Light-dependent transducin activation by an ultraviolet-absorbing rhodopsin mutant.
The photoactivation pathway of an ultraviolet-absorbing rhodopsin mutant was studied. The mutant pigment, in which the retinylidene Schiff base counterion, Glu113, was replaced by glutamine (E113Q), was known to exist in a pH-dependent equilibrium between spectral forms absorbing at about 380 and 490 nm. The 380-nm form contains an unprotonated Schiff base chromophore linkage, whereas the 490-nm form contains a protonated Schiff base chromophore linkage. The role of the Schiff base proton in photoactivation was investigated by measuring transducin activation as a function of photoactivation wavelength. The transducin activation action spectra of rhodopsin and of mutant E113Q were found to be very similar to their UV-visible absorption spectra. Thus, the 380-nm UV form of the mutant E113Q could be activated directly by UV light to catalyze nucleotide exchange by transducin. The quantum efficiency of photoactivation of the UV-absorbing form of E113Q was similar to that of its visible-absorbing form. These results show that the presence of a protonated Schiff base in the ground state is not necessarily required for efficient photoactivation of visual pigments. They support the hypothesis that the key role of the protonated Schiff base in visible-absorbing pigments is to stabilize the ground state and to allow absorbance at wavelengths above about 420 nm. The findings are also consistent with transducin activation studies of mutant apoproteins regenerated with all-trans-retinal, or of mutant apoproteins alone, suggesting that the active state of rhodopsin can be formed via a number of pathways.(ABSTRACT TRUNCATED AT 250 WORDS)
Howard Hughes Medical Institute, Rockefeller University, New York, New York 10021.
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D000818:Animals; D016477:Artifacts; D002417:Cattle; D002478:Cells, Cultured; D004789:Enzyme Activation / Q000528:radiation effects*; D006358:Hot Temperature; D007536:Isomerism; D009154:Mutation; D011522:Protons; D011994:Recombinant Proteins / Q000378:metabolism; D012243:Rhodopsin / Q000235:genetics / Q000378:metabolism / Q000528:radiation effects*; D012545:Schiff Bases; D013056:Spectrophotometry, Ultraviolet; D015293:Transducin / Q000378:metabolism / Q000528:radiation effects*; D014466:Ultraviolet Rays
D011522:Protons; D011994:Recombinant Proteins; D012545:Schiff Bases; D012243:Rhodopsin; D015293:Transducin