Tight Asp-85-Thr-89 association during the pump switch of bacteriorhodopsin

Unidirectional proton transport in bacteriorhodopsin is enforced by the swi tching machinery of the active site. Threonine 89 is located in this region , with its O-H group forming a hydrogen bond with Asp-85, the acceptor for proton transfer from the Schiff base of the retinal chromophore. Previous I R spectroscopy of [3-O-18]threonine-labeled bacteriorhodopsin showed that t he hydrogen bond of the O-D group of Thr-89 in D2O is strengthened in the K photocycle intermediate. Here, we show that the strength and orientation o f this hydrogen bond remains unchanged in the L intermediate and through th e M intermediate. Furthermore, a strong interaction between Asp-85 and the O-H (O-D) group of Thr-89 in M is indicated by a shift in the C=O stretchin g vibration of the former because of O-18 substitution in the latter. Thus, the strong hydrogen bond between Asp-85 and Thr-89 in K persists through M , contrary to structural models based on x-ray crystallography of the photo cycle intermediates. We propose that, upon photoisomerization of the chromo phore, Thr-89 forms a tight, persistent complex with one of the side-chain oxygens of Asp-85 and is thereby precluded from participating in the switch ing process. On the other hand, the loss of hydrogen bonding at the other o xygen of Asp-85 in M may be related to the switching event.