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.
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