Phototransduction by vertebrate ultraviolet visual pigments: Protonation of the retinylidene Schiff base following photobleaching

The photochemical and subsequent thermal reactions of the mouse short-wavelength visual pigment (MUV) were studied by using cryogenic UV-visible and FTIR difference spectroscopy. Upon illumination at 75 K, MUV forms a batho intermediate (lambda(max) similar to380 nm). The batho intermediate thermally decays to the lumi intermediate (lambda(max) similar to440 nm) via a slightly blue-shifted intermediate not observed in other photobleaching pathways, BL (lambda(max) similar to375 nm), at temperatures greater than 180 K. The lumi intermediate has a significantly red-shifted absorption maximum at 440 rim, suggesting that the retinylidene Schiff base in this intermediate is protonated. The lumi intermediate decays to an even more red-shifted meta I intermediate (lambda(max) similar to480 nm) which in turn decays to meta H (lambda(max) similar to380 nm) at 248 K and above. Differential FTIR analysis of the 1100-1500 cm(-1) region reveals an integral absorptivity that is more than 3 times smaller than observed in rhodopsin and VCOP. These results are consistent with an unprotonated Schiff base chromophore. We conclude that the MUV-visual pigment possesses an unprotonated retinylidene Schiff base in the dark state, and undergoes a protonation event during the photobleaching cascade.