A spectrally silent transformation in the photolysis of octopus rhodopsin: a protein conformational change without any accompanying change of the chromophore's absorption

A spectrally silent transformation in the photolysis of octopus rhodopsin was detected by the time-resolved transient grating method. Our results showed that at least two photointermediates, which share the same chromophore absorption spectrum, exist after the final absorption changes. Previously, mesorhodopsin was thought to decay to the final photoproduct, acid metarhodopsin with a lifetime of 38 micros at 15 degrees C, but the present results show that there is at least one intermediate species (called transient acid metarhodopsin) with a lifetime of 180 micros at 15 degrees C, before forming acid metarhodopsin. This indicates that the parts of the protein distant from the chromophore are still changing even after the changes in microenvironment around the chromophore are over. From the signal intensity detected by the transient grating method, the volume change of the spectrally silent transformation was found to be DeltaV = 13 ml/mol. The activation energy of the spectrally silent transformation is much lower than those of other transformations of octopus rhodopsin. Since stable acid metarhodopsin has not been shown to activate the G protein, this transient acid metarhodopsin may be responsible for G protein activation.     

Effect of anion binding on the thermal reverse reaction of bathoiodopsin: anion stabilizes two forms of iodopsin

The thermal reactions of the bathoproduct of the long wavelength sensitive visual pigment iodopsin were investigated under various anionic and environmental conditions, to get an insight into the mechanism leading to the unusual thermal isomerization of the retinal chromophore from the trans to the 11-cis form at very low temperatures (-160 degrees C). The all-trans chromophore of the bathoiodopsin produced from iodopsin in the presence of chloride thermally reverted to the 11-cis form, while in the presence of nitrate it kept its all-trans configuration upon warming. Different protein environments, either in a detergent or in phosphatidylcholine (PC) liposomes, did not change the reaction characteristics of the bathoiodopsins under the two anionic conditions. However, reaction characteristics of bathoiodopsins produced in the absence of small anions were dependent on the environment. The trans-to-cis isomerization occurred upon warming of bathoiodopsin in the presence of detergent but not in liposomes. Spectral measurements revealed that iodopsin in the absence of small anions is a mixture of two spectrally distinct forms that exhibit absorption maxima and reaction characteristics similar to those of chloride-bound and nitrate-bound iodopsins, respectively. Thus, iodopsin exhibits two conformational states, each of which is stabilized by the binding of chloride and nitrate, respectively.     

Interaction of aromatic retinal analogues with apopurple membranes of Halobacterium halobium

Absorption spectral properties of aromatic analogues of retinal with apopurple membrane of Halobacterium halobium were studied. The spectra of the all-trans forms were composed of two or more absorption bands. During incubation at 20 degrees C, an absorption band above 500 nm increased in intensity gradually at the expense of an absorption band in the shorter wavelength region with no isomerization of the chromophore. The longer wavelength species was shown to be the protonated form of the shorter wavelength species by changing the pH of the medium. Upon irradiation with blue light, the bandwidth of the spectrum became smaller with isomerization of the chromophore to its 13-cis form. Irreversible binding of protons on the membrane occurred during this process. The rate of the increase in the longer wavelength absorption band was especially low in the reaction with the all-trans form of retinal analogues having a bulky substituent at the para or meta positions of the phenyl ring. In contrast, the 13-cis isomer of aromatic retinal analogues gave a single absorption peak. The extent of the spectral shift upon binding to apopurple membranes was compared over a series of aromatic retinals, and the results were explained in terms of steric interactions of the chromophore with the protein.     

Effective light-induced hydroxylamine reactions occur with C13 = C14 nonisomerizable bacteriorhodopsin pigments.

