DETECTION OF A WATER MOLECULE IN THE ACTIVE-SITE OF BACTERIORHODOPSIN- HYDROGEN-BONDING CHANGES DURING THE PRIMARY PHOTOREACTION

FTIR-difference spectroscopy in combination with site-directed mutagen esis has been used to investigate the role of water during the photocy cle of bacteriorhodopsin. At least one water molecule is detected whic h undergoes an increase in H-bonding during the primary bR-->K phototr ansition. Bands due to water appear in the OH stretch region of the bR -->K FTIR-difference spectrum which downshift by approximately 12 cm(- 1) when the sample is hydrated with (H2O)-O-18. In contrast to (H2O)-H -2, the (H2O)-O-18-induced shift is not complete, even after 24 h of h ydration. This indicates that even though water is still able to excha nge protons with the outside medium, it is partially trapped in the in terior of the protein. In the mutant Y57D, these bands are absent whil e a new set of bands appear at much lower frequencies which undergo (H 2O)-O-18-induced shifts. It is concluded that the water molecule we de tect is located inside the bR active-site and may interact with Tyr-57 . The change in its hydrogen-bonding strength is most likely due to th e photoinduced all-trans-->13-cis isomerization of the retinal chromop hore and the associated movement of the positively charged Schiff base during the bR-->K transition. In contrast, a second water molecule, w hose infrared difference bands are not affected by the Y57D mutation, appears to undergo a decrease in hydrogen bonding during the K-->L and L-->M transitions.