Photochemistry of the primary event in short-wavelength visual opsins at low temperature

Two short-wavelength cone opsins, frog (Xenopus laevis) violet and mouse UV , were expressed in mammalian COS1 cells, purified in delipidated form, and studied using cryogenic UV-vis spectrophotometry. At room temperature, the X. laevis violet opsin has an absorption maximum at 426 nm when generated with 11-cis-retinal and an absorption maximum of 415 nm when generated with 9-cis-retinal, The frog short-wavelength opsin has two different bathe int ermediates, one stable at 30 K (lambda(max) approximate to 446 nm) and the other at 70 K (lambda(max) approximate to 475 nm). Chloride ions do not aff ect the absorption maximum of the violet opsin. At room temperature, mouse UV opsin has an absorption maximum of 357 nm, while at 70 K, the pigment ex hibits a bathochromic shift to 403 nm with distinct vibronic structure and a strong secondary vibronic band at 380 nm. We have observed linear relatio nships when analyzing the energy difference between the initial and bathoch romic intermediates and the normalized difference spectra of the bathe-shif ted intermediates of rod and cone opsins. We conclude that the binding site s of these pigments change from red to green to violet via systematic shift s in the position of the primary counterion relative to the protonated Schi ff base. The mouse UV cone opsin does not fit this trend, and we conclude t hat wavelength selection in this pigment must operate via a different molec ular mechanism. We discuss the possibility that the mouse UV chromophore is initially unprotonated.