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The D1 protein of photosystem II is synthesized in precursor with a carboxyl-terminal extension. Interestingly, there is quite a range in chain length of the extension, which roughly depends upon the class of organisms. In cyanobacteria, e.g. in Synechocystis sp. PCC6803, the extension consists of 16 amino acid residues, seven residues longer than its counterpart in higher plants. In this study, we examined the D1 processing in Synechocystis sp. PCC6803 by pulse-chase experiments and detected a proteolytic intermediate of this process. This finding suggests that the elongated extension in this organism is excised with a sequential two-step proteolysis, which differs markedly from the manner observed in higher plants.

A cross-linked product between the D1 and D2 subunits was generated by treating isolated spinach PS II reaction center with a bifunctional cross-linker, 1,6-hexamethylene diisocyanate. To obtain information on the contact site(s) between D1 and D2 proteins, the cross-linked product was cleaved by CNBr, the resultant fragments were separated using a reverse-phase HPLC, and then the partial sequence of the cross-linked polypeptide fragments were determined. By comparing the results with the deduced amino acid sequence of the D1 and D2 proteins from spinach, it is concluded that the C-terminal domains of the D1 subunit (D308-A334) and that of the D2 subunit (Y297-L353) are in close proximity.

Synthetic oligopeptides of different chain lengths of 11 to 38 amino acids, corresponding to the carboxyl-terminal sequence of D1 precursor protein of the photosystem II reaction center, were subjected to a proteolytic cleavage by a processing enzyme isolated from spinach, in order to analyze the recognition signal. Practically the same K m and V max values were obtained for the oligopeptides consisting of more than 19 amino acids; a decrease in affinity, without affecting the V max value, was observed for the peptide consisting of 16 amino acids; no detectable activity was found for the peptide with 11 amino acids. When Asp-342 (12th residue from C-terminus) was replaced with Asn, for the peptide consisting of 16 amino acids, the enzymatic activity was completely abolished. In contrast, replacing Asp-342 with Glu had little effect. The efficiency of these oligopeptides as a substrate mentioned above, together with their effectiveness as an inhibitor, clearly demonstrated that the negative charge on Asp-342 plays a crucial role in the recognition, i.e. binding and cleavage, of the substrate by the processing enzyme, and suggested that the carboxyl-terminal extension consisting of 9 amino acids, by itself is not important in the binding.

Two distinct cross-linked products containing psbI gene product were generated in the photosystem II reaction center complex from spinach by treatment with bifunctional reagents directed to amino groups. The first product, which was generated by a 3,3'-dithiobis(succinimidyl propionate) treatment, is deduced to be formed between the epsilon-amino group of Lys3 of the psbI gene product, and a side-chain amino group present on the stromal extension of the D2 protein. The CNBr cleavage analysis of the cross-linked product predicted that the amino group of the D2 protein engaged in the cross-linking is either one of the three lysine residues on the N-terminal fragment (from N-terminus to Met19) or the Lys268 on the 6th fragment (from Val248 to Met275). The second product, which was also generated on the stromal side by a 1,6-hexamethylene diisocyanate treatment preferentially in alkaline conditions, is predicted to be formed between the epsilon-amino group of Lys3 of the psbI gene product and the N-terminal alpha-amino group of the alpha-subunit of cytochrome b559.

In the presence of externally added ATP, in the dark, isolated pea chloroplasts accumulate two proteins of molecular masses of about 22 and 24 kDa which precipitate with specific antibodies raised against the D1 protein. By chasing in the light, these proteins disappeared on the fluorogram concomitant with the appearance of the precursor- and mature-sized D1 proteins. Polysome analysis indicated that the 22-kDa component is associated with membrane-bound ribosomes and is thus ascribed to a translation intermediate of the D1 protein. On the other hand, the 24-kDa component could not be found in the polysome fraction under the experimental condition used, suggesting the possibility that this component is a degradation product of the D1 protein. The conclusion from this analysis is that the synthesis and/or stable accumulation of the D1 protein requires factor(s) caused by illumination, in addition to ATP, in isolated pea chloroplasts.