The light-driven proton pump bacteriorhodopsin (bR) undergoes a bleaching reaction with hydroxylamine in the dark, which is markedly catalyzed by light. The reaction involves cleavage of the (protonated) Schiff base bond, which links the retinyl chromophore to the protein. The catalytic light effect is currently attributed to the conformational changes associated with the photocycle of all-trans bR, which is responsible for its proton pump mechanism and is initiated by the all-trans --> 13-cis isomerization. This hypothesis is now being tested in a series of experiments, at various temperatures, using three artificial bR molecules in which the essential C13==C14 bond is locked by a rigid ring structure into an all-trans or 13-cis configuration. In all three cases we observe an enhancement of the reaction by light despite the fact that, because of locking of the C13==C14 bond, these molecules do not exhibit a photocycle, or any proton-pump activity. An analysis of the rate parameters excludes the possibility that the light-catalyzed reaction takes place during the approximately 20-ps excited state lifetimes of the locked pigments. It is concluded that the reaction is associated with a relatively long-lived (micros-ms) light-induced conformational change that is not reflected by changes in the optical spectrum of the retinyl chromophore. It is plausible that analogous changes (coupled to those of the photocycle) are also operative in the cases of native bR and visual pigments. These conclusions are discussed in view of the light-induced conformational changes recently detected in native and artificial bR with an atomic force sensor.     

A nonbleachable rhodopsin analogue with a slow photocycle

Archaeal rhodopsins, e.g. bacteriorhodopsin, all have cyclic photoreactions. Such cycles are achieved by a light-induced isomerization step of their retinal chromophores, which thermally re-isomerize in the dark. Visual pigment rhodopsins, which contain in the dark state an 11-cis retinal Schiff base, do not share such a mechanism, and following light absorption, they experience a bleaching process and a subsequent release of the photo-isomerized all-trans chromophore from the binding pocket. The pigment is eventually regenerated by the rebinding of a new 11-cis retinal. In the artificial visual pigment, Rh(6.10), in which the retinal chromophore is locked in an 11-cis geometry by the introduction of a six-member ring structure, an activated receptor may be formed by light-induced isomerization around other double bonds. We have examined this activation of Rh(6.10) by UV-visible and FTIR spectroscopy and have revealed that Rh(6.10) is a nonbleachable pigment. We could further show that the activated receptor consists of two different subspecies corresponding to 9-trans and 9-cis isomers of the chromophore. Both subspecies relax in the dark via separate pathways back to their respective inactive states by thermal isomerization presumably around the C(13)=C(14) double bond. This nonbleachable pigment can be repeatedly photolyzed to undergo identical activation-relaxation cycles. The rate constants of these photocycles are pH-dependent, and the half-times vary between several hours at acidic pH and about 1.5 min at neutral to alkaline pH, which is several orders of magnitude longer than for bacteriorhodopsin.     

Kinetics of proton uptake and dye binding by photoactive yellow protein in wild type and in the E46Q and E46A mutants

We studied the kinetics of proton uptake and release by photoactive yellow protein (PYP) from Ectothiorhodospira halophila in wild type and the E46Q and E46A mutants by transient absorption spectroscopy with the pH-indicator dyes bromocresol purple or cresol red in unbuffered solution. In parallel, we investigated the kinetics of chromophore protonation as monitored by the rise and decay of the blue-shifted state I(2) (lambda(max) = 355 nm). For wild type the proton uptake kinetics is synchronized with the fast phase of I(2) formation (tau = 500 micros at pH 6.2). The transient absorption signal from the dye also contains a slower component which is not due to dye deprotonation but is caused by dye binding to a hydrophobic patch that is transiently exposed in the structurally changed and partially unfolded I(2) intermediate. This conclusion is based on the wavelength, pH, and concentration dependence of the dye signal and on dye measurements in the presence of buffer. SVD analysis, moreover, indicates the presence of two components in the dye signal: protonation and dye binding. The dye binding has a rise time of about 4 ms and is coupled kinetically with a transition between two I(2) intermediates. In the mutant E46Q, which lacks the putative internal proton donor E46, the formation of I(2) is accelerated, but the proton uptake kinetics remains kinetically coupled to the fast phase of I(2) formation (tau = 100 micros at pH 6.3). For this mutant the protein conformational change, as monitored by the dye binding, occurs with about the same time constant as in wild type but with reduced amplitude. In the alkaline form of the mutant E46A the formation of the I(2)-like intermediate is even faster as is the proton uptake (tau = 20 micros at pH 8.3). No dye binding occurred in E46A, suggesting the absence of a conformational change. In all of the systems proton release is synchronized with the decay of I(2). Our results support mechanisms in which the chromophore of PYP is protonated directly from the external medium rather than by the internal donor E46.     

