectra for S2/S1as well as for S2QA-/S1QAmeasured at cryogenic temperatures using PS II membranes in the presence of various cryoprotectants, and monohydric alcohols did not show any specific differences except for intensities of amide I bands, which were larger when ethylene glycol or glycerol was present in addition to sucrose. This result was interpreted due to more flexible movement of the protein backbones upon S2formation with a higher cryoprotectant content. Light-induced difference spectra measured at 150 K using either blue light without near-infrared light or red plus near-infrared light also did not show any detectable difference. In addition, a different spectrum upon near-infrared illumination at 150 K of the PS II sample in which the S2state had been photogenerated at 200 K exhibited no meaningful signals. These results indicate that the two S2forms that give rise to the multiline and g= 4.1 signals have only minor differences, if any, in the structures of amino-acid ligands and polypeptide backbones. This conclusion suggests that conversion between the two spin states is caused by a spin-state transition in the Mn(III) ion rather than valence swapping within the Mn-cluster that would considerably affect the vibrations of ligands.

A theoretical method to extract structural information on spin-exchange-coupled manganese tetramers from the EPR spectroscopy data is presented. This method has been applied to two EPR data, i.e. the ground-state spin (S= 1/2) and the first excitation energy (30—37 cm−1), for the S2-state Mn tetramer in the photosynthetic oxygen-evolving complex, which exhibits a “g= 2 multiline” EPR signal. Based on the EXAFS data and the manganese chemistry, a simplified model spin Hamiltonian to describe the S2-state Mn cluster will be presented, such that two spin-exchange interactions due to 2.7—2.8 Å and/or 3.2—3.5 Å Mn–Mn bonds can vary from weak to strong coupling, sensitively, depending upon the bridge structure, except for a strong-antiferromagnetic interaction due to a 2.7 Å Mn(III)–Mn(IV) bond and the other weak ones. By computer-search of the possible spin-exchange structures with respect to these two parametric interactions, it was found that (1) a dimer of di-μ2-oxo bridged Mn dimers, a propeller-type tetramer in which the central Mn ion is chelated by three di-μ-oxo Mn cores and some other models are highly unlikely, (2) the most promising cluster is a trimer-plus-monomer type of distorted cubane, and (3) S*= 5/2 excited states are higher than the first excited state with S*= 3/2 (majority) or S*= 1/2 (minority).

Divalent salt-washing of O 2-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 O 2evolution 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 O 2-evolving center.

The re‐reduction course of P‐680+, the photooxidized PS II primary donor, was measured as a function of excitation number in Cl−‐depleted PS II membranes. After the 1st and 2nd excitations the signal amplitude of P‐680+is small, indicating a submicrosecond reduction of P‐680+by Z, the secondary donor of PS II. After the 3rd excitation, however, a larger P‐680+signal with a 40–50 μs half‐life is observed. The slow decay of this signal is attributed to a back‐reaction with a reduced acceptor in the presence of the Z+S2state on the donor side. The state Z+S2has a lifetime longer than 300 ms and its formation was found to depend on the presence of the abnormal S2state created by the 1st excitation. The P‐680 data and thermoluminescence measurements show that the S‐state advancement beyond S2is blocked in the absence of Cl−and that the Cl−‐free abnormal S2state has a lifetime about 10‐times longer than the normal S2state.

A range of 4-hydroxypyridines were synthesized and their activity as PET inhibitors were investigated with regard to their structural resemblance to plastoquinone in photosynthetic electron transport (PET). The activity of these compounds was markedly enhanced upon modifying their structures: introduction of halogens into both the 3-and 5-positions of the pyridine ring and additional substitution at the a-position of the side chain at 6-position were effective among others in enhancing the activity. Insertion of a phenyl ring into the side chain at 6-position of the pyridine ring also increased the activity. Substituents on the phenyl ring greatly affected the activity: when substituted with an appropriate functional group, the compounds became 10-to 100-fold more active. The mode of action of both halogenated and non-halogenated 4-hydroxypyridines were investigated by means of thermoluminescence measurements and cross resistance examination against atrazine-resistant thylakoids having mutation in D 1 protein. It was inferred that upon halogenation, 4-hydroxypyridines changed their mode of action from plastoquinone-pool inhibitors to phenol-type inhibitors.

