Tetsuo Hiyama

The gene for the 9 kd polypeptide, a possible apoprotein for the iron-sulfur centers A and B of the photosystem I complex, in tobacco chloroplast DNA

SummaryThe gene for the 9 kd polypeptide (a possible apoprotein for the iron-sulfur centers A and B) of photosystem I has been located in the small single-copy region of tobacco chloroplast DNA. This gene (psaC) was identified by comparing the N-terminal amino acid sequence of the spinach 9 kd polypeptide with the entire sequence of tobacco chloroplast genome. The gene organization is ndhE (101 codons) — 263 by spacer — psaC (S1 codons) — 94 by spacer - ndhD (509 codons). Northern blot hybridization revealed that psaC is transcribed in the chloroplasts. The deduced amino acid sequence and secondary structure are presented. The predicted polypeptide is rich in cysteine residues and contains a unique repeated sequence.

The mechanism of the degradation of psaB gene product, one of the photosynthetic reaction center subunits of photosystem I, upon photoinhibition

The psaB gene product (PsaB protein), one of the reaction center subunits of Photosystem I (PS I), was specifically degraded by light illumination of spinach thylakoid membranes. The degradation of the protein yielded N-terminal fragments of molecular mass 51 kDa and 45 kDa. The formation of the 51 kDa fragment was i) partially suppressed by the addition of phenylmethylsulfonyl fluoride or 3,4-dichloroisocoumarin, which are inhibitors of serine proteases, and ii) enhanced in the presence of hydrogen peroxide during photoinhibitory treatment, but iii) not detected following hydrogen peroxide treatment in the dark. These results suggest that the hydroxyl radical produced at the reduced iron-sulfur centers in PS I triggers the conformational change of the PS I complex, which allows access of a serine-type protease to PsaB. This results in the formation of the 51 kDa N-terminal fragment, presumably by cleavage on the loop exposed to the stromal side, between putative helices 8 and 9. On the other hand, the formation of the 45 kDa fragment, which was enhanced in the presence of methyl viologen but did not accompany the photoinhibition of PS I, was not affected by the addition of hydrogen peroxide or protease inhibitors. Another fragment of 18 kDa was identified as a C-terminal counterpart of the 45 kDa fragment. N-terminal sequence analysis of the 18 kDa fragment revealed that the cleavage occurred between Ala500 and Val501 on the loop exposed to the lumenal side, between putative helices 7 and 8 of the PsaB protein.

Chlorophyll a′/P-700 and pheophytin a/P-680 stoichiometries in higher plants and cyanobacteria determined by HPLC analysis

Abstract The chlorophyll (CHl) a ′ and pheophytin (Pheo) a contents in photosynthetic organs have been reinvestigated by means of silica HPLC. Previously reported data (Watanabe, T. et al. (1985) BBA 807, 110–117 and FEBS Lett. 191, 252–256) were found to contain errors from two sources: slight epimerization and pheophytinization of Chl a during pigment extraction and PS I particle preparation, and an accidental overlap of a Chl a alteration product ( meso -chlorinated Chl a ) with Chl a ′ on the HPLC trace. The pigment molar ratios determined with the improved extraction/HPLC procedure were Chl a /Chl a ′ ÷ 460 and Chl a /Pheo a ÷ 120 in more than 100 samples from 13 different higher plants, and Chl a /Chl a ′ ÷ 125 and Chl a /Pheo a ÷ 100 in three cyanobacteria. These values, when combined with the PS I particle composition (Chl a ′/P-700 = 1) and the widely accepted molar ratio Pheo a /P-680 of 2, yield P-680/P-700 = 1.9 ± 0.5 and 0.65 ± 0.20 as reaction center stoichiometries in higher plants and cyanobacteria, respectively. The present results correspond well with the recently published Chl a /reaction center stoichiometry data acquired by (photo-)redox titrations of key components.

Identification of photosystem I components from the cyanobacterium, Synechococcus vulcanus by N‐terminal sequencing

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.

Polypeptide composition of higher plant photosystem I complex: Identification of psaI, psaJ and psaK gene products

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.