An energy-based approach to packing the 7-helix bundle of bacteriorhodopsin.

Based on the heavy-atom coordinates determined by the electron microscopy for the seven main helical regions of bacteriorhodopsin with the all-trans retinal isomer, energy optimizations were carried out for helix bundles containing the all-trans retinal and 13-cis retinal chromophores, respectively. A combination of simulated annealing and energy minimization was utilized during the process of energy optimization. It was found that the 7-helix bundle containing the all-trans isomer is about 10 kcal/mol lower in conformational energy than that containing the 13-cis isomer. An energetic analysis indicates that such a difference in energy is consistent with the observation that absorption of a 570-nm proton is required for the conversion of a bacteriorhodopsin from its all-trans to 13-cis form. It was also found that the above conversion process is accompanied by a significant conformational perturbation around the chromophore, as reflected by the fact that the beta-ionone ring of retinal moves about 5.6 A along the direction perpendicular to the membrane plane. This is consistent with the observation by Fodor et al. (Fodor, S.P.A., Ames, J.B., Gebhard, R., van der Berg, E.M.M., Stoeckenius, W., Lugtenburg, J., & Mathies, R.A., 1988, Biochemistry 27, 7097-7101). Furthermore, it is interesting to observe that although the retinal chromophore undergoes a significant change in its spatial position, the orientation of its transition dipole changes only slightly, in accord with experimental observations. In other words, even though orientation of the retinal transition dipole is very restricted, there is sufficient room, and degrees of freedom, for the retinal chromophore to readjust its position considerably. This finding provides new insight into the subtle change of the retinal microenvironment, which may be important for revealing the proton-pumping mechanism of bacteriorhodopsin. The importance of electrostatic and nonbonded interactions in stabilizing the 7-helix bundle structure has also been analyzed. Electrostatic interactions favor an antiparallel arrangement among adjacent helices. Nonbonded interactions, however, drive most of the closely packed helices into an arrangement in which the packing angles lie around -160 degrees, a value very near the -154 degrees value computed earlier as the most favorable packing arrangement of two poly(Ala) alpha-helices (Chou, K.-C., Némethy, G., & Scheraga, H.A., 1983, J. Phys. Chem. 87, 2869-2881). The structural features of the 7-helix bundle and their relationship to those found in typical 4-helix bundle proteins are also discussed.(ABSTRACT TRUNCATED AT 400 WORDS)     

Bathoproducts of rhodopsin, isorhodopsin I, and isorhodopsin II.

Bathorhodopsins were prepared by partially (10--15%) photoconverting bovine rhodopsin (11-cis chromophore) or isorhodopsin I (9-cis chromophore) at 77 degrees K; care was taken to avoid establishing photostationary states. The absorption spectra calculated for the bathorhodopsins derived from the two parent pigments are identical in their lambda max 'S, bandwidths, and extinction coefficients. This result provides further support for the hypothesis that bathorhodopsin is a common intermediate between an 11-cis pigment (rhodopsin) and a 9-cis one (isorhodopsin I) and thus probably has an all-trans chromophore. This in turn is strong evidence for the cis-trans isomerization model of the primary event in vision. The spectrum of the bathoproduct of isorhodopsin II (9,13-dicis chromophore) is different from the other pigments' bathoproducts.     

Chromophore configuration of pharaonis phoborhodopsin and its isomerization on photon absorption.