The pH dependence of emission peak temperature and decay time of thermoluminescence arising from S2QB- and S2QA- recombinations demonstrates that a stabilization of S2QB- occurs at low pH whereas stabilization of S2QA- occurs at high pH. Based on comparative analysis of thermoluminescence parameters of the two types of recombination, we suggest that in the pH range between 5.3 and 7.5, Em(S2/S1) and Em(QA/QA-) are constant, but Em(QB/QB-) gradually increases with decreasing pH, while in the pH range between 7.5 and 8.5, an unusual change occurs on S2QA- charge pair, which is interpreted as either a decrease in Em(S2/S1) or an increase in Em(QA/QA-).

Flash excitation of Tris-treated photosystem II (PSII) membranes generated a thermoluminescene band peaking -25°C. Indicating that a positively charged oxidizing equavalent is stably accumulated on donor side of PSII even in the Mn-cluster. This oxidizing equivalent was sensitive to low concentrations of exogenous Mn ???, and its stable accumulation was reversibly inhibited by diethylpyrocarbonate treatment that modifies histidine residues with high specificity. It was inferred that there is a photooxidizable histidine residue(s) on donor side of PSII and it provides a redox-active ligand for Mn

An O 2-evolving PS II reaction center complex retaining Mn and the extrinsic 33 kDa protein with almost the same relative abundance as in situ (denoted as RCII[33,Mn] complex) was prepared by a simple one-step sucrose density gradient centrifugation of octylglucopyranoside (OGP)-solubilized PS II membranes. The complex evolved O 2at a high rate of 550–850 μmolmgChl per h with ferricyanide as electron acceptor in the presence of CaCl 2and digitonin. The protein composition analysis of this complex revealed that OGP treatment discretely dissects the PS II membranes into RCII[33,Mn] complex and LHCP, and the resulting RCII[33,Mn] complex preserves the distinct structural integrity for O 2evolution in situ.

Photosystem II (PS II) reaction center complex of higher plants was revealed to contain a new low‐molecular‐mass component of 4.8 kDa encoded by a chloroplast gene, psbI [(1988) FEBS Lett. 241, 99–104], in addition to D1, D2 and two subunits of cytochrome b‐559. Here we isolated a PS II reaction center complex from the thermophilic cyanobacterium, Synechococcus vulcanus, by column chromatography using Triton X‐100 and urea, and attempted to identify the same gene product. SDS gel electrophoresis of the cyanobacterial reaction center complex resolved three protein bands of 8, 5.4 and 5 kDa in addition to D1, D2 and a partially remaining 47 kDa protein. N‐terminal sequencing revealed that the 5 kDa band consisted of two components: one, having an open N‐terminus, was homologous to the psbL gene product of higher plants, and the other, having a blocked N‐terminus, was homologous to the psbI gene product of higher plants. The 8 kDa and 5.4 kDa were the large and small subunits of cytochrome b‐559, respectively. Based on these results, we conclude that the subunit structure of cyanobacterial PS II reaction center complex is essentially the same as that of higher plants except that the cyanobacterial psbL product is more tightly associated with the reaction center.

High resolution gel electrophoresis in the low‐molecular‐mass region combined with electroblotting using polyvinylidene difluoride membranes enabled us to sequence the low‐molecular‐mass proteins of photosystem II membrane fragments from spinach and wheat. The determined N‐terminal sequences, all showing considerable homology between the two plants, involved two newly determined sequences for the 4.1 kDa protein and one for the 5 kDa proteins. The sequence of the 4.1 kDa protein did not match any part of the chloroplast DNA sequence from tobacco or liverwort, suggesting that it is encoded by the nuclear genome. In contrast, the sequence of the 5 kDa protein matched ORF38, which is located just downstream of psbE and psbF in the chloroplast DNA and is assumed to be co‐transcribed with them. These two components were associated with the O2‐evolving core complex. Sequences of other low‐molecular‐mass proteins confirmed the previous identification as photosystem II components.

