3.4.1 Classical photoisomerization pathway
The UV/Vis photointermediates of rhodopsin may be arranged in a (simplified) reaction scheme, which includes the approximate lifetime at room temperature and absorption maxima (in nm):
R (498) B (540) ^ BSI (477) L (497) MI (478) ^ Mil (380) ^ Mill (465)
i min opsin + all-mms-retinal (387)
After illumination of rhodopsin (R), bathorhodopsin (B) is trapped below -140°C. Lumirhodopsin (L) is obtained by warming above this temperature, and metarhodopsin I (MI) begins to form above -40°C (see ref. ). Above -15°C, MI is in thermal equilibrium with metarhodopsin II (Mil) [44,45], which decays slowly to metarhodopsin III (Mill) and/or to opsin and all-trans-retinal [46,47]. The blue-shifted intermediate (BSI), which does not accumulate at low temperatures, is only obtained in time-resolved measurements at room temperature .
As with other photoreceptors, photoproducts are denoted by their UV/Vis spectrophotometric properties. According to this convention, any 380 nm absorbing species (indicative of a deprotonated retinal Schiff base bond, see below) will be termed Mil, and isochromic forms of Mil will be denoted as subforms, as for example Mlla, Mllb, etc. An exception to this is the early isochromic Mil-like species, which is termed MI380 . It can be observed in detergent solution in considerable amounts, and is discussed in branched reaction schemes (for a review see ). A Mil-like species with an absorption maxima at 470 nm due to a reprotonated Schiff base can be obtained at low pH and high anion concentrations .
3.4.2 Early events - storage of photon energy in bathorhodopsin
Photon absorption provides rhodopsin with approximately 55kcalmol-1. Two-thirds of this energy is stored in the photo-activated chromophore (all-iraras-retinylidene) opsin complex , lifting the receptor from the dormant 11-cw-retinal/opsin conformation, via photorhodopsin, to bathorhodopsin. The dynamics of isomerization of the ll-c/s-retinal protonated Schiff base (PSB) have been elucidated by femtosecond pump-probe experiments. In rhodopsin, the \\-cis —► al\-trans photoisomerization occurs on an ultrafast (femtosecond) timescale yielding the first photoproduct after only 200 fs [52-54]. In solution, the photoisomerization reaction of 11-as-retinal-PSB is completed on the picosecond time scale , Steric interaction of the chromophore with the protein and chromophore distortion is believed to be responsible for the extremely fast kinetics and the high photoreaction quantum yield of rhodopsin of-0.67. Upon excitation of the chromophore, it is assumed that the molecule undergoes a nonadiabatic barrierless motion along a coordinate, which leads from the 1 l-cw-retinal-PSB excited state to the all-trans-retinal-PSB ground state. This appears to happen during a single torsional vibration . The observed vibrational coherence in the photoproduct  argues that the isomerization coordinate on the excited state continues directly onto the ground state potential energy surface thus avoiding excited state equilibration. Ab initio methods have been used to study excited state dynamics of 3,5-pentadienal PSB as a model chromophore for 11-cw-retinal-PSB [56,57, and references therein]. Based on experimental data and computations it appears that during the first 25 fs the excited state 11-cw-retinal-PSB chromophore moves out of the Franck-Condon region along a mode which involves primarily stretching of the polyene chain [54,56]. It is assumed that the initial motion consists of an elongation of the double bond in the middle of the polyene chain associated with the change in bond order. Then, the 11 -cis-retinal-PSB relaxes along a different coordinate towards a S, region where the excited (S^ and ground (S0) states conically intersect. The subsequent S[ to S0 decay occurs within 60 fs , The intersection point has a twisted central double bond that provides a route for efficient nonadiabatic cis-^ trans isomer-ization. Thus, torsion around the central m-configured double bond would set in only after the bond stretching has been completed. The computations on the 3,5-pentadienal PSB model chromophore provide information on the changes of charge distribution along the photoisomerization path and suggest that the S^So intersection point is influenced by the charge distribution around the retinal chromophore. The x-ray structure reveals that Glu181, in the extracellular plug domain, points towards the 11,12-ene of the chromophore and may be responsible for the exclusive isomerization around the Cn=C12 double bond. A corresponding external point charge, which can perturb the electron distribution of the polyene chain, was postulated earlier .
