LIGHT-DEPENDENT TRANSDUCIN ACTIVATION BY AN ULTRAVIOLET-ABSORBING RHODOPSIN MUTANT

The photoactivation pathway of an ultraviolet-absorbing rhodopsin muta nt was studied. The mutant pigment, in which the retinylidene Schiff b ase counterion, Glu113, was replaced by glutamine (E113Q), was known t o exist in a pH-dependent equilibrium between spectral forms absorbing at about 380 and 490 nm. The 380-nm form contains an unprotonated Sch iff base chromophore linkage, whereas the 490-nm form contains a proto nated Schiff base chromophore linkage. The role of the Schiff base pro ton in photoactivation was investigated by measuring transducin activa tion as a function of photoactivation wavelength. The transducin activ ation 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 t o catalyze nucleotide exchange by transducin. The quantum efficiency o f photoactivation of the UV-absorbing form of E113Q was similar to tha t of its visible-absorbing form. These results show that the presence of a protonated Schiff base in the ground state is not necessarily req uired for efficient photoactivation of visual pigments. They support t he hypothesis that the key role of the protonated Schiff base in visib le-absorbing pigments is to stabilize the ground state and to allow ab sorbance at wavelengths above about 420 nm. The findings are also cons istent with transducin activation studies of mutant apoproteins regene rated with all-trans-retinal, or of mutant apoproteins alone, suggesti ng that the active state of rhodopsin can be formed via a number of pa thways. UV-absorbing mutants of rhodopsin can be used as a model syste m for the study of naturally occurring vertebrate UV photoreceptors.