The O2‐evolving photosystem II core complex was isolated from a thermophilic cyanobacterium, Synechococcus vulcanusCopeland. Analysis by SDS‐polyacrylamide gel electrophoresis revealed that the complex contained at least seven low‐molecular‐mass proteins in addition to the well characterized CP47 apoprotein, CP43 apoprotein, 33 kDa extrinsic protein, D1 protein, D2 protein and large subunit of cytochrome b‐559. The separation profiles of these low‐molecular‐mass proteins were very similar between cyanobacterial and higher plant PS II. N‐terminal sequences of the 6.5 kDa and 3.9 kDa proteins of cyanobacterial core complex were determined after blotting to a polyvinylidene difluoride membrane. The sequence of the 6.5 kDa protein showed high homology with an internal sequence of plant psbH gene product, so‐called 10 kDa phosphoprotein, but did not conserve the Thr residue which is specifically phosphorylated in plants. The sequence of the 3.9 kDa protein corresponded to the K protein of higher plants (mature form of psbK gene product). These results indicate that the products of both psbH and psbK genes are present in cyanobacterial PS II as well as being associated with the O2‐evolving core complex.

High resolution gel electrophoresis of the native photosystem I complex retaining light‐harvesting chlorophyll complex revealed the presence of three low‐molecular‐mass proteins of 7, 4.1 and 3.9 kDa in spinach, and 6.8, 4.4 and 4.1 kDa in pea, in addition to the other well‐characterized higher‐molecular‐mass components. Upon further detergent treatment to deplete light‐harvesting chlorophyll complex, the 7 kDa and 4.1 kDa proteins were removed from the photosystem I core complex of spinach, while the 3.9 kDa protein was retained. N‐terminal sequencing demonstrated that the 4.1 kDa proteins from both spinach and pea correspond to the gene product of ORF42/44 in chloroplast genome of liverwort and higher plants, which was previously hypothesized as a photosystem I gene (psaJ) based on sequence homology with the cyanobacterial photosystem I component of 4.1 kDa [(1989) FEBS Lett. 253, 257‐263]. N‐terminal sequence of the spinach 3.9 kDa and pea 4.4 kDa proteins fitted with chloroplast ORF36/40 (psaI) although no homologue has been found in cyanobacteria. The spinach 7 kDa and pea 6.8 kDa proteins correspond to the nuclear‐encoded psaK product and significantly matched with the N‐terminal sequence of the cyanobacterial 6.5 kDa subunit. The evolutional conservation of the psaJ and psaK seems to suggest their intrinsic role(s) in photosystem I.

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 O2evolution 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.

On extracting the O2‐evolving Photosystem II (PS II) particles with CaCl2solution, three proteins of 33, 24 and 16 kDa were completely liberated from the particles concomitant with total inactivation of O2evolution, but most of the Mn atoms present in the initial particles were preserved unaffected being still associated with the particles. On reading the extracted proteins to the extracted particles, a considerable amount of 33 kDa protein rebound to the particles concomitant with significant reconstitution of O2‐evolving activity.

