THREONINE-89 PARTICIPATES IN THE ACTIVE-SITE OF BACTERIORHODOPSIN - EVIDENCE FOR A ROLE IN COLOR REGULATION AND SCHIFF-BASE PROTON-TRANSFER

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 b ase and amino acid residues Asp-85, Asp-212, and Arg-82. This active s ite participates in proton transfers and regulates the visible absorpt ion of bacteriorhodopsin and its photointermediates. In this work we f ind evidence that Thr-89 also participates in this active site. The su bstitution Thr-89 Asn (T89N) results in changes in the properties of t he all-trans retinylidene chromophore of light-adapted bR including a redshift of the visible lambda(max) and a downshift in C=N and C=C str etch frequencies. Changes are also found in the M and N intermediates of the T89N photocycle including shifts in lambda(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 c ontrast, the properties of the 13-cis retinylidene chromophore of dark -adapted T89N as well as the K and L intermediates of the T89N photocy cle are similar to the wildtype bacteriorhodopsin. These results are c onsistent with an interaction of the hydroxyl group of Thr-89 with the protonated Schiff base of light-adapted bR and possibly the N interme diate 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 ba se proton transfer to Asp-85 during formation of the M intermediate, p ossibly through an interaction with Asp-85. In contrast, the hydroxyl group of Thr-89 is not obligatory for proton transfer from Asp-96 to t he Schiff base during formation of the N intermediate.