SIMULATION ANALYSIS OF THE PHOTOCONVERSION PROCESS OF SQUID RHODOPSIN AT LIQUID HELIUM TEMPERATURE

The photoconversion process among squid rhodopsin, bathorhodopsin isorhodopsin and hypsorhodopsin was studied at liquid helium temperature. We evaluated the relative quantum yields of the photoconversion among four pigments by analysing the time-dependent change of absorption spectra. The result suggests that hypsorhodopsin is the common intermediate of rhodopsin and isorhodopsin, and there is no direct conversion between rhodopsin and isorhodopsin. Furthermore, rhodopsin converts to hypsorhodopsin or bathorhodopsin much more efficiently than does isorhodopsin, and bathorhodopsin does not convert directly to hypsorhodopsin. It was also found that rhodopsin and isorhodopsin convert to bathorhodopsin more efficiently than to hypsorhodopsin. In particular, the quantum yield of conversion from rhodopsin to bathorhodopsin was found to be about twice as large as that from rhodopsin to hypsorhodopsin. This result is somewhat in disagreement with the result obtained from the laser flash experiments at room and liquid nitrogen temperatures. The reason for the difference between the two experimental results is discussed.     

SQUID HYPSORHODOPSIN

Abstract— Squid hypsorhodopsin is produced by irradiating rhodopsin or isorhodopsin with yellow light (>480nm) at liquid He temperature (4K). Compared with cattle rhodopsin, squid rhodopsin easily converts to a photosteady state mixture composed of rhodopsin, isorhodopsin, hypsorhodopsin and bathorhodopsin at this temperature and the amount of hypsorhodopsin in the mixture is high. Hypsorhodopsin has a main absorption peak at 446 nm, and its extinction coefficient is 1.16 times larger than that of rhodopsin. On warming above 35 K, squid hypsorhodopsin converts to bathorhodopsin. A kinetic analysis indicates that the hypsorhodopsin can be formed not only from rhodopsin but also from isorhodopsin. On absorption of light. both squid bathorhodopsin and hypsorhodopsin convert to a mixture of rhodopsin and isorhodopsin.     

THE FORMATION OF TWO FORMS OF BATHORHODOPSIN AND THEIR OPTICAL PROPERTIES

Abstract— Using two kinds of rhodopsin preparations (digitonin extract and rod outer segments suspension), we measured changes in absorption spectra during the conversion of rhodopsin or isorhodopsin to a photosteady state mixture composed of rhodopsin, isorhodopsin and bathorhodopsin by irradiation with blue light (437 nm) at 77 K and during the reversion of bathorhodopsin to a mixture of rhodopsin and isorhodopsin by irradiation with red light (> 650 nm) at 77 K. The reaction kinetics could be expressed with only one exponential in the former case and with two exponentials in the latter case. These data suggest that both rhodopsin and isorhodopsin are composed of a single molecular species, while bathorhodopsin is composed of two molecular species, designated as bathorhodopsin₁ and bathorhodopsin₂. The absorption spectra of these bathorhodopsin were calculated by two different methods (kinetic method and warming-cooling method). The former was based on the kinetics of the conversion of two forms of bathorhodopsin by irradiation with the red light. The spectra obtained by this method were consistent with those obtained by the warming-cooling method. Bathorhodopsin₁ and bathorhodopsin₂ have Λ_max at 555 and 538 nm, respectively. The two forms of bathorhodopsin are interconvertible in the light, but not in the dark. Thus, we suggest that a rhodopsin molecule in the excited state relaxes to either bathorhodopsin₁ or bathorhodopsin₂ through one of the two parallel pathways.     

