Rhodopsin, proton transfer

Example 2—The first stage in this process of vision has been the excitation of rhodopsin. Rhodopsin partially gets deactivated forming an intermediate, prelumirhodopsin or bathorhodopsin. Picosecond spectroscopy reveals that prelumirhodopsin gets formed because of an intramolecular proton transfer—a jump of a proton from one position to another. 

Fig. 20. Model for the primary event in vision. Isomerization of the 11.12 bond leads to charge separation at Ihe Schiff base site. This process, as shown, can possibly be followed by proton transfer, the latter resulting from the charge separation. In rhodopsin, the second negative charge responsible for wavelength regulation is shown close to the 11,12 bond of the polyene chain. This model assumes that hypsorhodopsin is the unprotonated form of the Schiff base, and that it is formed possibly by proton transfer from the Schiff base nitrogen in some pigments. From Honig ct al. [207].

The process entails shifting of double bonds along the polyene chain, with the formation of a "retro-retinal" structure. Peters et al. (301) interpreted their observations by identifying PBAT with an excited state of rhodopsin, where single proton transfer toward the Schiff base nitrogen leads to the formation of bathorhodopsin. This approach has been supported by the theoretical interpretation of the spectrum of rhodopsin in terms of a nonprotonated Schiff base (214-216). A mechanism involving deprotonation of the Schiff base has also been suggested (310). All these models do not require cis-trans isomerization as a primary event in the chromo-phore. 

Both in the case of sensory rhodopsin in humans and of bacteriorhodopsin (a heptahelical membrane protein in halobacteria which is not coupled to a G protein) translocation of a Schiff-base proton is the essential step in making the protein functional (reviewed in ref 58). In rhodopsin the conversion of the inactive AH state to the AHI state that binds to the G protein is coupled to proton transfer from the Schiff base to the counterion, Glul 13, and proton uptake from the medium to the highly conserved Glul34, which serves as proton acceptor. Based on that similarity, one could consider sensory rhodopsin as an incomplete proton pump. Furthermore, a property shared by all G-protein-coupled receptors is a triplet, formed by residues 134-136 in rhodopsin, consisting of Glu-Arg-Tyr. The consequences of mutational replacement of Glul34 supports the notion that the state of protonation of this amino add is crudal for activity, and that its protonation triggers the conformational transition of the receptor from the inactive to the active state. 

Even though the number of biological processes amenable to study by time-resolved spectroscopy is extremely large and range from hemoglobin and vision to photosynthesis,this chapter shall be restricted to energy-transfer processes and specifically to the primary proton transfer in the visual pigments, the chromophore of vision, rhodopsin. 

There are several other results that also support the proton-transfer mechanism. The absorption spectrum of bathorhodopsin is red shifted with respect to the spectrum of rhodopsin itself, therefore, bathorhodopsin may be a more tightly protonated Schiff base than rhodopsin. Model studies by Sandorfy " show explicitly that translocating the proton toward the Schiff base nitrogen could account for the spectral red shift. 

Time-resolved Raman spectroscopy has proved to be a very useful tool to elucidate fast processes in biological molecules, for instance, to follow the fast structural changes during the visual process where, after photoexcitation of rhodopsin molecules, a sequence of energy transfer processes involving isomerization and proton transfer takes place. This subject is treated in more detail in Chap. 6 in comparison with other time-resolved techniques. 

A more complex and very interesting situation arises in the dark reactions of rhodopsin. More detail of the whole cascade of reactions involving rhodopsin is presented in section 4.2. There are at least two, but probably three, proton linked steps in the transition from metarhodopsin-I (MR-I) to metarhodopsin-II (MR-II) and to the state in which metarho-dopsin interacts with the G-protein, transducin, for its activation. The chromophore of rhodopsin, 11-civ-retinal, is bound to the protein via a Schiff s base linked to the e-amino group of a lysine residue. After the photochemical isomerization of 11-cis to all tronv-retinal, deprotonation of the Schiff s base (which may be an intramolecular proton transfer and not result in a free H ion) results in the spectral change (approximately 100 nm red shift of absorption peak) associated with the transition MR-I to MR-II. The very large temperature dependence of the equilibrium constant for this transition, discussed in section 7.2 permits its adjushnent to near unity at S °C and pH 7. This makes it possible to study the efifects of pH and thus of proton uptake, on the relaxation time of the transition. The equilibrium... 

Rafferty, C.N. and Shichi, H., Xhe involvement of water at the retinal binding site in rhodopsin and early Hght-induced intramolecular proton transfer, Photochem. Photobiol, 33, 229,1981. 

I to Meta II. It was shown that when Hght initiates events that lead to Schiff base deprotonation, the proton is transferred to its counterion, GIull3. The Meta I-Meta II transition is identified with this proton transfer. More recently, the x-ray structure of bovine rhodopsin has shown that another carbox-yhc acid, GIulSl, is also close to the Schiff base. Studies on an invertebrate pigment, retinochrome, suggest it can be in its anionic form so that it can, in some circumstances, act as a counterion. 

There is httle doubt that fast photoelectric signals are electrical manifestation of Hght-induced charge separation and recombination. For a macromolecule with a multistep reaction sequence as complex as rhodopsin or bacteriorhodopsin, there are many candidates for generating a fast photoelectric signal component. In principle, each step of reversible reaction can contribute a component. Fast photokinetic measurements have a tendency to pick up faster processes. We identified three fast components in reconstituted bacteriorhodopsin membranes Bl, B2, and a B2-like signal, which we called B2 component. Like B2, the B2 component is generated by interfacial proton transfer — the proton release and reuptake at the extracellular surface. This does not mean that slower components do not exist. In fact. 

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