High resolution gel electrophoresis of the native photosystem I complex retaining light-harvesting chlorophyll complex revealed the presence of three low-molecular-mass proteins of 7, 4.1 and 3.9 kDa in spinach, and 6.8, 4.4 and 4.1 kDa in pea, in addition to the other well-characterized higher-molecular-mass components. Upon further detergent treatment to deplete light-harvesting chlorophyll complex, the 7 kDa and 4.1 kDa proteins were removed from the photosystem I core complex of spinach, while the 3.9 kDa protein was retained. N-terminal sequencing demonstrated that the 4.1 kDa proteins from both spinach and pea correspond to the gene product of ORF42/44 in chloroplast genome of liverwort and higher plants, which was previously hypothesized as a photosystem I gene ( psaJ) based on sequence homology with the cyanobacterial photosystem I component of 4.1 kDa [(1989) FEBS Lett. 253, 257-263]. N-terminal sequence of the spinach 3.9 kDa and pea 4.4 kDa proteins fitted with chloroplast ORF36/40 ( psaI) although no homologue has been found in cyanobacteria. The spinach 7 kDa and pea 6.8 kDa proteins correspond to the nuclear-encoded psaK product and significantly matched with the N-terminal sequence of the cyanobacterial 6.5 kDa subunit. The evolutional conservation of the psaJ and psaK seems to suggest their intrinsic role(s) in photosystem I.

Photosystem I (PSI) complex of Anabaena variabilisATCC 29413 consists of at least 11 subunits, 9 of which are resolved by high resolution gel electrophoresis. N-terminal amino acid sequences of the four subunits with molecular masses of 6.8, 5.2, 4.8 and 3.5 kDa were determined. Based on the sequence homology, the 3.5 kDa subunit was revealed to correspond to PSI-I (the gene product of psaI), which had so far been detected only in higher plant PSI complexes. The 6.8 kDa protein and 4.8 kDa protein were identified as gene products of psaK and psaJ, respectively. The 5.2 kDa protein was homologous to a 4.8 kDa subunit of PSI of the thermophilic cyanobacterium Synechococcus vulcanus, suggesting that this protein is a component of PSI in cyanobacteria.

The O 2-evolving photosystem II core complex was isolated from a thermophilic cyanobacterium, Synechococcus vulcanusCopeland. Analysis by SDS-polyacrylamide gel electrophoresis revealed that the complex contained at least seven low-molecular-mass proteins in addition to the well characterized CP47 apoprotein, CP43 apoprotein, 33 kDa extrinsic protein, D1 protein, D2 protein and large subunit of cytochrome b-559. The separation profiles of these low-molecular-mass proteins were very similar between cyanobacterial and higher plant PS II. N-terminal sequences of the 6.5 kDa and 3.9 kDa proteins of cyanobacterial core complex were determined after blotting to a polyvinylidene difluoride membrane. The sequence of the 6.5 kDa protein showed high homology with an internal sequence of plant psbH gene product, so-called 10 kDa phosphoprotein, but did not conserve the Thr residue which is specifically phosphorylated in plants. The sequence of the 3.9 kDa protein corresponded to the K protein of higher plants (mature form of psbK gene product). These results indicate that the products of both psbH and psbK genes are present in cyanobacterial PS II as well as being associated with the O 2-evolving core complex.

On extracting the O 2-evolving Photosystem II (PS II) particles with CaCl 2solution, three proteins of 33, 24 and 16 kDa were completely liberated from the particles concomitant with total inactivation of O 2evolution, but most of the Mn atoms present in the initial particles were preserved unaffected being still associated with the particles. On reading the extracted proteins to the extracted particles, a considerable amount of 33 kDa protein rebound to the particles concomitant with significant reconstitution of O 2-evolving activity.

CaCl 2-washing of O 2-evolving PS II particles liberated 33, 24 and 16 kDa proteins concomitant with inactivation of O 2evolution, whereas almost all Mn remained associated with membranes [FEBS Lett. 164 (1983) 252–260], and the lost O 2evolution 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 CaCl 2-washed particles were unstably associated with the membrane, being gradually released during incubation in the absence of Ca 2+, whereas in the presence of Ca 2+the release of Mn was suppressed concomitant with partial reactivation of O 2evolution. These results were interpreted as indicating that Ca 2+as well as 33 kDa protein maintains the conformation around the Mn-binding sites as required for O 2evolution.