PICOSECOND LASER PHOTOLYSIS OF SQUID RHODOPSIN AT ROOM AND LOW TEMPERATURES

Abstract. Squid rhodopsin extracted with 2% digitonin (pH 10.5 or 7.0) was excited with a 347 nm light pulse from a mode-locked ruby laser at room temperature. Within 19 ps after the excitation, absorbance at 430 nm due to hypsorhodopsin increased and subsequently decreased with a decay time of 45 ± 10 ps. Absorbance at 550 nm due to bathorhodopsin increased with a rise time of 50 ± 10 ps. These results are the first observations of hypsorhodopsin at room temperature and clearly show that hypsorhodopsin is a precursor of bathorhodopsin which has been considered to be the earliest photoproduct in the photobleaching process of rhodopsin.
Hypsorhodopsin appeared with a rise time of 70 ± 10 ps at 421 nm at liquid nitrogen temperature without any bathorhodopsin being observed during the formation of hypsorhodopsin. An experiment using an N₂ laser showed that squid bathorhodopsin converted to lumirhodopsin with a decay time of about 300 ns at room temperature.     

Absorbance Changes by Aromatic Amino Acid Side Chains in Early Rhodopsin Photointermediates

Abstract— Absorbance changes were monitored from 250 to 650 nm during the first microsecond after photolysis of detergent suspensions of bovine rhodopsin at 20°C. Global analysis of the resulting data produced difference spectra for bathorhodopsin, BSI and lumirhodopsin which give the change in absorbance of the aromatic amino acid side chains in these photointermediates relative to rhodopsin. These spectra show that the significant bleaching of absorbance near 280 nm, which has been seen previously for the lumirhodopsin, metarhodopsin I and metarhodopsin II intermediates, extends to times as early as bathorhodopsin. Because no corresponding absorbance increase is observed in the 250-275 nm region, the earliest bleaching of the 280 nm absorbance in rhodopsin is attributed to disruption of a hyperchromic interaction affecting Trp²⁶⁵. Partial decay of this 280 nm bleaching as bathorhodopsin converts to BSI takes place maximally near 290 nm, where Trp²⁶⁵ has been shown to absorb, and could be due to the ring of the retinylidene chromophore resuming a position at the BSI stage that reestablishes the hyperchromic interaction with Trp²⁶⁵. A subsequent change in the 250-300 nm region, which has no counterpart in the visible chromophore bands, indicates the possible presence of a protein-localized process as lumirhodopsin is formed.     

EXISTENCE OF HYPSORHODOPSIN AS THE FIRST INTERMEDIATE IN THE PRIMARY PHOTOCHEMICAL PROCESS OF CATTLE RHODOPSIN

It has long been believed that bathorhodopsin is the first intermediate of visual process for cattle rhodopsin. In the present paper hypsorhodopsin is shown to be the first intermediate by the use of picosecond spectropic technique. The main first intermediate, hypsorhodopsin, is formed with the time constant of 15 ± 5 ps. The time constant of the formation of bathorhodopsin from hypsorhodopsin is 50 ± 20 ps. Bathorhodopsin intermediates formed directly from excited state rhodopsin and those formed indirectly through hypsorhodopsin are 71/2^#% and 93%, respectively, of total bathorhodopsin intermediates in octylglucoside buffered solution. Batho intermediates formed directly and indirectly are 0% and 100%. respectively, of total batho intermediates in LDAO buffered solution.     

NANOSECOND LASER PHOTOLYSIS OF RHODOPSIN AND ISORHODOPSIN

Kinetic and spectral measurements have been carried out on the primary intermediate in the photolysis of rhodopsin and isorhodopsin, initiated by a 457 nm, 6 ns (FWHM) laser pulse. In rhodopsin the kinetic decay of bathorhodopsin was found to be 140 ± 15 ns at 20°C. The decay of bathorhodopsin to lumirhodopsin has an activation energy of 51 ± 4 kJ/mol (12.2 ± 1 kcal/mol). The decay kinetics of bathorhodopsin were found to be the same for rhodopsin in membrane and detergent solubilized suspensions. The kinetic decay of the batho product in the photolysis of isorhodopsin was found to be the same as rhodopsin.
The corrected transient spectrum 50 ns following excitation in rhodopsin has two peaks near 560 and 440 nm. A peak was also observed in isorhodopsin near 550 nm at 50 ns following excitation but no transient was observed in the blue. The 550 nm peak in isorhodopsin has an intensity similar to that in rhodopsin indicating that the quantum yields for the formation of batho products of rhodopsin and isorhodopsin are similar under the irradiation conditions used here. Transient spectra for rhodopsin and isorhodopsin 1 μs following excitation are also different. In isorhodopsin the corrected transient spectrum has a peak at 500 nm, similar to low temperature steady state irradiation spectra. The 1 μs transient spectrum in rhodopsin is more intense than in isorhodopsin and shows a peak at 475 nm.     

