Origin of the unique topology of the triangular water cluster in Rubrobacter xylanophilus rhodopsin

Abstract

The crystal structure of Rubrobacter xylanophilus rhodopsin (RxR) reveals a triangular cluster of three water molecules (W413, W415, and W419) at the extracellular proton-release site, near Glu187 and Glu197. Using a quantum mechanical/molecular mechanical approach, we identified the structural nature of this unique water cluster. The triangular shape is best reproduced when all three water molecules are neutral H₂O with protonated Glu187 and deprotonated Glu197. Attempts to place H₃O⁺ at any of these water molecules result in spontaneous proton transfer to one of the acidic residues and significant distortion from the crystal structure. The plane defined by the triangular water cluster extends into the guanidinium plane of Arg71, with both aligned along the W413...W419 axis. This extended plane lies nearly perpendicular to a five-membered, ring-like H-bond network involving two carboxyl oxygen atoms from Glu187 and one from Glu197. The resulting bipartite planar architecture, defined by the water triangle, Arg71, and the Glu187/Glu197 network may reflect the exceptional thermal stability in RxR.

Caption of Graphical Abstract

A compact, triangular water cluster at the proton-release site of Rubrobacter xylanophilus rhodopsin (RxR), stabilized by Glu187, Glu197, and Arg71. Quantum mechanical/molecular mechanical (QM/MM) calculations indicate that three neutral water molecules form this geometry, without involving H₃O⁺. This architecture differs from that of bacteriorhodopsin and may relate to the exceptional thermal stability of RxR.