Elucidation of Molecular Properties of Water-dehydrogenase in Chloroplasts

本课题旨在揭示叶绿体中水脱氢酶（water-dehydrogenase）的分子特性，该酶是地球上最丰富的向需氧生物提供双氧的反应。(1)分离出由D1、D2和Cyt b-559组成的反应中心- PSII反应中心核心配合物，显示PSII反应，说明D1和/或D2为反应中心。由D1、D2、Cyt b-559、47-kDa和43-kDa肽段组成的核心复合物在与33-kDa肽段结合后可以氧化水。(2)外周肽和Mn-分离到参与水氧化的3个外周肽（33、23和18 kDa）。免疫学方法揭示了它们的拓扑结构。23-kDa肽参与Ca的结合。18-kDa的n端残基是其与核心结合所必需的。测定了33-kDa肽的氨基酸序列。33- kDa肽已被证明参与Mn的结合，其缺失导致s-3到s-0状态的进展受到抑制。靶大小分析表明，Mn结合所需的分子大小为65 kDa，表明至少需要两个肽才能结合。(3)钙、氯和反应机理——一个Ca原子与反应中心结合，在P680和z之间的反应中是必不可少的。氯离子的缺失抑制了s-1向s-2态的推进。过氧化氢在PSII膜中的析氧是由闪光灯诱导的催化活性催化的，其中S-0/s-2循环参与。证实了细胞色素c氧化酶反应中的质子摄取步骤。

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.