Elucidation of Molecular Properties of Water-dehydrogenase in Chloroplasts

本研究旨在揭示叶绿体中向有氧生物供氧的水脱氢酶的分子性质。(1)分离到由D1、D2和Cytb-559组成的反应中心-PSII反应中心核心复合体，显示出PSII反应，表明D1和/或D2是反应中心。由D_1、D_2、Cyt b-559、47-kDa和43-kDa组成的核心复合体与33-kDa的多肽结合后能氧化水样。(2)分离到了参与水氧化的外周多肽和Mn-3个外周多肽(33、23和18 kDa)。用免疫学方法揭示了它们的拓扑结构。23 kDa的多肽参与钙离子的结合。18-kDa的N-末端残基是其与核心结合所必需的。测定了33 kDa多肽的氨基酸序列。33 kDa的多肽参与了锰的结合，它的耗竭抑制了S-3向S-0状态的推进。靶分子大小分析表明，结合锰所需的分子大小为65 kDa，表明结合至少需要两个多肽。(3)钙、氯和反应机制--一个钙原子与反应中心结合，是P680与Z反应所必需的。氯的耗竭抑制了S-1向S-2状态的推进。在PSII膜上过氧化氢放氧是由闪光诱导的过氧化氢活性催化的，S-0/S-2循环参与其中。细胞色素C氧化酶反应中的质子吸收步骤已得到证实。

The present research project has been organized to reveal molecular properties of water-dehydrogenase in chloroplasts which donates dioxygen to aerobic organisms and is the most abundant reaction in the globes.(1) Reaction center - PSII reaction center core complex which is composed of D1, D2 and Cyt b-559 was isolated and showed the PSII reaction, indicating that D1 and/or D2 is the reaction center. The core complex composed of D1, D2, Cyt b-559, 47-kDa and 43-kDa peptides can oxidize water after binding of the 33-kDa peptide.(2) Peripheral peptides and Mn- Three peripheral peptides (33, 23 and 18 kDa) participating in the water oxidation have been isolated. Their topological arrangement was revealed by the immunoloical method. The 23-kDa peptide participates in the binding of Ca. The N-terminal residues of the 18-kDa are is necessary to its binding to the core. Amino acid sequence of the 33-kDa peptide was determined. The 33- kDa peptide has been shown to participate in the binding of Mn and its depletion caused the inhibition of the advancement of the s-3 to s-0 states. The target size analysis indicates that molecular size required for the binding of Mn is 65 kDa, suggesting that at least two peptides are necessary to the binding.(3) Calcium, chloride and reaction mechanism - One atom of Ca binds to the reaction center and is essential to the reaction between P680 and Z. Chloride depletion resulted in the inhibition of the advancement of the s-1 to s-2 states. Oxygen evolution from hydrogen peroxide in PSII membranes is catalyzed by the flashinduced catalatic activity in which the S-0/s-2 cycle participates. Proton uptake step in the cytochrome c oxidase reaction has been confirmed.