Participation of glutamate-354 of the CP43 polypeptide in the ligation of manganese and the binding of substrate water in photosystem II.
Participation of glutamate-354 of the CP43 polypeptide in the ligation of manganese and the binding of substrate water in photosystem II.
复制标题
CP43多肽的谷氨酸354参与光系统II中锰的连接和底物水的结合。
DOI：
10.1021/bi1015937
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发表时间：
2011
期刊：
影响因子：
2.9
通讯作者：
Debus,RichardJ
中科院分区：
文献类型：
--
作者：
Service,RachelJ;Yano,Junko;McConnell,Iain;Hwang,HongJin;Niks,Dimitri;Hille,Russ;Wydrzynski,Tom;Burnap,RobertL;Hillier,Warwick;Debus,RichardJ
在目前光系统II的X射线晶体结构模型中，CP43多肽的Glu354是放氧Mn4Ca簇中唯一不由D1多肽提供的氨基酸配体。为了进一步探索这个结构独特的残基对Mn4Ca簇性质的影响，蓝藻聚胞藻的CP43-E354Q突变体。用多种生物物理和光谱方法对PCC 6803进行了表征，包括极谱、电子顺磁共振、X射线吸收、FTIR和质谱学。突变体的释氧动力学与野生型基本一致。此外，突变体的氧闪产量表现出正常的周期4振荡，具有正常的S状态参数，尽管产量较低，这与突变体较低的稳态率(与野生型相比约为20%)有关。用H_(18)O进行的实验表明，在S3状态下，CP43-E354Q类囊体膜基质水交换的快相和慢相分别是野生型的8.5倍和1.8倍。纯化的放氧CP43-E354Q PSII核心络合物表现出轻微的S1态Mn-EXAFS光谱，轻微改变的S2态多线EPR信号，显著改变的S2-负-S1FTIR差谱，以及在相当一部分反应中心中S2态(>；10h)的异常长的寿命。相反，S2态的Mn-EXAFS谱与野生型几乎无法区分。S2-负-S1FTIR差示光谱显示，整个酰胺和羧酸伸缩区域都发生了变化。15N全局标记和1-[1-13C]丙氨酸特异性标记显示，该突变扰乱了D1-Ala344的α-CoO−基团的酰胺II和羧酸伸展模式，并使其对称的羧酸伸展模式在S1和S2状态下都向更高的频率移动了3−4 cM−1。电子顺磁共振和傅里叶变换红外光谱数据表明，76个−82%的CP43-E354Q PSII中心可以达到S2状态，其中大部分可以达到S3状态，但在FTIR数据中没有观察到超过S3状态的证据，至少在大多数PSII中心不是这样。虽然X射线吸收和EPR数据表明CP43-E354Q突变只是微妙地扰乱了处于S2状态的Mn4Ca团簇的结构和自旋态，但FTIR和H_(18)O交换数据表明，该突变强烈地影响了Mn4Ca团簇的其他性质，改变了许多羧基和酰胺基团对S1到S2转变过程中团簇上增加的正电荷的反应，并削弱了两种底物水分子(或水基配体)的结合，特别是在S3状态下快速交换的分子。FTIR数据提供了CP43-Glu354作为两个金属离子之间的桥联配体与S1态的Mn4Ca团簇配位的证据，但没有提供令人信服的证据表明该残基在S1态到S2态的配位方式发生了变化。H_218O交换数据提供了CP43-Glu354与连接底物水分子(或水衍生配体)的锰离子相互作用的证据，该底物水分子(或水基配体)在S3状态下快速交换。
In the current X-ray crystallographic structural models of photosystem II, Glu354 of the CP43 polypeptide is the only amino acid ligand of the oxygen-evolving Mn4Ca cluster that is not provided by the D1 polypeptide. To further explore the influence of this structurally unique residue on the properties of the Mn4Ca cluster, the CP43-E354Q mutant of the cyanobacteriumSynechocystissp. PCC 6803 was characterized with a variety of biophysical and spectroscopic methods, including polarography, EPR, X-ray absorption, FTIR, and mass spectrometry. The kinetics of oxygen release in the mutant were essentially unchanged from those in wild type. In addition, the oxygen flash yields exhibited normal period four oscillations having normal S state parameters, although the yields were lower, correlating with the mutant’s lower steady-state rate (approximately 20% compared to wild type). Experiments conducted with H218O showed that the fast and slow phases of substrate water exchange in CP43-E354Q thylakoid membranes were accelerated 8.5- and 1.8-fold, respectively, in the S3state compared to wild type. Purified oxygen-evolving CP43-E354Q PSII core complexes exhibited a slightly altered S1state Mn-EXAFS spectrum, a slightly altered S2state multiline EPR signal, a substantially altered S2-minus-S1FTIR difference spectrum, and an unusually long lifetime for the S2state (>10 h) in a substantial fraction of reaction centers. In contrast, the S2state Mn-EXAFS spectrum was nearly indistinguishable from that of wild type. The S2-minus-S1FTIR difference spectrum showed alterations throughout the amide and carboxylate stretching regions. Global labeling with15N and specific labeling withl-[1-13C]alanine revealed that the mutation perturbs both amide II and carboxylate stretching modes and shifts the symmetric carboxylate stretching modes of the α-COO−group of D1-Ala344 (the C-terminus of the D1 polypeptide) to higher frequencies by 3−4 cm−1in both the S1and S2states. The EPR and FTIR data implied that 76−82% of CP43-E354Q PSII centers can achieve the S2state and that most of these can achieve the S3state, but no evidence for advancement beyond the S3state was observed in the FTIR data, at least not in a majority of PSII centers. Although the X-ray absorption and EPR data showed that the CP43-E354Q mutation only subtly perturbs the structure and spin state of the Mn4Ca cluster in the S2state, the FTIR and H218O exchange data show that the mutation strongly influences other properties of the Mn4Ca cluster, altering the response of numerous carboxylate and amide groups to the increased positive charge that develops on the cluster during the S1to S2transition and weakening the binding of both substrate water molecules (or water-derived ligands), especially the one that exchanges rapidly in the S3state. The FTIR data provide evidence that CP43-Glu354 coordinates to the Mn4Ca cluster in the S1state as a bridging ligand between two metal ions but provide no compelling evidence that this residue changes its coordination mode during the S1to S2transition. The H218O exchange data provide evidence that CP43-Glu354 interacts with the Mn ion that ligates the substrate water molecule (or water-derived ligand) that is in rapid exchange in the S3state.