Flash excitation of Tris‐treated photosystem II (PSII) membranes generated a thermoluminescene band peaking ‐25°C. Indicating that a positively charged oxidizing equavalent is stably accumulated on donor side of PSII even in the Mn‐cluster. This oxidizing equivalent was sensitive to low concentrations of exogenous Mn???, and its stable accumulation was reversibly inhibited by diethylpyrocarbonate treatment that modifies histidine residues with high specificity. It was inferred that there is a photooxidizable histidine residue(s) on donor side of PSII and it provides a redox‐active ligand for Mn

Isogenic strains of Synechococcus PCC 7942 were genetically engineered so that copy I of the gene psbA was mutated at specific sites. These mutations resulted in replacements of Ser 264 by Gly or Ala and of Phe 255 by Tyr or Leu in the D1 protein. The mutants were resistant to herbicides inhibiting electron transfer in photosystem II. All mutants exhibited alterations in the stability of QB-as demonstrated by a temperature downshift, to various extents, of the in vivo thermoluminescence emission. Measurements of the light-dependent turnover of D1 showed a marked decrease in the t 1/2 of this protein in the mutants as compared to wild-type, under low to medium light intensities. A correlation was found between the degree of pertur­ bation in the QB-stability and the rate of acceleration in the turnover of D1. These data pro­ vide a direct evidence for the overlapping binding sites for the plastoquinone B and herbicides in the D1 protein. In addition these data indicate a close link between QB-destabilization in reaction center II and the mechanism controlling the light-dependent turnover of D1. Based on these results and previous work we suggest that destabilization of the semireduced quinone, facilitates a light-induced damage in D1 which triggers its degradation.

The effect of unoccupancy of the QBsite by plastoquinone on the photoinactivation of reaction center II in a Cyt b6/f-less mutant of Chlamydomonas reinhardtii, B6, was investigated. In these cells the oxidation of plastoquinol generated by electron flow via RC II to plastoquinone and thus the turnover of PQH2/PQ via the QBsite are drastically reduced. Reaction center II of the mutant cells was resistant to photoinactivation relative to the control cells as demonstrated by measurements of light-induced destabilization of S2-QBcharge recombination, rise in in­ trinsic fluorescence and loss of variable fluorescence. These parameters relate to functions in­ volving the reaction center II D1 protein. The light-induced degradation of D1 in the mutant cells was also considerably reduced, with a t 1/2 value of 7 h as compared, under similar conditions, to about 1.5 h for the control cells. These results indicate that the photoinactivation of RC II and turnover of the D1 protein are related and require occupancy of the QBsite by PQ and its light-driven reduction.

Studies on structure/activity relationships of phloroglucinol derivatives that had been designed based on the structures of grandinol and homograndinol. potent photosystem II (PS II) inhibitors in Eucalyptus grandis, revealed that two electron-withdrawing groups which differ by their electron-withdrawing power on a phloroglucinol nucleus were essential for activity. A larger difference in the electron-withdrawing power between the two groups enhanced the activity, and 3-nitro-phloroglucinecarboxamides and the corresponding thioamides were the most active ones when they had proper lipophilic side chains. Their binding domain seems to overlap with those of DCMU and atrazine, whereas they may approach to the site in a similar manner to that of phenol type inhibitors. Accordingly, the phloroglucinol derivatives represent a new type of PS II inhibitors.

Treatment of spinach PS II membranes with a citrate solution at pH 3.0 totally inactivated O 2evolution 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 O 2at a high rate in the presence of exogenous Ca 2+, but the activity was sensitive to EDTA. However, when the treated membranes were incubated with Ca 2+for a few tens of minutes, the O 2-evolving activity became EDTA-resistant, suggesting a firm re-ligation of Ca 2+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 O 2evolution, even in the presence of all three extrinsic proteins.

High resolution gel electrophoresis in the low-molecular-mass region combined with electroblotting using polyvinylidene difluoride membranes enabled us to sequence the low-molecular-mass proteins of photosystem II membrane fragments from spinach and wheat. The determined N-terminal sequences, all showing considerable homology between the two plants, involved two newly determined sequences for the 4.1 kDa protein and one for the 5 kDa proteins. The sequence of the 4.1 kDa protein did not match any part of the chloroplast DNA sequence from tobacco or liverwort, suggesting that it is encoded by the nuclear genome. In contrast, the sequence of the 5 kDa protein matched ORF38, which is located just downstream of psbE and psbF in the chloroplast DNA and is assumed to be co-transcribed with them. These two components were associated with the O 2-evolving core complex. Sequences of other low-molecular-mass proteins confirmed the previous identification as photosystem II components.

