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 group s in the resting state of bacteriorhodopsin have been carried out using the recently available 1.55 Angstrom resolution X-ray crystallographic structu re. The calculations are in reasonable agreement with the available experim ental data for groups on or near the ion transport chain (the retinal Schif f base; Asp85, 96, 115, 212, and Arg82). In contrast to earlier studies usi ng lower-resolution structural data, this agreement is achieved without man ipulations of the crystallographically determined heavy-atom positions or a d hoc adjustments of the intrinsic pK(a) of the Schiff base. Thus, the theo retical 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 mult i-conformational treatment of hydrogen atom placements, or retaining the cr ystallographically 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 a s pH-titratable sites not only Glu204 and Glu194, residues near the extrace llular side that have been proposed as the release group, but also an H5O2 molecule in a nearby cavity. The latter represents the recently proposed s torage of the release proton in an hydrogen-bonded water network. In all ca lculations where this possibility is included, the proton is stored in the H5O2+ rather than on either of the glutamic acids, thus establishing the pl ausibility on theoretical grounds of the storage of the release proton in b acteriorhodopsin in a hydrogen-bonded water network. The methods used here may also be applicable to other proteins that may store a proton in this wa y, such as the photosynthetic reaction center and cytochrome c oxidase. (C) 2001 Academic Press.