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Photochemical study of a cyanobacterial chloride-ion pumping rhodopsin

Hasemi, Takatoshi, Kikukawa, Takashi, Watanabe, Yumi, Aizawa, Tomoyasu, Miyauchi, Seiji, Kamo, Naoki, Demura, Makoto
Biochimica et biophysica acta 2019 v.1860 no.2 pp. 136-146
amino acids, binding capacity, binding sites, chlorides, electrodes, indium tin oxide, mutants, mutation, pH, photochemistry, protons, pumps, rhodopsin, schiff bases, spectral analysis
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.