Proton matrix ENDOR of the manganese multiline in the S 2, state of photosystem II membranes from spinach has been investigated. The spectral structure over a range of frequencies of ± 2 MHz centered on the position for a free proton was analyzed through employing a simulation method based on the dipolar interaction between electron and proton magnetic moments. The 6 pairs of lines resolved were attributed to the surrounding proton populations at distances varying within the range 2.7–6.0 Å from the putative manganese center. Two of the 6 pairs, namely those corresponding to protons at distances of 2.7 and 3.2 Å from the manganese center were eliminated on washing of the membranes with deuterated buffer. This suggests that these protons belong to the water molecules coordinating to the manganese cluster. The presence of ethylene glycol instead of glycerol and sucrose in the buffer broadened both EPR and ENDOR spectra. This suggests that ethylene glycol molecules are also accessible to the two-manganese cluster. A possible model of water association to the manganese ions in the photosynthetic water-oxidation system is proposed.

CaCl2‐washing of O2‐evolving PS II particles liberated 33, 24 and 16 kDa proteins concomitant with inactivation of O2evolution, whereas almost all Mn remained associated with membranes [FEBS Lett. 164 (1983) 252–260], and the lost O2evolution 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 O2evolution. 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 O2evolution.

Nitrile hydratase (NHase) from Rhodococcussp. N771 is a photosensitive enzyme that catalyzes hydration of nitriles to the corresponding amides. Light‐induced Fourier transform infrared difference spectra between the inactive and active forms of NHase were measured with both the natural (14N) and 15N‐labeled NHases. The results showed, for the first time, that NHase intrinsically possesses nitric oxide (NO) molecules bound to the non‐heme iron center. The possible role of NO in the photoactivation process of NHase is discussed.

3-Aminoalkylidene-2H-pyran-2,4(3H)-diones (APs), possessing a conjugated enamino moiety which is common to cyanoacrylates and 2-aminoalkylidenecyclohexane-1,3-diones (ACs), were found as a new class of photosynthetic electron transport inhibitors. Although the structural requirement of APs for photosynthetic electron transport inhibition was very similar to that of cyanoacrylates and ACs, thermoluminescence measurements indicated that the binding manner of APs to D 1 protein was totally different from that of other inhibitors.

Bioconversion of atmospheric carbon dioxide to ethylene was studied in a recombinant cyanobacterium. The gene for the ethylene-forming enzyme ofPseudomonas syringaepv.phaseolicolaPK2 was cloned and expressed in the cyanobacteriumSynechococcusPCC7942 R2-SPc by use of a shuttle vector pUC303. The ethylene-forming activityin vivoofSynechococcusPCC7942 R2-SPc that carried the gene for the ethylene-forming enzyme ofP. syringaepv.phaseolicolaPK2 was one-fifth of that ofE. coliJM109 that harbored the same plasmid. The enzyme accounted for 0.021% by weight of the total soluble protein inSynechococcusPCC7942 R2-SPc.

The nucleotide sequence was determined for the psaD gene of a thermophilic cyanobacterium, Synechococcus vulcanus, which encoded the PsaD subunit (Subunit II) of the Photosystem I reaction center complex. Except for some differences in the peripherals, the nucleotide sequence of the gene encoding PsaD was identical to that of another thermophilic cyanobacterium Synechococcus elongatus reported previously. Relationship between these primary structures and thermostability was also discussed.

