Transient spectra of intermediates in the photolytic sequence of octopus rhodopsin

The intermediate photolytic sequence of octopus rhodopsin was studied at different temperatures and different pH values by means of a flash photolysisrapid scan spectrophotometry near physiological temperature.The first photoproduct in the photolysis of rhodopsin was lumirhodopsin. Transformation of lumirhodopsin → mesorhodopsin took place independently of the pH of the solution. Mesorhodopsin was transformed to acid metarhodopsin in acid solution. In alkaline solution, mesorhodopsin was transformed to transient acid metarhodospsin whose absorption spectrum was similar to acid metarhodopsin. Transient acid metarhodopsin was then transformed to alkaline metarhodopsin reaching a tautomeric equilibrium which was determined by the pH of the solution.     

Light-induced protein conformational changes in the photolysis of octopus rhodopsin.

Light-induced protein conformational changes in the photolysis of octopus rhodopsin were measured with a highly sensitive time-resolved transient UV absorption spectrophotometer with nanosecond time resolution. A negative band around 280 nm in the lumirhodopsin minus rhodopsin spectra suggests that alteration of the environment of some of the tryptophan residues has taken place before the formation of lumirhodopsin. A small recovery of the absorbance at 280 nm was observed in the transformation of lumirhodopsin to mesorhodopsin. Kinetic parameters suggest that major conformational changes have taken place in the transformation of mesorhodopsin to acid metarhodopsin. In this transformation, drastic changes of amplitude and a shift of a difference absorption band around 280 nm take place, which suggest that some of the tryptophan residues of rhodopsin become exposed to a hydrophilic environment.     

Energetics and volume changes of the intermediates in the photolysis of octopus rhodopsin at a physiological temperature

Enthalpy changes (Delta H) of the photointermediates that appear in the photolysis of octopus rhodopsin were measured at physiological temperatures by the laser-induced transient grating method. The enthalpy from the initial state, rhodopsin, to bathorhodopsin, lumirhodopsin, mesorhodopsin, transient acid metarhodopsin, and acid metarhodopsin were 146 +/- 15 kJ/mol, 122 +/- 17 kJ/mol, 38 +/- 8 kJ/mol, 12 +/- 5 kJ/mol, and 12 +/- 5 kJ/mol, respectively. These values, except for lumirhodopsin, are similar to those obtained for the cryogenically trapped intermediate species by direct calorimetric measurements. However, the Delta H of lumirhodopsin at physiological temperatures is quite different from that at low temperature. The reaction volume changes of these processes were determined by the pulsed laser-induced photoacoustic method along with the above Delta H values. Initially, in the transformation between rhodopsin and bathorhodopsin, a large volume expansion of +32 +/- 3 ml/mol was obtained. The volume changes of the subsequent reaction steps were rather small. These results are compared with the structural changes of the chromophore, peptide backbone, and water molecules within the membrane helixes reported previously.     

Flash photolysis of rhodopsin in rabbit

Flash photolysis of rhodopsin in rabbit's retina has been analysed theoretically, and the results are found to be in good agreement with the experimental results of Hagins (1957). We have also obtained the variation of relative concentrations of rhodopsin, lumirhodopsin, isorhodopsin and metarhodopsin I during the period of the flash corresponding to two different intensities of the flash. It has been found that the quantum efficiencies of conversion of lumirhodopsin into rhodopsin and isorhodopsin will lie in the range 0.24–0.45 and 0.20–0.44 respectively; quantum efficiencies of conversion of metarhodopsin I into rhodopsin and isorhodopsin are found to have values greater than 0.52 and 0.45 respectively and the quantum efficiency of conversion of isorhodopsin into lumirhodopsin has been found to be approximately 0.865. Also the maximum value of the rate constant of the reaction metarhodopsin Imetarhodopsin II at 37 C has been determined in decerebrated eye and it has been found that it is of the same order as found by Pugh (1975) in the case of human eye.Work partially supported by Department of Science and Technology     

Circular dichroism of cephalopod rhodopsin and its intermediates in the bleaching and photoregeneration process.

