PHOTOCYCLE OF HALORHODOPSIN FROM HALOBACTERIUM-SALINARIUM

The light-driven chloride pump, halorhodopsin, is a mixture containing all-trans and 13-cis retinal chromophores under both light and dark-a dapted conditions and can exist in chloride-free and chloride-binding forms. To describe the photochemical cycle of the all-trans, chloride- binding state that is associated with the transport, and thereby initi ate study of the chloride translocation mechanism, one must first diss ect the contributions of these species to the measured spectral change s. We resolved the multiple photochemical reactions by determining fla sh-induced difference spectra and photocycle kinetics in halorhodopsin -containing membranes prepared from Halobacterium salinarium, with lig ht- and dark-adapted samples at various chloride concentrations. The h igh expression of cloned halorhodopsin made it possible to do these me asurements with unfractionated cell envelope membranes in which the ch romophore is photostable not only in the presence of NaCl but also in the Na2SO4 solution used for reference. Careful examination of the fla sh-induced changes at selected wavelengths allowed separating the spec tral changes into components and assigning them to the individual phot ocycles. According to the results, a substantial revision of the photo cycle model for H. salinarium halorhodopsin, and its dependence on chl oride, is required. The cycle of the all-trans chloride-binding form i s described by the scheme, HR-(hv) --> K double left right arrow L(1) double left right arrow L(2) double left right arrow N --> HR, where H R, K, L, and N designate halorhodopsin and its photointermediates. Unl ike the earlier models, this is very similar to the photoreaction of b acteriorhodopsin when deprotonation of the Schiff base is prevented (e .g., at low pH or in the D85N mutant). Also unlike in the earlier mode ls, no step in this photocycle was noticeably affected when the chlori de concentration was varied between 20 mM and 2 M in an attempt to ide ntify a chloride-binding reaction.