The configuration of the retinylidene chromophore in pharaonis phoborhodopsin (ppR) and its changes during the photoreaction cycle were investigated by means of a chromophore extraction method followed by HPLC analysis. The ppR has an all-trans chromophore, and unlike bacteriorhodopsin, it exhibits no dark isomerization of the chromophore. Irradiation of a ppR sample in the presence of 10 mM hydroxylamine, at which concentration a negligible amount of ppR was bleached, caused the formation of 90% 13-cis- and 10% all-trans-retinal oximes. Because the ppR sample under the continuous irradiation was a mixture containing original ppR, ppRM, and a small amount of ppRO, the above results showed that the chromophores of ppRM and ppRO are in a 13-cis form and an all-trans form, respectively. Therefore, the all-trans chromophore of ppR is isomerized to the 13-cis form on photon absorption, and it is thermally reisomerized to the all-trans form on the conversion process from ppRM to ppRO. The extracted retinal oximes from ppR and ppRO were mainly the 15-syn form, while that from ppRM was mainly the 15-anti form. This fact indicated that the attack of hydroxylamine on the chromophore is stereoselective owing to the unique structure of the chromophore binding site near the Schiff base region of the chromophore.     

High-pressure near-infrared Raman spectroscopy of bacteriorhodopsin light to dark adaptation.

Near-infrared (NIR) Raman spectroscopy is employed as an in situ probe of the chromophore conformation to study the light to dark-adaptation process in bacteriorhodopsin (bR) at variable pressure and temperature in the absence of undesired photoreactions. In dark-adapted bR deconvolution of the ethylenic mode into bands assigned to the all-trans (1526 cm-1) and 13-cis (1534 cm-1) isomers yields a 13-cis to all-trans ratio equal to 1 at ambient pressure (Schulte et al., 1995, Appl. Spectrosc. 49:80-83). Detailed spectroscopic evidence is presented that at high pressure the equilibrium is shifted toward the 13-cis isomers and that the light to dark adaptation kinetics is accelerated. The change in isomeric composition with temperature and pressure as well as the kinetics support a two-state model activation volumes of -16 ml/mol for the transition of 13-cis to all-trans and -22 ml/mol for the reverse process. These compare with a conformational volume difference of 6.6 ml/mol, which may be attributed to the ionization of one or two residues or the formation of three hydrogen bonds.     

Spectroscopic study of the batho-to-lumi transition during the photobleaching of rhodopsin using ring-modified retinal analogues

Photochemical and subsequent thermal reactions of rhodopsin containing 9-cis-retinal [Rh(9)] or one of four analogues with 9-cis geometries formed from ring-modified retinals, alpha-retinal [alpha Rh(9)], acyclic retinal [AcRh(9)], acyclic alpha-retinal [Ac alpha Rh(9)], and 5-isopropyl-alpha-retinal [P alpha Rh(9)] were investigated by low-temperature spectrophotometry and nanosecond laser photolysis. Irradiation of each pigment at -180 degrees C produced a photosteady-state mixture containing the original 9-cis pigment, its 11-cis pigment, and a photoproduct, indicating that the primary process of each pigment is a photoisomerization of its chromophore. The photoproduct produced by the irradiation of AcRh(9) had an absorption spectrum red shifted from the original AcRh(9) and was identified as the batho intermediate of AcRh(9). It was converted to the lumi intermediate through a metastable species, the BL intermediate, which has never been detected in Rh(9) at low temperature and whose absorption maximum was at shorter wavelengths than that of the batho intermediate. In contrast, the absorption maxima of the photoproducts produced from the other analogue pigments were at shorter wavelengths than those of the original pigments. They were identified as BL intermediates on the basis of their absorption maxima and thermal stabilities. The formation time constant of the lumi intermediate at room temperature was found to be dependent on the extent of modification of the ring portion of the chromophore, decreasing with the complete truncation of the cyclohexenyl ring [Ac alpha Rh(9)] and increasing with the attachment of the isopropyl group to the ring [P alpha Rh(9)].(ABSTRACT TRUNCATED AT 250 WORDS)     

