PROTON TRANSPORT BY HALORHODOPSIN

In halorhodopsin from Natronobacterium pharaonis, a light-driven chlor ide pump, the chloride binding site also binds azide. When azide is bo und at this location the retinal Schiff base transiently deprotonates after photoexcitation with light > 530 nm, like in the light-driven pr oton pump bacteriorhodopsin. As in the photocycle of bacteriorhodopsin , pyranine detects the release of protons to the bulk. The subsequent reprotonation of the Schiff: base is also dependent on azide, but with different kinetics that suggest a shuttling of protons from the surfa ce as described earlier for halorhodopsin from Halobacterium salinariu m. This azide-dependent, bacteriorhodopsin-like photocycle results in active electrogenic proton transport in the cytoplasmic to extracellul ar direction, detected in cell envelope vesicle suspensions both with a potential-sensitive electrode and by measuring light-dependent pH ch ange. We conclude that in halorhodopsin an azide bound to the extracel lular side of the Schiff base, and another azide shuttling between the Schiff base and the cytoplasmic surface, fulfill the functions of Asp -85 and Asp-96, respectively, in bacteriorhodopsin. Thus, although hal orhodopsin is normally a chloride ion pump, it evidently contains all structural requirements, except an internal proton acceptor and a dono r, of a proton pump. This observation complements our earlier finding that when a chloride binding site was created in bacteriorhodopsin thr ough replacement of Asp-85 with a threonine, that protein became a chl oride ion pump.