Revisiting the Proposed Protonated Water Cluster at the Exit Site of the Proton Transfer Pathway in Bacteriorhodopsin

The existence of a protonated water species, H3O+ or H5O2+, at the extracellular terminus of the proton transfer pathway in bacteriorhodopsin was proposed based on spectroscopic studies. Here, we revisit this hypothesis using the high-resolution crystal structure employed in the original proposal, combined with quantum mechanical/molecular mechanical (QM/MM) calculations. When H3O+ is modeled at the proposed site (W403), the QM/MM-optimized geometry under constraints on the three O–H-bond lengths deviates from the typical planar configuration and adopts a pyramidal shape, indicating incompatibility with the protein environment. H3O+ is unstable and readily decomposes into a neutral water molecule at the W403 site, with the proton relocating to either Glu194 or Glu204, resulting in energetically nearly equivalent states. The potential-energy surfaces lack the symmetric funnel-like shape required to stabilize H3O+ and reveal that W403 and W404 do not form a low-barrier H-bond, ruling out H5O2+ as well. These results indicate that neither H3O+ nor H5O2+ is energetically feasible at this site, emphasizing that such species are unlikely to form within efficient proton transfer pathways.