RELATIONSHIP OF RETINAL CONFIGURATION AND INTERNAL PROTON-TRANSFER ATTHE END OF THE BACTERIORHODOPSIN PHOTOCYCLE

In the last step of the bacteriorhodopsin photocycle the initial state is regenerated from the O intermediate in an essentially unidirection al reaction. Comparison of the rate of this photocycle step and the ra te of deprotonation of Asp-85 in pH jump experiments with various site -specific mutants indicates that recovery of the initial state is infl uenced by (1) residues such as Glu-204 that affect deprotonation of As p-85 and (2) residues such as Leu-93 that contact the retinal and ther efore must affect its thermal reisomerization from 13-cis to all-trans as suggested by Delaney, Schweiger, and Subramaniam (Proc. Natl. Acad . Sci. U.S.A. 92, 11120-11124, 1995). These results, together with FTI R spectra (Kandori, Hatanaka, Yamazaki; Needleman, Brown, Richter, Lan yi, & Maeda, manuscript in preparation) of the last intermediate in th e photocycles of representatives of the two kinds of mutants, E204Q an d L93M, suggest the following sequence of events: reisomerization of t he retinal from 13-cis to an all-trans configuration that contains a t wisted chain (with high amplitude hydrogen out-of-plane vibrational ba nds) triggers proton transfer from Asp-85 to Glu-204 or directly to th e extracellular surface, and the proton transfer in turn triggers rela xation of the twist in the retinal. The involvement of the proton tran sfer in the kinetics of this sequence suggests the reason for the unid irectionality of the overall reaction: upon reisomerization of the ret inal the very low pK(a) of Asp-85 in the unphotolyzed protein is reest ablished and this residue thereby becomes a good proton donor.