In contrast, ring-constrained 1 1-ds-locked analogs, i.e. 11-m-retinal analogs, with a bridge between C10 and C13 of 1-4 carbons which prevents isomerization around the Cn=C12 bond, stabilize opsin in its inactive conformation (or minimally active), even under light conditions [59-61]. Rhodopsin, regenerated with a locked 11 -ene via a six-membered ring (two-carbon bridge between C10 and C13), readily undergoes photoisomerization, albeit not around the C—C]2 double bond. Several isomers (9-cis- and/or 13-cw-forms of locked 11-cw-retinal) can be extracted from the retinal binding pocket , However, this photoisomerization does not lead to significant activation of Gt [61-63] or chromophore-induced conformational changes of the opsin moiety, as investigated by FTIR spectroscopy . These results suggest that the main effect of the native chromophore cis—*trans isomerization around Cu-C|2 is to impose strain on the chromophore, confining energy in the interaction of the chromophore with nearby residues. This allows transformation and storage of photon energy into chemical free energy, which is used to change the protein conformation into the active state. In a possible scenario compatible with the crystal structure data, isomerization by rotation around the Schiff base side of the C,,-C12 double bond would lead to relocation of the polyene chain to a closer position to the side chain of Ser186. Steric restriction should limit the degree of rotation. This may explain part of the distortion in the all-trans configuration in bathorhodopsin and the unchanged Schiff base environment, in agreement with the spectroscopic data [64,65].
3.4.3 Relaxation and steric trigger — lumirhodopsin and metarhodopsin I
A large positive reaction enthalpy and reaction volume relative to the ground state accompanies the formation of the lumi (L) intermediate , The enthalpy of reaction depends strongly on the hydrophobic environment (90 vs.
11 kJ mol-1 for washed membranes and dodecyl maltoside solution, respectively ). In the B—>L transition (via BSI), stored energy is released rapidly through changes in the chromophore and its local environment and thermal dissipation. In L, most of the photon energy absorbed by rhodopsin has already transferred to the apoprotein ,
Several spectroscopic investigations using cyclohexenyl ring ((3-ionone)-modified retinal analogues suggested that, in this transition, the ring portion of the chromophore changes its interaction with nearby amino acid residues (for details see ). Photolabelling studies have indicated that the [3-ionone ring, whose position is largely constrained in the ground state, is relocated in the B—>L transition , thereby releasing chromophore distortion as seen in the largely reduced hydrogen out-of-plane modes in this intermediate . It is thought that in this new configuration the (3-ionone ring triggers the formation of the later, protonation- and G-protein-dependent MI and Mil states, which form on the timescale of micro- and milliseconds. Mil formation coincides with the eventual activation of the receptor. Accordingly, ring-modified rhodopsins showed a decrease in Gt activation . The L—>MI transition occurs with a decrease in enthalpy and entropy, while the MI—>MII transition occurs with an increase in enthalpy and entropy , suggesting that the molecular events occurring in the L—>MI transition are opposite in nature to those in the MI—»Mil transition. This may be explained by specific interactions built up in the chromophore region leading to a thermodynamically stable MI conformation. Conversely, loss of these interactions leads to an increased coupling between rhodopsin's hydrophobic core and its cytoplasmic domain and allows the MI—>MII transition, which results in relaxation of the whole protein into the thermodynamically stable Mil state [43,73,74], Rhodopsin, regenerated with 11-m-9-demethyl-retinal, forms MI but shows a significant shift in the MI^Mil equilibrium towards the MI side with severe consequences for the ability of 9-demethyl rhodopsin to activate Gt. The activity of this pigment is reduced four- to five-fold, because fewer molecules enter the active Mil state due to less constrainting chromophore-protein interactions and increased entropy in 9-demethyl MI [50,75]. Correct chromophore-protein interactions in MI are decisive for transition to Mil, especially for accompanying proton transfer reactions which depend on the scaffolding function of alWra/w-retinal in the MI state . Transitions L—>MI and MI—»Mil are influenced by rhodopsin's lipid environment. Detergents such as alkyl-glycosides and alkylmaltosides increase the entropy of the ground state and intermediates, driving rhodopsin's light-induced conformational changes to Mil ,
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