pH-Dependence of Anabaena Sensory Rhodopsin: Retinal Isomer Composition, Rate of Dark Adaptation and pKa of Asp217 Side Chain

Rinat Belitzky Rozin, Department of Organic Chemistry, Weizmann Institute of Science, Rehovot , Israel
Mordechai Sheves, Department of Organic Chemistry, Weizmann Institute of Science, Rehovot, Israel

Anabaena sensory rhodopsin (ASR) is a microbial rhodopsin discovered in the last decade in the freshwater cyanobacteria. Microbial rhodopsins are photoactive proteins, and their binding site can accommodate either all-trans or 13-cis retinal chromophores of which only the all-trans isomer is biologically active. In contrast to microbial rhodopsins, ASR exhibits a number of unusual characteristics. The assumption is that ASR is a photochromic color sensor that functions as a photoreceptor for chromatic adaptation. However, the mechanism of its photoactivation and interaction with its transducer is far from being established. We have studied the effect of pH on the protein properties. The main findings include: (a) Using spectroscopic titration experiments and calculated absorption spectra of the protein, three pKa values of 4.0, 6.5 and 8.5 of three side chains in proximity to the retinal binding site were observed, whereas the second is associated with the cytoplasmic Asp217 side chain. The protonation states of these protein residues affect the absorption spectrum of the pigment and most probably the isomerization process of the retinal chromophore. (b) The isomeric composition of ASR is determined over a wide pH range, and found to be almost pH-independent in the dark (>96% AT isomer), but highly pH-dependent in the light-adapted form. This observation is in keeping with ASRs photochromic features. (c) The kinetics of dark adaptation is recorded over a wide pH range, showing that the thermal isomerization from 13C to AT retinal occurs much faster at high pH rather than in acidic conditions.