Photosensitivities of rhodopsin mutants with a displaced counterion

Visual pigments consist of a protein moiety opsin and an 11-cis-retinal chromophore that is covalently bound to the opsin via a Schiff base linkage. They have a high photosensitivity, which can be attributed to the high probability of photon absorption and the high photoisomerization quantum yield of the retinal chromophore. Both of these parameters are regulated by the opsin, though the precise mechanism is unknown. We previously found that counterion residue E113, which stabilizes the proton on the Schiff base, is involved in the efficient photoisomerization in vertebrate visual pigments. To test the positional effect of the counterion on the photon absorption and the photoisomerization, we measured the photosensitivities of a set of mutants of bovine rhodopsin in which the counterion was displaced to position 90, 94, 117, or 292. The molar extinction coefficient was reduced in many of the mutants, leading to reductions in the photosensitivity for monochromatic lights. However, the oscillator strength, the probability of photon absorption integrated over the entire wavenumber range of the absorption band, was relatively similar among the mutants and the wild type. In addition, the quantum yields of the mutants were not markedly different from that of the wild type. These results indicate that the counterion does not need to be located at position 113 for a high photosensitivity for natural light. Interestingly, all of the mutants exhibited greatly increased hydroxylamine sensitivity. This result suggests that the counterion in vertebrate visual pigments is optimally located for the stability of the Schiff base linkage.