The 110-kDa reaction center protein of photosystem-I, P700-chlorophyll A-protein-1, is an iron-sulfur protein

Germination and growth of barley (Hordeum vulgare L.) in the presence of 59Fe2+ or 35SO4(2-) allows heavy incorporation of both isotopes into the thylakoid membranes and into isolated photosystem I particles. Analysis of 59Fe-labeled preparations by sodium dodecyl sulfate-polyacrylamide gel electrophoresis under mild conditions demonstrates that a minimum of four iron atoms/P700 is carried on P700-chlorophyll a-protein 1. When isolated from 35S-labeled preparations, P700-chlorophyll a-protein 1 binds zero valence 35S, which is converted into acid-labile [35S]sulfide by dithiothreitol reduction. Isolated photosystem I particles contain 14 acid-labile sulfide atoms and 10 iron atoms for each molecule of P700 and are composed of polypeptides of 110, 18, 15, 10, and 8 kDa of which the 10-kDa component is loosely bound. Under the electrophoretic conditions used, none of the low molecular weight polypeptides could be shown to be specifically associated with iron or acid-labile sulfide. Carboxymethylation of cysteine residues shows a high cysteine content in the 8-kDa polypeptide and an intermediate content in the 110- and 18-kDa polypeptides, whereas the 15-kDa polypeptide is devoid of sulfur amino acids. The experiments with the 59Fe-labeled thylakoids reveal other labeled polypeptides not associated with photosystem I, namely cytochrome f and possibly cytochromes b6 and b559

Evolution of polysaccharide hydrolase substrate-specificity: catalytic amino-acids are conserved in barley 1,3-1,4-glucanase and 1,3-beta-glucanase

Catalytic amino acid residues in a 1,3-beta-D-glucan 3-glucanohydrolase (EC 3.2.1.39) and a homologous 1,3-1,4-beta-D-glucan 4-glucanohydrolase (EC 3.2.1.73) from barley have been investigated. To identify amino acids responsible for protonation of the glycosidic oxygen during hydrolysis, carbodiimide-mediated labeling of the enzymes with [14C]glycine ethyl ester was performed. This resulted in loss of activity and specific modification of the Glu288 residues in both enzymes. The stoichiometry of labeling was approximately 1:1, and modification was reduced in the presence of substrate analogues. Based on these data, the Glu288 residues are likely to be present at the active sites of the respective enzymes and may represent the catalytic acids in the hydrolytic reaction. The catalytic nucleophiles of the two enzymes were investigated by labeling with specific, mechanism-based epoxyalkyl-beta-oligoglucosides. Amino acid residues Glu232 and Glu231 were identified as the likely catalytic nucleophiles in the 1,3-1,4- and 1,3-beta-glucanases, respectively. Thus the position of the catalytic nucleophile and the putative proton donating amino acids in the two classes of beta-glucan endohydrolases are conserved. The acquisition of distinct substrate specificities in the evolution of these related enzymes may therefore not require the recruitment of novel catalytic amino acids but rather differences in their positioning at the active site and/or changes in substrate binding residues

Identification of a chloroplast-encoded 9-Kda polypeptide as a 2[4Fe-4S] protein carrying center-A and center-B of photosystem-I

An improved procedure is reported for large-scale preparation of photosystem I (PS-I) vesicles from thylakoid membranes of barley (Hordeum vulgare L.). The PS-I vesicles contain polypeptides of molecular masses 82, 18, 16, 14, and 9 kDa in an apparent molar ratio of 4:2:2:1:2. The 18-, 16-, and 9-kDa polypeptides were purified to homogeneity after exposure of the PS-I vesicles to chaotropic agents. The isolated 9-kDa polypeptide binds 65-70% of the zero-valence sulfur of denatured PS-I vesicles, and the remaining 30-35% is bound to P700-chlorophyll a-protein 1. The N-terminal amino acid sequence (29 residues) of the 9-kDa polypeptide was determined. Comparison with the nucleotide sequence of the chloroplast genome of Marchantia polymorpha (Ohyama, K., Fukuzawa, H., Kohchi, T., Shirai, H., Sano, T., Sano, S., Umesono, K., Shiki, Y., Takeuchi, M., Chang, Z., Aota, S.-i., Inokuchi, H., and Ozeki, H. (1986) Nature 322, 572-574) and of Nicotiana tabacum (Shinozaki, K., Ohme, M., Tanaka, M., Wakasugi, T., Hayashida, N., Matsubayashi, T., Zaita, W., Chunwongse, J., Obokata, J., Yamaguchi-Shinozaki, K., Ohto, C., Torazawa, K., Meng, B. Y., Sugita, M., Deno, H., Kamogashira, T., Yamada, K., Kusuda, J., Takaiwa, F., Kato, A., Tohdoh, N., Shimada, H., and Sugiura, M. (1986) EMBO J. 5, 2043-2049) identified the chloroplast gene encoding the 9-kDa polypeptide. We designate this gene psaC. The complete amino acid sequence deduced from the psaC gene identifies the 9-kDa PS-I polypeptide as a 2[4Fe-4S] protein. Since P700-chlorophyll a-protein 1 carries center X, the 9-kDa polypeptide carries centers A and B. A hydropathy plot permits specific identification of the cysteine residues which coordinate centers A and B, respectively. Except for the loss of the N-terminal methionine residue, the primary translation product of the psaC gene is not proteolytically processed. P700-chlorophyll a-protein 1 binds 4 iron atoms and 4 molecules of acid-labile sulfide/molecule of P700. Each of the two apoproteins of P700-chlorophyll a-protein 1 contains the sequence Phe-Pro-Cys-Asp-Gly-Pro-Gly-Arg-Gly-Gly-Thr-Cys (Fish, L. E., Kück, U., and Bogorad, L. (1985) J. Biol. Chem. 260, 1413-1421). The stoichiometry of the component polypeptides of PS-I indicates the presence of four copies of this sequence per molecule of P700. Center X may be composed of two [2Fe-2S] centers bound to the 8 cysteine residues contained in these four segments

The complete primary structure of rat chaperonin-10 reveals a putative-beta-alpha-beta nucleotide-binding domain with homology to P21(ras)

The first complete amino-acid sequence of a mitochondrial chaperonin 10 is reported. The amino-terminal alanine residue is acetylated, a modification that may be required for the interaction with heptameric chaperonin 60. Part of the sequence constitutes a potential dinucleotide binding motif and is identical with 7 out of 10 residues in the GTP-binding site of p21ras. This similarity may be the structural basis for the recently discovered complex between p21ras and chaperonin 60 in intact cell

Partial amino-acid sequences of 2 nuclear-encoded photosystem-I polypeptides from barley

Partial amino acid sequences of two nuclear-encoded Photosystem I polypeptides of barley (Hordeum vulgare L.) have been determined. The sequence data include the N- and C-terminal parts of the polypeptides. The N-terminal parts of the polypeptides are homologous and rich in proline and alanine. In one polypeptide, the homologous region is repeated. The two polypeptides are peripheral membrane proteins