Høj P B, Svendsen I, Scheller H V, Møller B L
J Biol Chem. 1987 Sep 15;262(26):12676-84.
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.
本文报道了一种改进的方法，用于从大麦（Hordeum vulgare L.）类囊体膜大规模制备光系统I（PS-I）囊泡。PS-I囊泡含有分子量分别为82、18、16、14和9 kDa的多肽，其表观摩尔比为4:2:2:1:2。将PS-I囊泡暴露于离液剂后，18 kDa、16 kDa和9 kDa的多肽被纯化至均一。分离得到的9 kDa多肽结合了变性PS-I囊泡中65 - 70%的零价硫，其余30 - 35%与P700 - 叶绿素a - 蛋白1结合。测定了9 kDa多肽的N端氨基酸序列（29个残基）。与多形藻（Marchantia polymorpha）（大宫，K.，福泽，H.，小池，T.，白井，H.，佐野，T.，佐野，S.，梅园，K.，志木，Y.，竹内，M.，张，Z.，青田，S.-i.，井口，H.，和小泽，H.（1986）《自然》322，572 - 574）和烟草（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.，岛田，H.，和杉浦，M.（1986）《欧洲分子生物学组织杂志》5，2043 - 2049）叶绿体基因组的核苷酸序列进行比较，确定了编码9 kDa多肽的叶绿体基因。我们将此基因命名为psaC。从psaC基因推导的完整氨基酸序列表明9 kDa的PS-I多肽是一种2[4Fe - 4S]蛋白。由于P700 - 叶绿素a - 蛋白1携带中心X，9 kDa多肽携带中心A和B。亲水性图谱可以特异性鉴定分别配位中心A和B的半胱氨酸残基。除了N端甲硫氨酸残基的缺失外，psaC基因的初级翻译产物未经过蛋白水解加工。P700 - 叶绿素a - 蛋白1结合4个铁原子和4分子酸不稳定硫化物/分子P700。P700 - 叶绿素a - 蛋白1的两个脱辅基蛋白各自含有序列Phe - Pro - Cys - Asp - Gly - Pro - Gly - Arg - Gly - Gly - Thr - Cys（菲什，L. E.，屈克，U.，和博戈拉德，L.（1985）《生物化学杂志》260，1413 - 1421）。PS-I组成多肽的化学计量表明每个P700分子存在该序列的四个拷贝。中心X可能由两个[2Fe - 2S]中心组成，它们与这四个片段中含有的8个半胱氨酸残基结合。
