Phototransduction by Vertebrate Ultraviolet Visual Pigments:  Protonation of the Retinylidene Schiff Base following Photobleaching

Abstract

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) approximately 380 nm). The batho intermediate thermally decays to the lumi intermediate (lambda(max) approximately 440 nm) via a slightly blue-shifted intermediate not observed in other photobleaching pathways, BL (lambda(max) approximately 375 nm), at temperatures greater than 180 K. The lumi intermediate has a significantly red-shifted absorption maximum at 440 nm, 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) approximately 480 nm) which in turn decays to meta II (lambda(max) approximately 380 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.

Vertebrate ultraviolet visual pigments: Protonation of the retinylidene Schiff base and a counterion switch during photoactivation

“…Cryogenic. Low-temperature spectroscopy was performed as described (14,17). Spectra of visual pigment samples (A max Ϸ 0.1) were collected in a solution containing 67% glycerol in buffer A (50 mM Hepes, pH 6.6͞140 mM NaCl͞3 mM MgCl 2 ͞0.05% N-dodecyl-␤-D-maltoside).…”

“…Spectra above 230 K were collected at the indicated temperature. Retinal oximes were extracted and analyzed as reported (17). Acid denaturation.…”

“…Calculations were based on the homology model of MUV reported in ref. 17, and unless noted otherwise, the backbone atoms in the transmembrane helices and the EII loop were fixed at the corresponding rhodopsin coordinates. Additional details of the modeling methods and results, as well as additional data on MUV-E108Q relating to the biochemical, photophysical, and spectroscopic properties of the photobleaching sequence, are provided in Supporting Methods, which is published as supporting information on the PNAS web site.…”

“…Fourier transform infrared (FTIR) spectroscopic studies indicate that the darkadapted form of the MUV pigment has an unprotonated SB (17). Thus, the sensitivity to UV light is accomplished by changing the pK a of the SB.…”

“…The photoactivation of MUV is remarkable in that the retinylidene SB is protonated during the formation of lumi and meta I (17). Subsequently, during the formation of the active species, meta II, the SB reverts to the unprotonated state.…”