In the photocycle of bacteriorhodopsin at pH 7, a proton is ejected to the extracellular medium during the protonation of Asp-85 upon formation of the M intermediate. The group that releases the ejected proton does not become reprotonated until the prephotolysis state is restored from the N and O intermediates. In contrast, at acidic pH, this proton release group remains protonated to the end of the cycle. Time-resolved Fourier transform infrared measurements obtained at pH 5 and 7 were fitted to obtain spectra of kinetic intermediates, from which the spectra of M and N/O versus unphotolyzed state were calculated. Vibrational features that appear in both M and N/O spectra at pH 7, but not at pH 5, are attributable to deprotonation from the proton release group and resulting structural alterations. Our results agree with the earlier conclusion that this group is a protonated internal water cluster, and provide a stronger experimental basis for this assignment. A decrease in local polarity at the N-C bond of the side chain of Lys-216 resulting from deprotonation of this water cluster may be responsible for the increase in the proton affinity of Asp-85 through M and N/O, which is crucial for maintaining the directionality of proton pumping.
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