Antibodies raised against each of the constituent proteins of the photosystem II core complex, i.e. against the 47 kDa protein ( psb B product), the 43 kDa protein ( psb C product), cytochrome b -559 ( psb E and psb F products), the psb A product expressed in E. coli or against a synthetic oligopeptide corresponding to a part of the sequence of psb D gene, were utilized to determine the origin of higher molecular mass bands exhibited by SDS-polyacrylamide gel electrophoresis of photosystem II preparations, and to identify the two medium-size polypeptides in the 30 kDa region of the recently isolated photosystem II reaction center [(1987) Proc. Natl. Acad. Sci. USA 84, 109–112]. The result of analysis clearly indicated that the photosystem II reaction center consists entirely of D-1 ( psb A product), D-2 ( psb D product) and cytochrome b -559.

EPR signals in the high-spin region were studied at 10 K in photosystem II (PS II) particles and in a purified oxygen-evolving PS II reaction center complex under oxidizing conditions. PS II particles showed EPR peaks at g = 8.0 and 5.6, confirming the recent report by Petrouleas and Diner [(1986) Biochim. Biophys. Acta 849, 264-275]. Addition of 3-(3',4'-dichlorophenyl)-1,1-dimethylurea (DCMU) or o -phenanthroline shifted the peaks to be closer to g = 6.0 depending on the medium pH. On the other hand, the PS II reaction center complex showed peaks at g = 6.1 and 7.8, and at g = 6.1 and 6.4, in the absence and presence of o -phenanthroline, respectively. All these peaks were found to be decreased by the illumination at 10 K. These results suggest that the high-spin signals are due to Q 400 , Fe(III) atom interacting with the PS II primary electron acceptor quinone Q A as reported and that the Fe atom also interacts with the secondary acceptor quinone Q B . This interaction seems to induce the highly asymmetric ligand coordination of the Fe atom and to be affected by DCMU and o -phenanthroline in a somewhat different manner.

The enzyme involved in the processing of D1 precursor protein was solubilized from spinach thylakoids by Triton X-100 treatment and then partially purified in the presence of the detergent by Sephadex G-75 gel-filtration chromatography. The apparent molecular mass of the enzyme was estimated via this procedure to be about 34 kDa. The D1 precursor protein translated from the extracted spinach chloroplast RNA by a wheat germ cell-free system was used here as a substrate in measurements of the activity.

Effects of silicomolybdate on the charge recombination between P680+ and the reduced pheophytin were studied by absorption and electron paramagnetic resonance spectroscopies in the photosystem II D1/D2/cytochrome b-559 reaction center complex. This preparation lacks the primary and secondary quinone acceptors, QA and QB, and exhibits the charge recombination which produces the triplet state of P680 to a large extent. In the presence of silicomolybdate, the light-induced triplet signal of P680 was almost completely eliminated at cryogenic temperatures as well as at 4°C. Under these conditions, two types of signals, one reversible and the other irreversible, which are ascribable to P680+ and the cation radical of antenna chlorophyll a, respectively, were generated upon illumination at cryogenic temperatures. These results indicate that silicomolybdate, which is known to be an artificial electron acceptor of QA, rapidly receives electrons from the reduced pheophytin even at cryogenic temperatures and thus suppresses the radical pair recombination which occurs in the time range of nanosecond. P680+ formed by flash excitation in the presence of silicomolybdate relaxed mainly with a long half decay time of 74 ms at 4°C. This indicates that the reduction of P680+ by the secondary electron donor, Z, is significantly decreased.

The highly purified PS II reaction center complex with 47, 43, 32, 30 and 9 kDa (and 4.5 kDa) polypeptides was subjected to iodo-labeling to identify the polypeptide(s) participating on the donor side of the photosystem. The 32 kDa component (D2) and the aggregate(s) of about 60 kDa were exclusively labeled in the dark, suggesting the presence of a dark-stable oxidizing component on this polypeptide. Illumination of the complex in the presence of 2,6-dichlorophenolindophenol and iodide resulted in the preferential iodination of the 30 kDa component (D1) and the aggregate(s), but not the 47 kDa polypeptide. This iodo-labeling was completely inhibited by the addition of an electron donor, 1,5-diphenylcarbazide. From these results, it is concluded that the site of photooxidation of iodide, and presumably the site of Z binding, is located on or near the herbicide-binding polypeptide, D1, in the PS II reaction center complex.