In the bleaching process of cephalopod rhodopsin, a new intermediate was found in the conversion process from lumirhodopsin to metarhodopsin. This intermediate of octopus has an absorption peak at about 475 nm and has been named as M475. The circular dichroism value of M475 is too small to be evaluated. On the other hand, lumirhodopsin shows a negative CD at 470 nm, a positive CD at 350 nm and a large positive CD band with three peaks at 280, 287 and 295 nm. Such a large CD band in the ultraviolet region is not observed in rhodopsin, M475 and metarhodopsin. This CD seems to be mainly due to tryptophan and tyrosine residues restricted in free rotation in the protein moiety of lumirhodopsin. The intermediate in the photoregeneration process of cephalopod rhodopsin, P380, has a positive CD band at the main peak, 380 nm, and also a large positive CD band in the ultraviolet region like lumirhodopsin.     

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.     

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.     

Removal of the 9-methyl group of retinal inhibits signal transduction in the visual process. A Fourier transform infrared and biochemical investigation

The photoreaction of opsin regenerated with 9-demethylretinal has been investigated by UV-vis spectroscopy, flash photolysis experiments, and Fourier transform infrared difference spectroscopy. In addition, the capability of the illuminated pigment to activate the retinal G-protein has been tested. The photoproduct, which can be stabilized at 77 K, resembles more the lumirhodopsin species, and only minor further changes occur upon warming the sample to 170 K (stabilizing lumirhodopsin). UV-vis spectroscopy reveals no further changes at 240 K (stabilizing metarhodopsin I), but infrared difference spectroscopy shows that the protein as well as the chromophore undergoes further molecular changes which are, however, different from those observed for unmodified metarhodopsin I. UV-vis spectroscopy, flash photolysis experiments, and infrared difference spectroscopy demonstrate that an intermediate different from metarhodopsin II is produced at room temperature, of which the Schiff base is still protonated. The illuminated pigment was able to activate G-protein, as assayed by monitoring the exchange of GDP for GTP gamma S in purified G-protein, only to a very limited extent (approximately 8% as compared to rhodopsin). The results are interpreted in terms of a specific steric interaction of the 9-methyl group of the retinal in rhodopsin with the protein, which is required to initiate the molecular changes necessary for G-protein activation. The residual activation suggests a conformer of the photolyzed pigment which mimics metarhodopsin II to a very limited extent.     

pH dependence of photolysis intermediates in the photoactivation of rhodopsin mutant E113Q

Glutamic acid at position 113 in bovine rhodopsin ionizes to form the counterion to the protonated Schiff base (PSB), which links the 11-cis-retinylidene chromophore to opsin. Photoactivation of rhodopsin requires both Schiff base deprotonation and neutralization of Glu-113. To better understand the role of electrostatic interactions in receptor photoactivation, absorbance difference spectra were collected at time delays from 30 ns to 690 ms after photolysis of rhodopsin mutant E113Q solubilized in dodecyl maltoside at different pH values at 20 degrees C. The PSB form (pH 5. 5, lambda(max) = 496 nm) and the unprotonated Schiff base form (pH 8. 2, lambda(max) = 384 nm) of E113Q rhodopsin were excited using 477 nm or 355 nm light, respectively. Early photointermediates of both forms of E113Q were qualitatively similar to those of wild-type rhodopsin. In particular, early photoproducts with spectral shifts to longer wavelengths analogous to wild-type bathorhodopsin were seen. In the case of the basic form of E113Q, the absorption maximum of this intermediate was at 408 nm. These results suggest that steric interaction between the retinylidene chromophore and opsin, rather than charge separation, plays the dominant role in energy storage in bathorhodopsin. After lumirhodopsin, instead of deprotonating to form metarhodopsin I(380) on the submillisecond time scale as is the case for wild type, the acidic form of E113Q produced metarhodopsin I(480), which decayed very slowly (exponential lifetime = 12 ms). These results show that Glu-113 must be present for efficient deprotonation of the Schiff base and rapid visual transduction in vertebrate visual pigments.     

