Photocycle of halorhodopsin from Halobacterium salinarium

Abstract

The light-driven chloride pump, halorhodopsin, is a mixture containing all-trans and 13-cis retinal chromophores under both light and dark-adapted 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 initiate study of the chloride translocation mechanism, one must first dissect the contributions of these species to the measured spectral changes. We resolved the multiple photochemical reactions by determining flash-induced difference spectra and photocycle kinetics in halorhodopsin-containing membranes prepared from Halobacterium salinarium, with light- and dark-adapted samples at various chloride concentrations. The high expression of cloned halorhodopsin made it possible to do these measurements with unfractionated cell envelope membranes in which the chromophore is photostable not only in the presence of NaCl but also in the Na2SO4 solution used for reference. Careful examination of the flash-induced changes at selected wavelengths allowed separating the spectral changes into components and assigning them to the individual photocycles. According to the results, a substantial revision of the photocycle model for H. salinarium halorhodopsin, and its dependence on chloride, is required. The cycle of the all-trans chloride-binding form is described by the scheme, HR-hv-->K<==>L1<==>L2<==>N-->HR, where HR, K, L, and N designate halorhodopsin and its photointermediates. Unlike the earlier models, this is very similar to the photoreaction of bacteriorhodopsin when deprotonation of the Schiff base is prevented (e.g., at low pH or in the D85N mutant). Also unlike in the earlier models, no step in this photocycle was noticeably affected when the chloride concentration was varied between 20 mM and 2 M in an attempt to identify a chloride-binding reaction.

A Unique Light-Driven Proton Transportation Signal in Halorhodopsin from Natronomonas pharaonis

“…HsHR served as a negative control because of the inability of the light-driven proton transportation to generate a protein-based photocurrent. In addition, the photocycle of HsHR was shown to lack the O state; alternatively, another possibility mentioned in a previous study is that the O state might have occurred too rapidly to be detected (16). Therefore, HsHR might undergo both Cl À release and reuptake processes during the N-state decay (30).…”

“…First, crystal structures have been solved for HsHR (12,13) and NpHR (14), and revealed that these HRs differ in the location of their secondary anion binding site (14)(15)(16). Second, substitution of chloride with azide was found to affect the photoreactions of the two proteins differently (17)(18)(19)(20).…”

Characterization of a Cyanobacterial Chloride-pumping Rhodopsin and Its Conversion into a Proton Pump

“…Ion-pumping rhodopsins transport one ion during a single turnover of photocycle and generally undergo fast photocycles (Ͻ100 ms), which enable the generation of a large transmembrane electrochemical potential under constant illumination. On the other hand, similar to HR and FR, no absorbance change at 400 nm was observed throughout the photocycle (14,34,36) The slow photocycle of MrHR might reflect some differences in the physiological role from other Cl Ϫ -pumping rhodopsins. Although the details are not fully resolved, the Cl Ϫ pumps are believed to play roles in light-driven ATP production and/or maintaining the cellular osmotic balance during the volume increase of the growing cell (38 -41).…”

Halorhodopsin pumps Cl–and bacteriorhodopsin pumps protons by a common mechanism that uses conserved electrostatic interactions

“…They show similar absorption spectral changes during their photocycles (17), indicating that the retinal is in a similar conformation and electrostatic environment (19). Five of six spectroscopically identified BR intermediates are found in HR (20).…”