Two-dimensional (2D) crystals of photosystem II (PSII) were obtained from n-heptyl-β-

The D1 protein of the photosystem (PS) II reaction center turns over very rapidly in a light-dependent manner initiated by its selective and specific cleavage. The cleavage of D1 was studied by using a PS II inhibitor, N-octyl-3-nitro-2,4,6-trihydroxybenzamide (PNO8), as a molecular probe. The following results were obtained. (i) D1 was selectively cleaved into 23-kDa N-terminal and 9-kDa C-terminal fragments in complete darkness by PNO8 at a single site in a D-E loop connecting membrane-spanning helices D and E. (ii) The cleavage was markedly enhanced when PS II was illuminated for a brief period before the addition of PNO8 in darkness. (iii) The effect of preillumination was slowly lost during incubation in the dark, with a decay half-time of ∼1 h at 25°C. (iv) The light intensity of preillumination required for the cleavage was much lower than that required for O2evolution. (v) The light-triggered cleavage of D1 was observed in thylakoids, PS II membranes, and PS II core particles, but not in purified PS II reaction centers. More than 60% of D1 was cleaved into the two fragments with no other by-products. (vi) The cleavage reaction revealed a marked pH dependence that was considerably different from that for inhibition of PS II activity. The results are interpreted as indicating that the binding of PNO8 to the QB-binding pocket triggers proteolytic cleavage of D1 that has been previously modified during illumination.

Removal of the 33 kDa extrinsic protein from photosystem (PS) II membranes resulted in markedly increased stabilization of the S 2Q A−charge pair as measured by thermoluminescence. The stabilization increase was specific for the S 2Q A−charge pair and did not require any special herbicide. The effect was fully reversed by reconstitution with the 33 kDa protein, but not at all by high concentrations of Cl −, as opposed to those effects known to be reversed by 200 mM Cl −. The data are interpreted as indicating a structural change of the donor and/or acceptor side of PS II dependent on association with the 33 kDa extrinsic protein.

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.

The photosystem I core complex isolated from a thermophilic cyanobacterium, Synechococcus vulcanus, is composed of eight low-molecular-mass proteins of 18, 14, 12, 9.5, 9, 6.5, 5 and 4.1 kDa in addition to the PS I chlorophyll protein. N-terminal amino acid sequences of all these components were determined and compared with those of higher plants. Clearly, the 9.5 kDa component corresponds to the protein which carries the non-heme iron-sulfur centers A and B. This protein is so poorly visualized by staining that it has probably been overlooked in gel electrophoresis analyses. The 18, 14, 12 and 9 kDa components show appreciable homology with respective subunits of higher plant PS I. In contrast, the 6.5, 5 and 4.1 kDa components do not correspond to any known proteins except that the sequence of the 4.1 kDa component matches an unidentified open reading frame (ORF) 42 (liverwort) or ORF44 (tobacco) of chloroplast DNA.

We recently reported the presence of several low-molecular-mass protein components in the PS II O 2-evolving core complex from the thermophilic cyanobacterium, Synechococcus vulcanus[(1989) FEBS Lett. 244, 391–396]. Here we have characterized the three components (4.1, 4.7, 5 kDa) of the same cyanobacterial core complex by N-terminal sequencing. There were two components in the 4.7 kDa region, both having a blocked N-terminus. One has a sequence highly homologous to open reading frame 34 of plant chloroplast DNA (tentatively designated psbM), while the other has a sequence partially homologous to open reading frame 43 of chloroplast DNA (designated psbN), although neither of the two gene products has yet been confirmed in chloroplasts. The cyanobacterial 4.1 kDa protein partially corresponds to the 4.1 kDa nuclear-encoded core component of higher plant PS II. The cyanobacterial 5 kDa component, however, shows a sequence that is unrelated to any other known proteins.

