BACKGROUND The availability of high-resolution X-ray structures has shown that proteins contain numerous water molecules, but their role is still not fully understood. Protonated and deprotonated water species are often involved in biochemical reactions. However protons are exceedingly difficult to detect directly because they are electron-poor species. METHODS The oxygen‑oxygen distance of the crystallographic water molecules was analyzed in a large high-resolution data set. Moreover, a detailed analysis was carried out on the protein-bound water in the available structures of carbonic anhydrase II and cytochrome c oxidase, chosen as protein models in which protonated and deprotonated water species play a significant role. RESULTS The analysis shows an excess of water-water distances below the expected value for hydrogen bond. In the cavities and on the surface of the considered model proteins, clusters of water molecules are found, whose structure suggests the presence of chemical species deriving from self-ionization of water. CONCLUSIONS The presence of a small maximum below the hydrogen bond threshold in the oxygen‑oxygen distance distribution of crystallographic water molecules, along with the location of many of these water clusters, suggest the presence of Zundel-like structures in, or near, the proteins. Particularly significant is the presence of such structures in protein regions which have been identified as proton antennae or channels. GENERAL SIGNIFICANCE This work shows the possibilities, still unexplored, offered by this type of analysis in detecting in structures obtained by X-ray diffraction the presence of aqueous protons or hydroxide ions, which are chemical species as important as elusive.

中文翻译：

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蛋白质结合的结晶水中的氧-氧距离表明存在质子化簇。

背景技术高分辨率X射线结构的可用性已经表明蛋白质包含许多水分子，但是它们的作用仍未被完全理解。质子化和去质子化的水物种经常参与生化反应。但是，质子极难直接检测，因为它们是电子贫乏的物种。方法在大型高分辨率数据集中分析了结晶水分子的氧-氧距离。此外，对碳酸酐酶II和细胞色素c氧化酶的可用结构中结合蛋白的水进行了详细分析，这些结构被选为在质子化和去质子化水种中起重要作用的蛋白模型。结果分析表明，水-水距离超出氢键的预期值。在所考虑的模型蛋白质的腔内和表面上，发现了水分子簇，其结构表明存在水自电离衍生的化学物质。结论结晶水分子的氧-氧距离分布中氢键阈值以下存在一个很小的最大值，并且其中许多水簇的位置表明蛋白质中或附近存在Zundel样结构。 。特别重要的是这种结构在已被鉴定为质子触角或通道的蛋白质区域中的存在。一般意义这项工作表明了这种分析方法在检测X射线衍射获得的结构中是否存在含水质子或氢氧根离子方面仍存在尚未探索的可能性，