The rhodopsin system of the squid

Squid rhodopsin (λ_max 493 mµ)—like vertebrate rhodopsins—contains a retinene chromophore linked to a protein, opsin. Light transforms rhodopsin to lumi- and metarhodopsin. However, whereas vertebrate metarhodopsin at physiological temperatures decomposes into retinene and opsin, squid metarhodopsin is stable. Light also converts squid metarhodopsin to rhodopsin. Rhodopsin is therefore regenerated from metarhodopsin in the light. Irradiation of rhodopsin or metarhodopsin produces a steady state by promoting the reactions, See PDF for Equation Squid rhodopsin contains neo-b (11-cis) retinene; metarhodopsin all-trans retinene. The interconversion of rhodopsin and metarhodopsin involves only the stereoisomerization of their chromophores. Squid metarhodopsin is a pH indicator, red (λ_max 500 mµ) near neutrality, yellow (λ_max 380 mµ) in alkaline solution. The two forms—acid and alkaline metarhodopsin—are interconverted according to the equation, Alkaline metarhodopsin + H⁺ acid metarhodopsin, with pK 7.7. In both forms, retinene is attached to opsin at the same site as in rhodopsin. However, metarhodopsin decomposes more readily than rhodopsin into retinene and opsin. The opsins apparently fit the shape of the neo-b chromophore. When light isomerizes the chromophore to the all-trans configuration, squid opsin accepts the all-trans chromophore, while vertebrate opsins do not and hence release all-trans retinene. Light triggers vision by affecting directly the shape of the retinene chromophore. This changes its relationship with opsin, so initiating a train of chemical reactions.     

Photolysis intermediates of human rhodopsin.

Photochemical studies were conducted on human rhodopsin at 20 degrees C to characterize the intermediates which precede the formation of metarhodopsin II, the trigger for the enzyme cascade mechanism of visual transduction. Human rhodopsin was prepared from eyes which had previously been used for corneal donations. Time resolved absorption spectra collected from 10(-8) to 10(-6) s after photolysis of human rhodopsin in detergent suspensions displayed biexponential decay kinetics. The apparent lifetimes obtained from the data are 65 +/- 20 and 292 +/- 25 ns, almost a factor of 2 slower than the corresponding rates in bovine rhodopsin. The spectra can be fit well using a model in which human bathorhodopsin decays toward equilibrium with a blue-shifted intermediate (BSI) which then decays to lumirhodopsin. Spectra and kinetic rate constants were determined for all these intermediates using a global analysis which showed that the spectra of the human intermediates are remarkably similar to bovine intermediates. Microscopic rate constants derived from this model are 7.4 x 10(6) s-1 for bathorhodopsin decay and 7.5 x 10(6) s-1 and 4.6 x 10(6) s-1 for the forward and reverse reactions of BSI, respectively. Decay of lumirhodopsin to later intermediates was studied from 10(-6) to 10(-1) s after photolysis of rhodopsin in human disk membrane suspensions. The human metarhodopsin I in equilibrium metarhodopsin II equilibrium appears to be more forward shifted than in comparable bovine studies.     

Two forms of intermediates of frog rhodopsin in rod outer segments

Using frog rod outer segments, we measured changes of the absorption spectrum during the conversion of rhodopsin to a photosteady-state mixture composed of rhodopsin, isorhodopsin and bathorhodopsin by irradiation with blue light (440 nm) at ? 190°C and during the reversion of bathorhodopsin to a mixture of rhodopsin and isorhodopsin by irradiation with red light (718 nm) at ? 190°C. The reaction kinetics was expressed by one exponential in the former case and by two exponentials in the latter. These results suggest that rhodopsin is composed of a single molecular species, while bathorhodopsin is composed of two kinds of molecular species designated as batho₁-rhodopsin and batho₂-rhodopsin. On warming the two forms of bathorhodopsin, each bathorhodopsin converted to its own lumirhodopsin, metarhodopsin I and finally a free all-trans-retinal plus opsin. The absorption spectra of the two forms of bathorhodopsin, lumirhodopsin and metarhodopsin I were measured at ? 190°C. We infer that a rhodopsin molecule in the excited state relaxes to either batho₁-rhodopsin or batho₂-rhodopsin, and then converts to its own intermediates through one of the two parallel pathways.     