(D1,D2) Photosystem II reaction centers were studied by flash absorption spectroscopy with microsecond resolution. Without addition, absorption transients in the 660–850 nm region are due to a triplet state, probably of P-680, with a decay half-time of 700 μs (no oxygen) or 40 μs (under air). With the addition of DBMIB, presumably acting as electron acceptor, new kinetic and spectral features appear, which are attributed to P-680. A 5 μs phase of decay is present, which is pH-dependent and is attributed to donation from Z to P-680+.

Flash-induced absorbance changes in the picosecond and nanosecond time range have been measured in the ‘D1-D2-cyt b-559 complex’ isolated from photosystem II membranes. The results indicate the efficient formation of the primary radical pair P680+ pheophytin−, which had a lifetime of about 36 ns, and the presence of unconnected chlorophyll in this preparation. It is concluded that the complex contains the active photosystem II reaction center, and that this reaction center contains at most 4 chlorophyll a molecules.

The stability of EPR Signal IIs in oxygen-evolving PS II particles in the different S-states has been studied as a function of storage time at 77 K. Signal IIs in dark-adapted samples retains the same intensity after 1 month storage at 77 K, while that in the S2 state produced by illumination at 195 K decays with a half-life time of about 5 days without decay in the manganese multiline intensity observed at 4.5 K. Signal IIs, once decayed in the S2 state, recovers its original intensity upon dark adaptation of the sample at 210 K accompanied by a decrease in the multiline intensity. A model for the charge recombination process for the S2 state involving Signal IIs is discussed.

Digitonin extracts of spinach thylakoid membranes obtained as 100000 × g supernatant have been shown to retain part of the oxygen-evolving activity if the extraction is carried out at acidic pH. The photosystem II reaction center complex with a uniform particle size of 300–500 kDa was purified from the extracts by ion-exchange/gel-permeation chromatography performed at pH 6.0 using DEAE-Toyopearl-650. The purified materials contained only two polypeptides of about 22 (not 24) and 33 kDa, in addition to those of the reaction center core complex, i.e., 47, 43, 30 and 10 kDa. The purified complex was fairly active in the photochemical oxygen evolution supported by phenyl-p-benzoquinone (~150μmol oxygen evolved·mg chl−1·h−1) and DCIP photoreduction with water. From these results it is concluded that the principal site of water oxidation is associated rather with the photosystem II core complex than with the water soluble peripheral complex consisting of 18,24 and 33 kDa polypeptides and that the thylakoid membrane structure is not essential for manifestation of oxygen-evolving activity.

In an in vitro translation experiment using spinach chloroplasts a novel protein band of about 17.5 kDa appeared by light to dark transition. The protein never accumulated in detectable amounts either in continuous illumination or in continuous darkness. The 17.5 kDa protein accumulated upon light—dark transition, on the other hand, disappeared by the subsequent illumination. Accumulation of the protein in light, however, was observed when stromal level of ATP in chloroplasts was lowered after preillumination by the addition of various chemical compounds which, irrespective of the mode of action, eventually decrease the ATP level, e.g. atrazine, carbonyl-cyanide-?-chlorophenyl hydrazone and glycerate. The dark-accumulated protein was concluded to be a translation intermediate of DI protein based on the facts that this component precipitates with specific antibodies and is resistant to lyxylendopeptidase treatment. The suppression by chloramphenicol of both appearance upon light-dark transition and disappearance by the subsequent illumination of the protein also supported this conclusion. The phenomenon was discussed in terms of pausing in the translation of psbA mRNA

A pigment-protein complex consisting of D1 and D2 proteins, but depleted in the two lower molecular mass components of photosystem II, i.e. cytochrome b-559 and psbI gene product, has been isolated by octyl-β-D-glucopyranoside treatment of the purified photosystem II reaction center complex from spinach [(1987) Proc. Natl. Acad. Sci. USA 84, 109-112], followed by separation by high performance liquid chromatography using a gel-permeation column (TSK G3000 SW). The isolated complex is photochemically active in the photoreduction of intrinsic pheophytin a under steady-state illumination, in the presence of dithionite and methyl viologen, and exhibits pigment stoichiometries similar to those in the untreated reaction center, indicating that the D1-D2 complex provides the site of primary photochemistry in photosystem II, as well as the principal binding sites of pigments in the reaction center.