Conformational motion in bacteriorhodopsin: the K to L transition

By comparison of the time dependence of linear dichroism and transient absorption in light-adapted bacteriorhodopsin over the first 10 microseconds following excitation, conformational motion in the protein has been detected. Time-resolved linear dichroism and transient absorption scans are reported for several wavelengths that probe the K610 and L550 intermediates in the bacteriorhodopsin photocycle. The transient absorption scans are insensitive to conformational motion and yield the lifetimes of the K610 and L550 intermediates. In contrast, the time-resolved linear dichroism scans demonstrate orientational motion of the chromophore with a 1.7-microsecond rotational time. The wavelength dependence of the least-squares fitting parameters establishes that this motion is associated with L550. This motion is discussed in relation to a protein conformational change in the course of the bacteriorhodopsin photocycle. No evidence is observed for orientational motion on the time scale of the L550----M410 transition.     

Squid retinochrome

Retinochrome is a photosensitive pigment located primarily in the inner portions of the visual cells of cephalopods. Its absorption spectrum resembles that of rhodopsin, but its chromophore is all-trans retinal, which light isomerizes to 11-cis, the reverse of the situation in rhodopsin. The 11-cis photoproduct of retinochrome slowly reverts to retinochrome in the dark. The chromophoric site of retinochrome is more reactive than that of most visual pigments: (a) Hydroxylamine converts retinochrome in the dark to all-trans retinal oxime + retinochrome opsin. (by Sodium borohydride reduces it to N-retinyl opsin. (c) Lambda max of retinochrome shifts from 500 to 515 nm as the pH is raised from 6 to 10, with a loss of absorption above pH 8; meanwhile above this PH a second band appears at shorter wavelengths with lambda max 375 nm. These changes are reversible. (d) If retinochrome is incubated with all-trans 3-dehydroretinal (retinal2) in the dark, some 3-dehydroretinochrome (retinochrome2, lambda max about 515 nm) is formed. Conversely, when retinochrome2, made by adding all-trans retinal2 to bleached retinochrome or retinochrome opsin, is incubated in the dark with all-trans retinal some of it is converted to retinochrome. Retinal and 3-dehydroretinal therefore can replace each other as chromophores in the dark.     

Characterization of the chromophore of the third rhodopsin-like pigment of Halobacterium halobium and its photoproduct.

Halobacterium halobium contains at least three retinal-containing pigments: bacteriorhodopsin, halorhodopsin, and a third rhodopsin-like pigment (tR) absorbing at approximately 590 nm, tR590. Illumination of tR590 gives rise to a very long-lived blue absorbing photoproduct, tR370. Using high-performance liquid chromatography we show that the chromophore of tR590 is primarily all-trans retinal and its conversion by light to tR370 causes the chromophore to isomerize primarily to the 13-cis conformation. Irradiation of the tR370 gives rise to a transient photoproduct absorbing at approximately 520 nm that decays back to the initial pigment tR590. In addition to all-trans retinal, the apomembrane of tR can also combine with 13-cis retinal but not with the 9- or 11-cis isomers.     

Photochromism of halorhodopsin. cis/trans isomerization of the retinal around the 13-14 double bond

