Biochemical and spectroscopic characterization of a new oxygen-evolving photosystem II core complex from the cyanobacterium Synechocystis PCC 6803.
Biochemical and spectroscopic characterization of a new oxygen-evolving photosystem II core complex from the cyanobacterium Synechocystis PCC 6803.
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来自蓝藻集胞藻 PCC 6803 的新型释氧光系统 II 核心复合物的生化和光谱表征。
DOI：
10.1021/bi00181a021
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发表时间：
1994
期刊：
影响因子：
2.9
通讯作者：
B. Diner
中科院分区：
文献类型：
--
作者：
Xiao;B. Diner
我们在此描述了一种新程序，可以从蓝藻集胞藻 PCC 6803 中快速（12-13 小时）分离纯放氧光系统 II (PSII) 核心复合物。该程序涉及类囊体膜的十二烷基麦芽糖苷提取，然后使用弱阴离子交换剂进行单步柱色谱。 SDS-PAGE 和免疫印迹显示该复合物由 5 种内在膜蛋白（CP47、CP43、D1、D1 和 cyt b559）、一种外在蛋白（MSP）和一种分子量约为 26 kDa 的未知蛋白组成。标准化为 2 个脱镁叶绿素 a 分子的化学和功能分析表明，该 PSII 核心复合物含有 1 个光活性质体醌、QA、4 个锰原子、38 个叶绿素 a 分子、1 个细胞色素 b559、2 个质体醌-9 和 9-10 个 β-胡萝卜素。该配合物表现出高析氧速率，在 2,5-二氯苯醌作为人工电子受体存在时，通常为 2400-2600 mumol O2 (mg of Chl)-1 h-1，最佳 pH 值为 6.5。在 198 K 照射后，在 10 K 下观察到由放氧复合物 (OEC) 的 S2 态产生的强光减去暗多线 EPR 信号。每饱和微秒闪光的绝对氧产量的测定表明，基本上所有 PSII 中心都含有功能性放氧复合物。室温光照下，OEC 的直接氧化剂氧化还原活性酪氨酸 YZ 不存在光累积，进一步支持了这一点。根据 EPR 光谱、氧化减去还原差光谱和 SDS-PAGE，该制剂在每摩尔 PSII 的基础上仅含有痕量的 PSI（约 0.04）、细胞色素 b6/f 复合物（< 或 = 0.01）和ATP酶（<或= 0.05）。所有这些结果表明，该 PSII 制剂是迄今为止来自集胞藻 6803 的纯度最高的析氧核心复合物，它保留了所有对析氧有活性的反应中心。由于集胞藻 6803 广泛用于 PSII 的定点诱变，因此该制剂对于野生型和定点突变体的 PSII 的光谱和生化分析特别有价值。
We describe here a new procedure permitting rapid (12-13 h) isolation of a pure oxygen-evolving photosystem II (PSII) core complex from the cyanobacterium Synechocystis PCC 6803. This procedure involves dodecyl maltoside extraction of thylakoid membranes followed by single-step column chromatography using a weak anion-exchanger. SDS-PAGE and immunoblotting show that the complex consists of five intrinsic membrane proteins (CP47, CP43, D1, D1, and cyt b559), one extrinsic protein (MSP), and one unknown protein with a molecular mass of approximately 26 kDa. A chemical and functional analysis, normalized to 2 molecules of pheophytin a, indicates that this PSII core complex contains 1 photoactive plastoquinone, QA, 4 manganese atoms, 38 chlorophyll a molecules, 1 cytochrome b559, 2 plastoquinone-9, and 9-10 beta-carotenes. The complex exhibits high rates of oxygen evolution, typically 2400-2600 mumol of O2 (mg of Chl)-1 h-1 in the presence of 2,5-dichlorobenzoquinone as an artificial electron acceptor with a pH optimum of 6.5. A strong light minus dark multiline EPR signal, arising from the S2 state of the oxygen-evolving complex (OEC), is observed at 10 K following illumination at 198 K. The determination of the absolute oxygen yield per saturating microsecond flash indicates that essentially all of the PSII centers contain functional oxygen-evolving complexes. This point is further supported by the absence of photoaccumulation, upon room temperature illumination, of the immediate oxidant of the OEC, redox-active tyrosine, YZ.. On the basis of EPR spectra, oxidized minus reduced difference spectra, and SDS-PAGE, the preparation contains on a per mole basis with PSII only trace amounts of PSI (approximately 0.04), cytochrome b6/f complex (< or = 0.01), and ATPase (< or = 0.05). All of these results indicate that this PSII preparation is to date the most highly purified oxygen-evolving core complex from Synechocystis 6803 that retains all of the reaction centers active for oxygen evolution. As Synechocystis 6803 is being used extensively for site-directed mutagenesis of PSII, this preparation is particularly valuable for spectroscopic and biochemical analyses of PSII from wild-type and from site-directed mutants.