Conformation changes of cuttlefish (Euprymna morsei) rhodopsin following photoconversion

Cuttlefish (Euprymna morsei) rhodopsin solubilized in lauryl ester of sucrose and its photoproduct, acid metarhodopsin, were examined by small-angle X-ray scattering and chromatofocusing to investigate the conformation changes of visual pigment following photoconversion. From spectroscopic studies, it was found that more than 93% of Euprymna rhodopsin could be converted to meta form under the condition of red light irradiation at neutral pH. Since almost pure acid metarhodopsin solution was prepared without changing the specimen concentration, the small-angle X-ray scattering intensities of both pigment-detergent complexes were directly compared. The radius of gyration increased on going from rhodopsin to acid metarhodopsin by approximately 1.5%. There were also discernible changes in the secondary peak intensities. The distribution function, derived by the Fourier transformation of intensity data, showed a significant change around 55 A. The maximum linear dimension of the rhodopsin-detergent complex was about 95 A and hardly changed after illumination. Intensity at zero angle did not change after illumination, suggesting that the aggregation did not occur. The change of the intensity profile could be due to the conformational change of the pigment-detergent monomers. The pI value of rhodopsin determined by chromatofocusing was 5.32 and that of acid metarhodopsin was 5.06, indicating that a few carboxyl groups are newly dissociated. The shift of the protein mass and the charge redistribution were observed following photoconversion.     

Molecular mechanism for the initial process of visual excitation. III. Theoretical studies of optical spectra and conformations of chromophores in visual pigments, their analogues and intermdiates based on the torsion model.

The torsion model with which we proposed to interpret the specific properties of the photoisomerization reaction of rhodopsin has been developed to apply to isorhodopsin I, isorhodopsin II and some intermediates. Based on this model, optical absorption wavelengths and oscillator strengths, as well as rotational strengths of visual pigments, analogues and intermediates at low temperatures are analyzed by varying twisted conformations of the chromophores. As a result, it was found that most of the optical data could be very well accounted for quantitatively by the torsion model. The twisting characters in the chromophore of rhodopsin are very similar to those of isorhodopsin. The obtained conformations of the chromophores are very similar in rhodopsin and its analogues, and in isorhodopsin and its analogues. Those of the chromophores of bathorhodopsin, lumirhodopsin and metarhodopsin I are similar to one another except that the conjugated chain of metarhodopsin I bends considerably when compared with the other intermediates.     

Optical activity of octopus metarhodopsins.

The optical activity of octopus rhodopsin, acid metarhodopsin and alkaline metarhodopsin was studied by a sensitive and rapid CD apparatus. For sometime it has been thought that cephalopod metarhodopsins do not have any optical activity associated with their main absorption band. However, the present work shows that acid metarhodopsin in digitonin has a positive CD band at 498 nm and a negative CD band at 436 nm and alkaline metarhodopsin has a negative CD band at 381 nm. Detergent affected the wavelength of the CD peak of the visual pigments though the pattern of the spectrum was similar. From these results it is concluded that the conformation of all-trans retinal in octopus metarhodopsin is influenced by the asymmetric conformation of the protein near the retinal and therefore inducing optical activity.     

Purification of squid rhodopsin and reassembly into lipid bilayers

Rhodopsin from squid photoreceptor membranes was solubilized in octyl glucoside and purified to a single band on SDS-polyacrylamide gels of $\text{M}{}_{\mathrm{<mtext>r</mtext>}}$ 46 000. Purified rhodopsin was recombined with phospholipids to form vesicles by detergent dialysis. Spectroscopic analysis of the rhodopsin-lipid vesicles showed that the interconversion between acid and basic metarhodopsin had a $\text{p}\text{K}$ of 8. Furthermore, rhodopsin in the vesicles could be photoregenerated from metarhodopsin in solutions of either neutral or alkaline pH. These two spectroscopic properties are comparable to those for rhodopsin in photoreceptor membranes. The results indicate that the native conformation of rhodopsin is preserved during purification and after recombination with phospholipids into vesicles. This preparation is, therefore, an active starting point for functional reconstitution studies.     