The isomeric composition of retinal in membrane-bound and in purified but detergent-free, dark-adapted halorhodopsin was found to be about 70% 13-cis and 30% all-trans. Any illumination increased the all-trans content relative to the dark-adapted state, but blue illumination shifted the isomeric composition more toward all-trans while red illumination of blue-adapted samples shifted it more toward 13-cis. In the presence of chloride this photoisomerization caused the kind of photochromic behavior reported earlier in Smith, S. O., Marvin, M. J., Bogomolni, R. A., and Mathies, R. A. (1984) J. Biol. Chem. 259, 12326-12329, i.e. blue light caused the absorption maximum to move toward longer wavelengths and red light reversed the shift. Only the all-trans chromophore exhibited the complete photocycle described earlier in detergent-solubilized halorhodopsin, and this was the form that could be associated with light-driven chloride transport activity in cell envelope vesicles. In the absence of chloride the spectroscopic changes caused by illumination were much smaller. Reconstitution of bleached preparations with 13-cis- and all-trans-retinal, in the presence and absence of chloride, confirmed that the difference between the absorption maxima of the two isomeric forms of the chromophore is affected by chloride: 13-cis-halorhodopsin absorbs at about 567-568 nm with and without chloride, and the all-trans pigment absorbs near 568 nm in the absence of chloride, but at 578 nm in its presence. The simplest explanation of this finding is that most of the red-shift which accompanies the 13-cis----all-trans transition originates from electrostatic interaction of the retinal with chloride bound in its vicinity.     

Squid retinochrome. Configurational changes of the retinal chromophore.

The configurations of the retinal chromophore in light and dark reactions of squid retinochrome were investigated by means of high-performance liquid chromatography. Orange light isomerized the chromophore of retinochrome, all-trans-retinal, mainly to the 11-cis configuration in metaretinochrome. Irradiation with shorter-wavelength lights not only accelerates the photoreversal of metaretinochrome to retinochrome but also leads to a slight production of isoretinochrome (13-cis-retinochrome), yielding a photoequilibrium mixture of three kinds of retinochrome. 13-cis- and 9-cis-retinochromes are photosensitive, and are converted into metaretinochrome upon irradiation with orange light. When steadily exposed to orange light in the presence of a trace of retinochrome-protein, all of the all-trans-, 13-cis-, and 9-cis-retinals are catalytically isomerized only to the 11-cis form, although the reaction rate is reduced in the order of the retinals listed above. In the dark, 9-cis-retinochrome, like retinochrome, remains unchanged, but both meta- and 13-cis-retinochromes slowly change to retinochrome. The chromophore of 13-cis-retinochrome changes directly to the all-trans form, whereas the 11-cis chromophore of metaretinochrome goes to all-trans mainly through the 13-cis form. The direct isomerization from 11-cis to all-trans hardly occurs at temperatures as low as 20 degrees C, and shows high values of the activation enthalpy and entropy changes. Based upon these findings, the role of retinochrome in the photoreception of the visual cells is discussed.     

Early steps of the intramolecular signal transduction in rhodopsin explored by molecular dynamics simulations

We present molecular dynamics simulations of bovine rhodopsin in a membrane mimetic environment based on the recently refined X-ray structure of the pigment. The interactions between the protonated Schiff base and the protein moiety are explored both with the chromophore in the dark-adapted 11-cis and in the photoisomerized all-trans form. Comparison of simulations with Glu181 in different protonation states strongly suggests that this loop residue located close to the 11-cis bond bears a negative charge. Restrained molecular dynamics simulations also provide evidence that the protein tightly confines the absolute conformation of the retinal around the C12-C13 bond to a positive helicity. 11-cis to all-trans isomerization leads to an internally strained chromophore, which relaxes after a few nanoseconds by a switching of the ionone ring to an essentially planar all-trans conformation. This structural transition of the retinal induces in turn significant conformational changes of the protein backbone, especially in helix VI. Our results suggest a possible molecular mechanism for the early steps of intramolecular signal transduction in a prototypical G-protein-coupled receptor.     

