Photochemical study of a cyanobacterial chloride-ion pumping rhodopsin

Mastigocladopsis repens halorhodopsin (MrHR) is a Cl^--pumping rhodopsin that belongs to a distinct cluster far from other Cl^- pumps. We investigated its pumping function by analyzing its photocycle and the effect of amino acid replacements. MrHR can bind I^- similar to Cl^- but cannot transport it. I^--bound MrHR undergoes a photocycle but lacks the intermediates after L, suggesting that, in the Cl^--pumping photocycle, Cl^- moves to the cytoplasmic (CP) channel during L decay. A photocycle similar to that of the I^--bound form was also observed for a mutant of the Asp200 residue, which is superconserved and assumed to be deprotonated in most microbial rhodopsins. This residue is probably close to the Cl^--binding site and the protonated Schiff base, in which a chromophore retinal binds to a specific Lys residue. However, the D200N mutation affected neither the Cl^--binding affinity nor the absorption spectrum, but completely eliminated the Cl^--pumping function. Thus, the Asp200 residue probably protonates in the dark state but deprotonates during the photocycle. Indeed, a H⁺ release was detected for photolyzed MrHR by using an indium‑tin oxide electrode, which acts as a good time-resolved pH sensor. This H⁺ release disappeared in the I^--bound form of the wild-type and Cl^--bound form of the D200N mutant. Thus, Asp200 residue probably deprotonates during L decay and then drives the Cl^- movement to the CP channel.