Vertebrate rhodopsin consists of the apoprotein opsin and the chromophore 11-cis-retinal covalently linked via a protonated Schiff base. Upon photoisomerization of the chromophore to all-trans-retinal, the retinylidene linkage hydrolyzes, and all-trans-retinal dissociates from opsin. The pigment is eventually restored by recombining with enzymatically produced 11-cis-retinal. All-trans-retinal release occurs in parallel with decay of the active form, metarhodopsin (Meta) II, in which the original Schiff base is intact but deprotonated. The intermediates formed during Meta II decay include Meta III, with the original Schiff base reprotonated, and Meta III-like pseudo-photoproducts. Using an intrinsic fluorescence assay, Fourier transform infrared spectroscopy, and UV-visible spectroscopy, we investigated Meta II decay in native rod disk membranes. Up to 40% of Meta III is formed without changes in the intrinsic Trp fluorescence and thus without all-trans-retinal release. NADPH, a cofactor for the reduction of all-trans-retinal to all-trans-retinol, does not accelerate Meta II decay nor does it change the amount of Meta III formed. However, Meta III can be photoconverted back to the Meta II signaling state. The data are described by two quasiirreversible pathways, leading in parallel into Meta III or into release of all-trans-retinal. Therefore, Meta III could be a form of rhodopsin that is storaged away, thus regulating photoreceptor regeneration.Phototransduction in vertebrate rods starts with the isomerization of the 11-cis-retinal bound to opsin and the formation of the active photoproduct, metarhodopsin (Meta) 1 II. In Meta II, the Schiff base linkage between the all-trans-retinal and Lys 296 is still intact but deprotonated. Catalytic activation of the Gprotein, G t or transducin, leads to a biochemical cascade of reactions, termed phototransduction. These reactions culminate in the hyperpolarization of the photoreceptor cells and ultimately in changes in the rate of neurotransmitter release at the synaptic terminus. The signaling state of Meta II is quenched rapidly by the action of rhodopsin kinase and arrestin. Equally important for vision is the metabolic cycle, which enables the visual system to take away the photolyzed chromophore, all-trans-retinal, and replace it with 11-cis-retinal, thus regenerating the pigment. The decay of Meta II thus provides an interlink among transduction, the quenching by phosphorylation and capping with arrestin, and regeneration (reviewed in Ref. 1).During the decay of Meta II, the Schiff base linkage between the all-trans-retinal and the opsin apoprotein (Lys 296 ) is hydrolyzed. A side product is the bright orange ( max ϳ 470 nm) Meta III, which slowly replaces the pale yellow color of the Meta II product ( max ϭ 380 nm). Although it is not clear whether Meta III represents one homogeneous species, one may define it as the late product in which the chromophore is still bound to its original binding site. In the isolated retina and in intact rod outer segment preparations...

Deactivation of light-activated rhodopsin (metarhodopsin II) involves, after rhodopsin kinase and arrestin interactions, the hydrolysis of the covalent bond of alltrans-retinal to the apoprotein. Although the long-lived storage form metarhodopsin III is transiently formed, all-trans-retinal is eventually released from the active site. Here we address the question of whether the release results in a retinal that is freely diffusible in the lipid phase of the photoreceptor membrane. The release reaction is accompanied by an increase in intrinsic protein fluorescence (release signal), which arises from the relief of the fluorescence quenching imposed by the retinal in the active site. An analogous fluorescence decrease (uptake signal) was evoked by exogenous retinoids when they non-covalently bound to native opsin membranes. Uptake of 11-cis-retinal was faster than formation of the retinylidene linkage to the apoprotein. Endogenous all-trans-retinal released from the active site during metarhodopsin II decay did not generate the uptake signal. The data show that in addition to the retinylidene pocket (site I) there are two other retinoidbinding sites within opsin. Site II involved in the uptake signal is an entrance site, while the exit site (site III) is occupied when retinal remains bound after its release from site I. Support for a retinal channeling mechanism comes from the rhodopsin crystal structure, which unveiled two putative hydrophobic binding sites. This mechanism enables a unidirectional process for the release of photoisomerized chromophore and the uptake of newly synthesized 11-cis-retinal for the regeneration of rhodopsin.During its function as a photoreceptor, the visual pigment rhodopsin undergoes changes through a cycle of uptake of 11-cis-retinal and release of photoisomerized chromophore, alltrans-retinal (reviewed in Ref. 1). In rhodopsin, 11-cis-retinal is bound to the opsin apoprotein by a Schiff base linkage to Lys 296 . In the dark ground state, the rhodopsin conformation with a max of 500 nm is stabilized by a salt bridge between the protonated Schiff base and the Glu 113 counterion. The activated state metarhodopsin II (Meta II, max ϭ 380 nm) arises from light-induced 11-cis/all-trans-retinal isomerization and conformational changes that break the salt bridge but retain the all-trans-retinylidene linkage in the original binding site (reviewed in Refs. 2-5). Thereby, photoisomerization is coupled to the protein conformational change that leads to a G-proteincoupled signal cascade, eventually setting off neuronal signaling (1).Initial deactivation of Meta II begins with the interaction of active rhodopsin with its receptor kinase, phosphorylation of the receptor, and a tight binding of arrestin to the still activated phosphorylated form of the receptor (6, 7). Full deactivation occurs when rhodopsin is regenerated. This requires the hydrolysis of the all-trans-retinylidene linkage and release of all-trans-retinal from the active site (1). Critical steps include the nucleophilic attack of wa...

To regenerate light-sensitive rhodopsin in rods from active metarhodopsin II (Meta II), all-trans-retinal must be removed from the retinal binding pocket and metabolically supplied 11-cis-retinal has to form a new retinylidene bond in the active site. Recent work from this laboratory has focused on Meta II decay and release and uptake of retinals in opsin employing intrinsic protein fluorescence. Here we summarize the results in the retinal channeling hypothesis, which describes a passage of the chromophore through the protein. 11-cis-retinal is taken up into an entrance site, and photolyzed all-trans-retinal is released from the active site into an exit site.

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A new dual-fluorescent compound, 5-(oxo)penta-2,4-dienyl-p-(N,N-dimethylamino)benzoate (1), a derivative of dimethylaminobenzoic acid, has been synthesised and studied photophysically. This compound continues the series of potential fluorescent probes for visual and proton-pumping opsin proteins. The photophysical behaviour of this molecule, including charge-transfer interaction in the ground state and dual-fluorescence emission, is similar to that of the previously studied analogue cis-3-(oxo)propenyl-p-(N,N-dimethylamino)benzoate (cis-2). The presence of several theoretically calculated conformers of compound 2 was suggested to be responsible for the observed strongly red-shifted absorption and excitation wavelength dependence. These photophysical anomalies were also observed for molecule 1, though the models put forward to explain them in the cases of 1 and 2 are rather different. Based on theoretical calculations and experimental results, we propose that some of the stable conformers might be connected with either a charge-transfer complex or mesomeric interactions in the ground state. Upon changing the electronic nature of the oxo-pentadienyl acceptor moiety, e.g. protonation, chemical or biochemical reaction, the charge-transfer absorption disappears, which leads to a dramatic increase in the fluorescence quantum yield.