The two photocycles of photoactive yellow protein from Rhodobacter sphaeroides

The absorption spectrum of the photoactive yellow protein from Rhodobacter sphaeroides (R-PYP) shows two maxima, absorbing at 360 nm (R-PYP(360)) and 446 nm (R-PYP(446)), respectively. Both forms are photoactive and part of a temperature- and pH-dependent equilibrium (Haker, A., Hendriks, J., Gensch, T., Hellingwerf, K. J., and Crielaard, W. (2000) FEBS Lett. 486, 52-56). At 20 degrees C, for PYP characteristic, the 446-nm absorbance band displays a photocycle, in which the depletion of the 446-nm ground state absorption occurs in at least three phases, with time constants of <30 ns, 0.5 micros, and 17 micros. Intermediates with both blue- and red-shifted absorption maxima are transiently formed, before a blue-shifted intermediate (pB(360), lambda(max) = 360 nm) is established. The photocycle is completed with a monophasic recovery of the ground state with a time constant of 2.5 ms. At 7 degrees C these photocycle transitions are slowed down 2- to 3-fold. Upon excitation of R-PYP(360) with a UV-flash (330 +/- 50 nm) a species with a difference absorption maximum at approximately 435 nm is observed that returns to R-PYP(360) on a minute time scale. Recovery can be accelerated by a blue light flash (450 nm). R-PYP(360) and R-PYP(446) differ in their overall protein conformation, as well as in the isomerization and protonation state of the chromophore, as determined with the fluorescent polarity probe Nile Red and Fourier Transform Infrared spectroscopy, respectively.     

Proton transfers in the photochemical reaction cycle of proteorhodopsin

The spectral and photochemical properties of proteorhodopsin (PR) were determined to compare its proton transport steps to those of bacteriorhodopsin (BR). Static and time-resolved measurements on wild-type PR and several mutants were done in the visible and infrared (FTIR and FT-Raman). Assignment of the observed C=O stretch bands indicated that Asp-97 and Glu-108 serve as the proton acceptor and donor, respectively, to the retinal Schiff base, as do the residues at corresponding positions in BR, but there are numerous spectral and kinetic differences between the two proteins. There is no detectable dark-adaptation in PR, and the chromophore contains nearly entirely all-trans retinal. Because the pK(a) of Asp-97 is relatively high (7.1), the proton-transporting photocycle is produced only at alkaline pH. It contains at least seven transient states with decay times in the range from 10 micros to 200 ms, but the analysis reveals only three distinct spectral forms. The first is a red-shifted K-like state. Proton release does not occur during the very slow (several milliseconds) rise of the second, M-like, intermediate, consistent with lack of the residues facilitating extracellular proton release in BR. Proton uptake from the bulk, presumably on the cytoplasmic side, takes place prior to release (tau approximately 2 ms), and coincident with reprotonation of the retinal Schiff base. The intermediate produced by this process contains 13-cis retinal as does the N state of BR, but its absorption maximum is red-shifted relative to PR (like the O state of BR). The decay of this N-like state is coupled to reisomerization of the retinal to all-trans, and produces a state that is O-like in its C-C stretch bands, but has an absorption maximum apparently close to that of unphotolyzed PR.     

Studies on cephalopod rhodopsin: photoisomerization of the chromophore.

Photoisomerization of the chromophore of squid rhodopsin is dependent upon the irradiation temperature. Above 0 degrees C, only 11-cis in equilibrium all-trans reaction proceeds and the all-trans leads to 9-cis reaction is limited to extremely low efficiency. At liquid nitrogen temperature, 11 cis in equilibrium all-trans in equilibrium 9-cis reaction takes place. At intermediary low temperatures (-80 degrees C to -15 degrees C) another isomer of retinal may be produced by the irradiation, which forms a pigment having an absorbance maximum at 465 nm (P-465). The formation of P-465 decreases remarkably in the narrow temperature range from -30 degrees C to 0 degrees C where mesorhodopsin converts to metarhodopsin. Medsorhodopsin is quite different from metarhodopsin in the photoisomerization of the chromophore because P-465 is produced from the former but not from the latter. No P-465 is produced both at liquid nitrogen temperature and above 0 degrees C. P-465 is more labile than any of the other photoproducts so far known, that is isorhodopsin, alkaline and acid metarhodopsins. P-465 is converted to metarhodopsin by irradiation.