Photosystem II (PS II) reaction center complex of higher plants was revealed to contain a new low-molecular-mass component of 4.8 kDa encoded by a chloroplast gene, psbI [(1988) FEBS Lett. 241, 99–104], in addition to D1, D2 and two subunits of cytochrome b-559. Here we isolated a PS II reaction center complex from the thermophilic cyanobacterium, Synechococcus vulcanus, by column chromatography using Triton X-100 and urea, and attempted to identify the same gene product. SDS gel electrophoresis of the cyanobacterial reaction center complex resolved three protein bands of 8, 5.4 and 5 kDa in addition to D1, D2 and a partially remaining 47 kDa protein. N-terminal sequencing revealed that the 5 kDa band consisted of two components: one, having an open N-terminus, was homologous to the psbL gene product of higher plants, and the other, having a blocked N-terminus, was homologous to the psbI gene product of higher plants. The 8 kDa and 5.4 kDa were the large and small subunits of cytochrome b-559, respectively. Based on these results, we conclude that the subunit structure of cyanobacterial PS II reaction center complex is essentially the same as that of higher plants except that the cyanobacterial psbL product is more tightly associated with the reaction center.

Proton matrix ENDOR of the manganese multiline in the S2, state of photosystem II membranes from spinach has been investigated. The spectral structure over a range of frequencies of ± 2 MHz centered on the position for a free proton was analyzed through employing a simulation method based on the dipolar interaction between electron and proton magnetic moments. The 6 pairs of lines resolved were attributed to the surrounding proton populations at distances varying within the range 2.7–6.0 Å from the putative manganese center. Two of the 6 pairs, namely those corresponding to protons at distances of 2.7 and 3.2 Å from the manganese center were eliminated on washing of the membranes with deuterated buffer. This suggests that these protons belong to the water molecules coordinating to the manganese cluster. The presence of ethylene glycol instead of glycerol and sucrose in the buffer broadened both EPR and ENDOR spectra. This suggests that ethylene glycol molecules are also accessible to the two‐manganese cluster. A possible model of water association to the manganese ions in the photosynthetic water‐oxidation system is proposed.

The extrinsic 33-kDa protein of photosystem II (PSII) was modified with various reagents, and the resulting proteins were checked for the ability to rebind to PSII and to reactivate oxygen evolution. While modification of more than eight carboxyl groups of aspartyl and glutamyl residues with glycine methyl ester did not affect the rebinding and reactivating capabilities, modification of amino groups of lysyl residues with either N-succinimidyl propionate or 2,4,6-trinitrobenzene sulfonic acid or modification of guanidino groups of arginyl residues with 2,3-butanedione resulted in a loss of rebinding and reactivating capabilities of the 33-kDa protein. Moreover, the number of lysyl and arginyl residues susceptible to modification was significantly decreased when the protein was bound to PSII as compared with when it was free in solution, whereas the number of carboxyl groups modified was little affected. These results suggested that positive charges are important for the electrostatic interaction between the extrinsic 33-kDa protein and PSII intrinsic proteins, whereas negative charges on the protein do not contribute to such interaction. By a combination of protease digestion and mass spectroscopic analysis, the domains of lysyl residues accessible to N-succinimidyl propionate or 2,4,6-trinitrobenzene sulfonic acid modification only when the 33-kDa protein is free in solution were determined to be Lys4, Lys20, Lys66-Lys76, Lys101, Lys105, Lys130, Lys159, Lys186, and Lys230-Lys236. These domains include those previously reported accessible to N-hydroxysuccinimidobiotin only in solution (Frankel and Bricker (1995) Biochemistry34, 7492-7497), and may be important for the interaction of the 33-kDa protein with PSII intrinsic proteins.

A Fourier‐transform infrared (FTIR) spectrum of the radical cation of β‐carotene photoinduced in photosystem II (PSII) membranes was obtained at 80K under oxidizing conditions, by utilizing the light‐induced FTIR difference technique. Formation of the β‐carotene cation was monitored with the electronic absorption band at 993 nm. An FTIR spectrum of a chemically‐generated β‐carotene cation in chloroform was also measured and compared with the spectrum of PSII. Since the FTIR bands of carotenoid cation have characteristic features with strong intensities, they can be useful markers in studying the reaction of carotenoid in PSII.

Oxygen evolutionPS IIReaction center33 kDa proteinOctylglucopyranosidePhotosynthesis