Cloning, characterization and functional analysis of groESL operon from thermophilic cyanobacterium Synechococcus vulcanus

Genes encoding 10914 Da and 58267 Da polypeptides homologous to groES and groEL of Escherichia coli were cloned and sequenced from a thermophilic cyanobacterium, Synechococcus vulcanus. The deduced amino acid sequence of the GroEL protein was much more homologous to GroELs of other cyanobacteria which accompany GroES than another GroEL homolog of S. vulcanus (GroEL2) reported previously (M. Furuki, N. Tanaka, T. Hiyama, and H. Nakamoto, Biochim. Biophys. Acta 1294 (1996) 106-110). We designate the gene as groEL1 to distinguish it from the non-operon forming groEL2 gene. A 9-base pair inverted repeat sequence (TTAGCACTC-N9-GAGTGCTAA) was located upstream of the promoter region of groEL1, which was absent in groEL2. Southern blot analysis indicated that only one groESL1 operon was present in the genomic DNA of S. vulcanus. The amount of the bicistronic, 2.3 kb transcript of groESL1 operon increased 30-fold within 30 min upon heat shock. The increase was completely inhibited by chloramphenicol, suggesting the involvement of heat-induced production of a polypeptide. Introduction of the cloned groEL1 gene into a groEL defective mutant of E. coli resulted in the complementation of heat sensitivity, which contrasted with the previous result with groEL2.

Cloning, characterization and functional analysis of groEL-like gene from thermophilic cyanobacterium Synechococcus vulcanus, which does not form an operon with groES☆

A gene encoding 57 102 Da polypeptide homologous to groEL of Escherichia coli but accompanying no groES, has been cloned and sequenced from a thermophilic cyanobacterium, Synechococcus vulcanus. The amount of the gene transcript increased several folds by heat shock. The gene was expressed as a minor component of two types of HSP60, and designated as groEL2. Although expressed and induced well upon heat shock treatment in the E. coli, introduction of the cloned groEL2 gene of S. vulcanus into an E. coli groEL-less mutant did not result in the complementation of heat sensitivity.

Changes in composition of membrane proteins accompanying the regulation of PS I/PS II stoichiometry observed with Synechocystis PCC 6803

Changes in composition of membrane proteins in Synechocystis PCC 6803 induced by the shift of light regime for photosynthetic growth were studied in relation to the regulation of PS I/PS II stoichiometry. Special attention was paid to the changes in abundance of proteins of PS I and PS II complexes. Composition was examined using a LDS-PAGE and a quantitative enzyme immunoassay. Abundance of PsaA/B polypeptides and the PsaC polypeptide of the PS I complex, on a per cell basis, increased under the light regime exciting preferentially PS II and decreased under the light regime exciting mainly PS I. Similar changes were observed with polypeptides of 18.5, 10 and 8.5 kDa. The abundance of other proteins associated with membranes, including PsbA polypeptide of the PS II complex, was fairly constant irrespective of light regime. These results are consistent with our previous observations with other strains of cyanophytes (Anabaena variabilis M2 and Synechocystis PCC 6714) that PS I is the variable component in changes in PS I/PS II stoichiometry in response to changing light regimes for photosynthesis.

Destruction of Photosystem I Iron-Sulfur Centers of Spinach and Anacystis nidulans by Mercurials

Several mercurials destroyed Photosystem I (PSI) Fe−S centers in thylakoids and PSI particles from spinach and fromAnacystis nidulans as revealed by EPR measurement and acid-labile sulfide determination. Of the mercurials tested, HgCl2 was the most effective, followed by phenylmercuric acetate (PMA), Mersalyl and pCMB in the order of decreasing effectiveness. Fe−S centers in thylakoids were much more labile than those in PSI particles. InA. nidulans thylakoids, Center B was more susceptible than Center A and X to PMA. P700 was less susceptible to PMA than these centers. For 50% inactivation of Fe−S centers inA. nidulans thylakoids, about 0.4 mM PMA was required for Center B, and about 1 mM was required for Center A and X. These differential susceptibilities of Fe−S centers were more pronounced with HgCl2 than with the other three mercurials.

15-Cis-β-carotene found in the reaction center of spinach Photosystem I

Abstractβ-Carotene was extracted from spinach Photosystem I reaction centers (one consisting of the Psa A, B, C, D and E subunits and the other consisting of the Psa A and B subunits alone), and the extract was subjected to high-pressure liquid chromatography using an apparatus equipped with a two-dimensional diode-array detector; all the procedures were performed at ≈ 4 °C in complete darkness. Both 15-cis and all-trans-β-carotene were identified in the extract by means of electronic absorption spectroscopy. Thus, universal presence of 15-cis carotenoid in the reaction centers of purple photosynthetic bacteria and of spinach Photosystem I and Photosystem II has been shown.