Purification and characterization of a stable oxygen-evolving Photosystem II complex from a marine centric diatom, Chaetoceros gracilis
Purification and characterization of a stable oxygen-evolving Photosystem II complex from a marine centric diatom, Chaetoceros gracilis
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DOI：
10.1016/j.bbabio.2009.09.008
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发表时间：
2010-02-01
影响因子：
4.3
通讯作者：
Enami, Isao
中科院分区：
文献类型：
--
作者：
Nagao, Ryo;Tomo, Tatsuya;Enami, Isao
已经从海洋中心硅藻，纤细角毛藻（Chaetoceros gracilis）制备了保留高放氧活性的放氧光系统II颗粒（粗PSII）（Nagao等人，2007年）。然而，粗PSII，含有大量的岩藻黄质叶绿素a/c结合蛋白（FCP）。在这项研究中，一个纯化的PSII复合物，这是剥夺了FCP的主要成分，通过一个步骤的阴离子交换色谱分离从粗PSII处理Triton X-100。纯化的PSII仍与PsbO、PsbQ ′、PsbV、Psb 31和PsbU这5种外源蛋白结合，在苯基对苯醌存在下表现出2135 μ mol O ~（-2）（mg Chl a）（-1）h（-1）的高放氧活性，这几乎不依赖于CaCl_2的加入。该活性比粗PSII的活性高2.5倍以上。3-（3，4）-二氯苯基-（1，1）-二甲基脲（DCMU）可完全抑制该酶的活性。纯化的PSII在2分子脱镁叶绿素a的基础上含有42分子Chl a、2分子硅甲藻黄素和2分子Chl c，其吸收光谱和荧光光谱与其他生物的PSII相似。在这项研究中，我们还发现，粗PSII是显着不稳定的，作为一个显着的失活的氧释放，叶绿素漂白和降解PSII亚基在25摄氏度在黑暗中孵育过程中观察。相反，这些失活，漂白和降解几乎没有检测到在纯化的PSII。因此，我们首次成功地从硅藻细胞中制备了稳定的PSII。(C)2009爱思唯尔有限公司版权所有。
Oxygen-evolving Photosystem II particles (crude PSII) retaining a high oxygen-evolving activity have been prepared from a marine centric diatom, Chaetoceros gracilis (Nagao et al., 2007). The crude PSII, however, contained a large amount of fucoxanthin chlorophyll a/c-binding proteins (FCP). In this study, a purified PSII complex which was deprived of major components of FCP was isolated by one step of anion exchange chromatography from the crude PSII treated with Triton X-100. The purified PSII was still associated with the five extrinsic proteins of PsbO, PsbQ', PsbV, Psb31 and PsbU, and showed a high oxygen-evolving activity of 2135 mu mol O-2 (mg Chl a)(-1) h(-1) in the presence of phenyl-p-benzoquinone which was virtually independent of the addition of CaCl2. This activity is more than 2.5-fold higher than the activity of the crude PSII. The activity was completely inhibited by 3-(3,4)-dichlorophenyl-(1,1)-dimethylurea (DCMU). The purified PSII contained 42 molecules of Chl a, 2 molecules of diadinoxanthin and 2 molecules of Chl c on the basis of two molecules of pheophytin a, and showed typical absorption and fluorescence spectra similar to those of purified PSIIs from the other organisms. In this study, we also found that the crude PSII was significantly labile, as a significant inactivation of oxygen evolution, chlorophyll bleaching and degradation of PSII subunits were observed during incubation at 25 degrees C in the dark. In contrast, these inactivation, bleaching and degradation were scarcely detected in the purified PSII. Thus, we succeeded for the first time in preparation of a stable PSII from diatom cells. (C) 2009 Elsevier B.V. All rights reserved.