Bacteriorhodopsin (bR) functions as a light-driven proton pump in the purple membrane of Halobacterium salinarium. A major feature of bR is the existence of an active site which includes a retinylidene Schiff base and amino acid residues Asp-85, Asp-212, and Arg-82. This active site participates in proton transfers and regulates the visible absorption of bacteriorhodopsin and its photointertmediates. In this work we find evidence that Thr-89 also participates in this active site. The substitution Thr-89 Ash (T89N) results in changes in the properties of the all-trans retinylidene chromophore of light-adapted bR including a redshift of the visible λ(max) and a downshift in C=N and C=C stretch frequencies. Changes are also found in the M and N intermediates of the T89N photocycle including shifts in λ(max), a downshift of the Asp-85 carboxylic acid C=O stretch frequency by 10 cm-1, and a 3-5-fold decrease in the rate of formation of the M intermediate. In contrast, the properties of the 13-cis retinylidene chromophore of dark-adapted T89N as well as the K and L intermediates of the T89N photocycle are similar to the wild-type bacteriorhodopsin. These results are consistent with an interaction of the hydroxyl group of Thr-89 with the protonated Schiff base of light-adapted bR and possibly the N intermediate but not the 13-cis chromophore of dark-adapted bR or the K and L intermediates. Thr-89 also appears to influence the rate of Schiff base proton transfer to Asp-85 during formation of the M intermediate, possibly through an interaction with Asp-85. In contrast, the hydroxyl group of Thr- 89 is not obligatory for proton transfer from Asp-96 to the Schiff base during formation of the N intermediate.
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