Manganese proteins isolated from spinach thylakoid membranes and their role in O2 evolution. II. A binuclear manganese-containing 34 kilodalton protein, a probable component of the water dehydrogenase enzyme.
Manganese proteins isolated from spinach thylakoid membranes and their role in O2 evolution. II. A binuclear manganese-containing 34 kilodalton protein, a probable component of the water dehydrogenase enzyme.
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从菠菜类囊体膜中分离出的锰蛋白及其在 O2 演化中的作用。
DOI：
10.1016/0005-2728(84)90172-5
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发表时间：
1984
期刊：
影响因子：
--
通讯作者：
Dismukes,GC
中科院分区：
文献类型：
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作者：
Abramowicz,DA;Dismukes,GC
已经发现了导致锰保留在33-34 kDa蛋白质上的提取条件，该蛋白质首先由Kuwabara和Murata作为脱辅基蛋白分离（Kuwabara，T.和Murata，N.（1979）生物化学。Biophys Acta 581，228-236）。通过在菠菜粒-类囊体膜的蛋白质提取期间用亲水性和亲脂性氧化还原缓冲液保持氧化溶液电势，观察到33-34 kDa蛋白质结合最多2 Mn/蛋白质，其不通过延长透析相对于缓冲液释放。该锰是通常与氧释放复合物相关的4 Mn/光系统II库的一部分。在分离过程中保留锰的蛋白质的机制似乎是通过抑制天然结合的，高价锰的化学还原到不稳定的锰（II）氧化态。这种蛋白质也以化学计量水平存在于含有4-5 Mn/PS II的高活性、释放O2、洗涤剂提取的PS-II颗粒中。导致Mn和O2从功能膜中释放活性损失的条件，例如在1.5 mM NH 2 OH或抗坏血酸盐加连二亚硫酸盐中孵育，也从蛋白质中释放Mn。蛋白质以单体形式存在，凝胶过滤为33 kDa，凝胶电泳为34 kDa，等电点为5.1 ± 0.1。该蛋白质的EPR谱仅在12 K以下，其延伸超过至少2000 G，以g = 2为中心，由非均匀分离的超精细跃迁组成，平均分裂为45-55 G。这种分裂的幅度名义上是在具有O或N供体配体的单体锰络合物中观察到的分裂的一半。这显然是由于电子耦合的两个55锰核在一个假定的双核网站。无论是铁磁耦合的双核锰2（III，III）网站或反铁磁耦合的混合价锰2（II，III）网站被认为是可能的氧化态占EPR谱。在具有偶数自旋基态的铁磁耦合双核锰配合物中观察到类似于质量的超精细结构分裂。极端的温度依赖性表明，人口的低位激发自旋态，如存在于弱耦合的二聚体和更高的集群的Mn离子，或可能，从有效的自旋弛豫，如发生在Mn（III）的氧化态。无论是1.5 mM NH 2 OH或与还原剂孵育废除低温EPR信号和释放两个Mn（II）离子的解决方案。这与分离的蛋白质中Mn（III）的存在一致。在模型化合物中观察到的固有不稳定的Mn 2（II，III）氧化态有利于将稳定的蛋白质氧化态分配给Mn 2（III，III）制剂。这种蛋白质表现出与长期寻求锰网站的光合O2进化的识别一致的特性。在黑暗适应的完整光合膜中可观察到具有定性相似特征的EPR光谱（Dismukes，G.C.，阿布拉莫维奇，D.A.，Ferris，F.K.，Mathur，P.，Upadrashta，B.和Watnick，P.（1983）在The Oxygen-Evolving System of Plant Photosynthesis（Inoue，Y.，ed.），pp. 145-158，Academic Press，Tokyo）和去污剂提取的释放O2的光系统-II颗粒（Abramowicz，D.A.，Raab，T.K.和Dismukes，G.C.（1984）第六届光合作用国际会议论文集（Sybesma，C.，编辑），Vol. I，pp. 349-354，Martinus Nijhoff/W.垃圾出版商，海牙，荷兰），从而建立了与O2进化的复杂的直接联系。
Extraction conditions have been found which result in the retention of managanese to the 33–34 kDa protein, first isolated as an apoprotein by Kuwabara and Murata (Kuwabara, T. and Murata, N. (1979) Biochim. Biophys Acta 581, 228–236). By maintaining an oxidizing-solution potential, with hydrophilic and lipophilic redox buffers during protein extraction of spinach grana-thylakoid membranes, the 33–34 kDa protein is observed to bind a maximum of 2 Mn/protein which are not released by extended dialysis versus buffer. This manganese is a part of the pool of 4 Mn/Photosystem II normally associated with the oxygen-evolving complex. The mechanism for retention of Mn to the protein during isolation appears to be by suppression of chemical reduction of natively bound, high-valent Mn to the labile Mn(II) oxidation state. This protein is also present in stoichiometric levels in highly active, O2-evolving, detergent-extracted PS-II particles which contain 4–5 Mn/PS II. Conditions which result in the loss of Mn and O2evolution activity from functional membranes, such as incubation in 1.5 mM NH2OH or in ascorbate plus dithionite, also release Mn from the protein. The protein exists as a monomer of 33 kDa by gel filtration and 34 kDa by gel electrophoresis, with an isoelectric point of 5.1 ± 0.1. The protein exhibits an EPR spectrum only below 12 K which extends over at least 2000 G centered atg= 2 consisting of non-uniformly separated hyperfine transitions with average splitting of 45–55 G. The magnitude of this splitting is nominally one-half the splitting observed in monomeric manganese complexes having O or N donor ligands. This is apparently due to electronic coupling of the two55Mn nuclei in a presumed binuclear site. Either a ferromagnetically coupled binuclear Mn2(III,III) site or an antiferromagnetically coupled mixed-valence Mn2(II,III) site are considered as possible oxidation states to account for the EPR spectrum. Qualitatively similar hyperfine structure splittings are observed in ferromagnetically coupled binuclear Mn complexes having even-spin ground states. The extreme temperature dependence suggests the population of low-lying excited spin states such as are present in weakly coupled dimers and higher clusters of Mn ions, or, possibly, from efficient spin relaxation such as occurs in the Mn(III) oxidation state. Either 1.5 mM NH2OH or incubation with reducing agents abolishes the low temperature EPR signal and releases two Mn(II) ions to solution. This is consistent with the presence of Mn(III) in the isolated protein. The intrinsically unstable Mn2(II,III) oxidation state observed in model compounds favors the assignment of the stable protein oxidation state to the Mn2(III,III) formulation. This protein exhibits characteristics consistent with an identification with the long-sought Mn site for photosynthetic O2evolution. An EPR spectrum having qualitatively similar features is observable in dark-adapted intact, photosynthetic membranes (Dismukes, G.C., Abramowicz, D.A., Ferris, F.K., Mathur, P., Upadrashta, B. and Watnick, P. (1983) in The Oxygen-Evolving System of Plant Photosynthesis (Inoue, Y., ed.), pp. 145–158, Academic Press, Tokyo) and in detergent-extracted, O2-evolving Photosystem-II particles (Abramowicz, D.A., Raab, T.K. and Dismukes, G.C. (1984) Proceedings of the Sixth International Congress on Photosynthesis (Sybesma, C., ed.), Vol. I, pp. 349–354, Martinus Nijhoff/Dr. W. Junk Publishers, The Hague, The Netherlands), thus establishing a direct link with the O2evolving complex.