pK(a) Calculations suggest storage of an excess proton in a hydrogen-bonded water network in bacteriorhodopsin.

Calculations of protonation states and pK(a) values for the ionizable groups in the resting state of bacteriorhodopsin have been carried out using the recently available 1.55 A resolution X-ray crystallographic structure. The calculations are in reasonable agreement with the available experimental data for groups on or near the ion transport chain (the retinal Schiff base; Asp85, 96, 115, 212, and Arg82). In contrast to earlier studies using lower-resolution structural data, this agreement is achieved without manipulations of the crystallographically determined heavy-atom positions or ad hoc adjustments of the intrinsic pK(a) of the Schiff base. Thus, the theoretical methods used provide increased reliability as the input structural data are improved. Only minor effects on the agreement with experiment are found with respect to methodological variations, such as single versus multi-conformational treatment of hydrogen atom placements, or retaining the crystallographically determined internal water molecules versus treating them as high-dielectric cavities. The long-standing question of the identity of the group that releases a proton to the extracellular side of the membrane during the L-to-M transition of the photocycle is addressed by including as pH-titratable sites not only Glu204 and Glu194, residues near the extracellular side that have been proposed as the release group, but also an H(5)O(2)(+) molecule in a nearby cavity. The latter represents the recently proposed storage of the release proton in an hydrogen-bonded water network. In all calculations where this possibility is included, the proton is stored in the H(5)O(2)(+) rather than on either of the glutamic acids, thus establishing the plausibility on theoretical grounds of the storage of the release proton in bacteriorhodopsin in a hydrogen-bonded water network. The methods used here may also be applicable to other proteins that may store a proton in this way, such as the photosynthetic reaction center and cytochrome c oxidase.