Treatment with 3 M urea of the oxygen-evolving PS II core complex previously described [(1985) FEBS Lett. 179, 60-64] resulted in approx. 80% release of 33 kDa polypeptide accompanied by partial (40%) inactivation of the water-splitting activity. On the other hand, the treatment only slightly decreased the amount of bound Mn from 2.7 to 2.0 atoms per 50 chlorophyll a . Readdition of purified 33 kDa polypeptide to the urea-treated sample almost completely restored the oxygen-evolving activity. These results suggest that the Mn atoms functional for oxygen-evolving activity are located in the PS II reaction center core complex, and that the 33 kDa polypeptide is not essential for water oxidation but is necessary for the full oxygenevolving activity. Photosystem II Reaction center Reconstitution Urea treatment Manganese binding 33 kDa polypeptide Oxygen evolution

A clear parallelism was demonstrated between the efficiency as substrate of the substituted oligopeptides corresponding to the carboxy-terminal (C-terminal) sequence of the precursor D1 protein (pD1) in the in vitro enzymatic assay and their competitive inhibitory capacity toward the proteolytic C-terminal processing of the full-length pD1 integrated in the intact photosystem II complex embedded in the thylakoid membrane of Scenedesmus obliquus LF-1 mutant, as shown e.g. by the influence of L343A, A345G and A345V substitutions and the effect of C-terminal fragments. This suggests that the basic mechanism for substrate recognition by the processing protease elucidated in the enzymatic analysis using synthetic oligopeptides is also effective in vivo, although it can sometimes be difficult to detect the consequence of amino acid substitution in the integrated systems.

Solvent extraction at approximately 4 degrees C in complete darkness, and subsequent analysis by high-pressure liquid chromatography (at approximately 4 degrees C) using an apparatus equipped with a two-dimensional diode-array detector, spectroscopically identified 15-cis-beta-carotene in the reaction center (RC) of spinach photosystem II (PS II). The result revises a previous conclusion that beta-carotene bound to the PS II RC takes the all-trans configuration [Fujiwara, H., Hayashi, H., Tasumi, M., Kanaji, M., Koyama, Y. and Satoh, K. (1987) Chem. Lett. 2005-2008], and generalizes the concept established for purple photosynthetic bacteria that 15-cis-carotenoid is naturally selected by the photosynthetic reaction centers.

Pigment stoichiometries in the D1/D2/cytochrome b-559 reaction center complex from spinach have been studied by spectrophotometry and normalphase silica HPLC of the solvent extracts. Based on the well-accepted molar ratio Pheo a/P680 = 2, the results are summarized by Chl a/P680 = 6.01 ± 0.37 (n = 24) and β-carotene/P680= 1.84 ± 0.11 (n = 4). These stoichiometries are significantly different from those of bacterial reaction center complexes.

A synthetic COOH-terminal oligopeptide of D1 protein deduced from the spinach psbA gene (Asn-325-Gly-353) was subjected to proteolytic digestion by purified processing enzyme of D1 protein [(1989) FEBS Lett. 246, 218–222] and the following two fragments were obtained as cleavage products: a COOH-terminal 9-amino-acid fragment (Ala-345-Gly-353) and an NH2-terminal 10-amino-acid fragment (Asn-325-Arg-334). It was concluded that: (i) the oligopeptide consisting of the COOH-terminal 29-amino-acid sequence deduced from the spinach psbA gene provides the recognition domain for the processing enzyme; (ii) the cleavage takes place at the predicted processing site of native DI precursor protein (COOH side of Ala-344); and (iii) another cleavage takes place at an additional site (COOH side of Arg-334) for the synthetic substrate, but not for the native D1 precursor protein.