Bleaching kinetics of artificial visual pigments with modifications near the ring-polyene chain connection

Absorbance difference spectra were recorded at 20 degrees C from 30 ns to milliseconds after photolysis of lauryl maltoside suspensions of artificial visual pigments derived from 9-cis isomers of 5-ethylretinal, 8,16-methanoretinal (a 6-s-trans-bicyclic analogue), or 5-demethyl-8-methylretinal. In all three pigments, the earliest intermediate that was detected had the characteristics of a mixture of bathorhodopsin and a blue-shifted intermediate, BSI, which is the first decay product of bathorhodopsin in bovine rhodopsin. The first decays resolved on the nanosecond time scale were the formation of the lumirhodopsin analogues. Subsequent decays were able to be fit with a mechanistic scheme which has been shown to apply to both membrane and detergent suspensions of rhodopsin. Large increases were seen in the amount of metarhodopsin I which appeared after photolysis of 5-ethylisorhodopsin and the bicyclic isorhodopsin analogue, while 5-demethyl-8-methylisorhodopsin more closely followed native rhodopsin in decaying through meta I380, a 380 nm absorbing precursor to metarhodopsin II. In addition to forming more metarhodopsin I, the bicyclic analogue stabilized the metarhodopsin I-metarhodopsin II equilibrium similarly to what has been previously reported for 9-demethylrhodopsin in detergent, introducing the possibility that the bicyclic analogue could similarly be defective in transducin activation. These observations support the idea that long after initial photolysis, structural details of the retinylidene chromophore continue to play a decisive role in processes leading to the activated form, metarhodopsin II.     

Photoenergetics of octopus rhodopsin

The enthalpy changes associated with each of the major steps in the photoconversion of octopus rhodopsin have been measured by direct photocalorimetry. Formation of the primary photoproduct (bathorhodopsin) involves energy uptake of about 130 kJ/mol, corresponding to storage of over 50% of the exciting photon energy, and is comparable to the energy storage previously observed in bovine rhodopsin. Subsequent intermediates involve the step-wise dissipation of this energy to give the physiological end-product (acid metarhodopsin) at a level only slightly above the parent rhodopsin. No significant differences in energetics are observed between rhodopsin in microvilli membrane suspensions or detergent dispersions. Use of different buffer systems in the calorimetric experiments shows that conversion of rhodopsin to acid metarhodopsin involves no light-induced protonation change, whereas alkali metarhodopsin photoproduction occurs with the release of one proton per molecule and an additional enthalpy increase of about 50 kJ/mol. Van't Hoff analysis of the effect of temperature on the reversible metarhodopsin equilibrium gives an enthalpy for the acid alkali transition consistent with this calorimetric result, and the proton release is confirmed by direct observation of light-induced pH changes. Acid-base titration of metarhodopsin yields an apparent pK of 9.5 for this transition, though the pH profile deviates slightly from ideal titration behaviour. We suggest that a high energy primary photoproduct is an obligatory feature of efficient biological photodetectors, as opposed to photon energy transducers, and that the similarity at this stage between cephalopod and vertebrate rhodopsins represents either convergent evolution at the molecular level or strong conservation of a crucial functional characteristic.     

Molecular mechanism for the initial process of visual excitation

The torsion model with which we proposed to interpret the specific properties of the photoisomerization reaction of rhodopsin has been developed to apply to isorhodopsin I, isorhodopsin II and some intermediates. Based on this model, optical absorption wavelengths and oscillator strengths, as well as rotational strengths of visual pigments, analogues and intermediates at low temperatures are analyzed by varying twisted conformations of the chromophores. As a result, it was found that most of the optical data could be very well accounted for quantitatively by the torsion model. The twisting characters in the chromophore of rhodopsin are very similar to those of isorhodopsin. The obtained conformations of the chromophores are very similar in rhodopsin and its analogues, and in isorhodopsin and its analogues. Those of the chromophores of bathorhodopsin, lumirhodopsin and metarhodopsin I are similar to one another except that the conjugated chain of metarhodopsin I bends considerably when compared with the other intermediates.A part of this work was performed while one of the authors (T.K.) was a Visiting Investigator of Japan Society for the Promotion of Science at Kyoto University from April, 1977 to March, 1978     

Studies on cephalopod rhodopsin: Photoisomerization of the chromophore

Photoisomerization of the chromophore of squid rhodopsin is dependent upon the irradiation temperature. Above 0°C, only 11-cis ? all-trans reaction proceeds and the all-trans → 9-cis reaction is limited to extremely low frequency. At liquid nitrogen temperature, 11-cis ? all-trans ? 9-cis reaction takes place. At intermediary low temperatures (?80°C to ?15°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°C to 0°C where mesorhodopsin converts to metarhodopsin. Mesorhodopsin is quite different from metharhodopsin 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°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.