THEORETICAL STUDY OF OPTICAL SPECTRA AND CONFORMATION OF THE CHROMOPHORE OF HYPSORHODOPSIN

Optical spectra of hypsorhodopsin were theoretically analyzed by assuming the unprotonated all-trans form of the Schiff base of the chromophore. The large bathochromic shift of the optical absorption of hypsorhodopsin from that of the retinylidene Schiff base in solution could be easily explained by twisting the double bond of the chromophore; it could not be explained by simple counter anion models. Using the same twisted chromophore conformation for hypsorhodopsin as that of bathorhodopsin obtained by the torsion model, we showed that the calculated absorption wavelength was in fairly good agreement with the experimental value. Our calculated oscillator strengths and rotational strengths were quite similar between hypsorhodopsin and bathorhodopsin. Those theoretical results will be useful when one examines the relation of the chromophore's conformation between hypsorhodopsin and bathorhodopsin experimentally.     

ORIENTATION OF RETINYLIDENE CHROMOPHORE OF HYPSORHODOPSIN IN FROG RETINA

Abstract— Hypsorhodopsin and bathorhodopsin were formed in the frog retina by irradiating rhodopsin at liquid He temperature (9 K) with orange light (> 520 nm) and blue light (437 nm), respectively. Hypsorhodopsin was converted to bathorhodopsin in the retina by warming above 32 K in the dark. Similar phenomena were observed in the rod outer segment suspension. A difference spectrum between hypsorhodopsin and bathorhodopsin in the retina produced by warming was almost identical with that in the rod outer segment suspension. This suggests that the transition dipole moment of hypsorhodopsin is parallel to the disk membrane plane which is also parallel to that of bathorhodopsin.     

A Resonance Raman Study Of the C=C Stretch Modes in Bovine and Octopus Visual Pigments with Isotopically Labeled Retinal Chromophores

Abstract— Previous resonance Raman spectroscopic studies of bovine and octopus rhodopsin and bathorhodopsin in the C–C stretch fingerprint region have shown drastically different spectral patterns, which suggest different chromophore-protein interactions. We have extended our resonance Raman studies of bovine and octopus pigments to the C=C stretch region in order to reveal a more detailed picture about the difference in retinal-protein interactions between these two pigments. The C=C stretch motions of the protonated retinal Schiff base are strongly coupled to form highly delocalized ethylenic modes located in the 1500 to 1650 cm⁻¹ spectral region. In order to decouple these vibrations, a series of 11,12-D₂-labeled retinals, with additional 13C labeling at C₈, C₁₀, C₁₁ and C₁₄, respectively, are used to determine the difference of specific C=C stretch modes between bovine and octopus pigments. Our results show that the C₉=C₁₀ and C₁₃=C₁₄ stretch mode are about 20 cm⁻¹ lower in the Raman spectrum of octopus bathorhodopsin than in bovine bathorhodopsin, while the other C=C stretch modes in these two bathorhodopsins are similar. In contrast, only the C₉=C₁₀ stretch mode in octopus rhodopsin is about 10 cm⁻¹ lower than in bovine rhodopsin, while other C=C stretches are similar.     

FEMTOSECOND STUDIES OF PRIMARY PHOTOPROCESSES IN OCTOPUS RHODOPSIN

Abstract— Femtosecond spectroscopy of octopus rhodopsin in H₂O and D₂O was performed over a very wide spectral region of 400–1000 nm. Transient gain and absorption from the excited state were observed for the first time around 650 and 700 nm, respectively, just after 300 fs pulse excitation. Bathorhodopsin was formed within 400 fs from the excited state; therefore, the cis-trans isomerization completes within 400 fs. The first intermediate "primerhodopsin" found in our previous paper is most likely "quasi-thermal" bathorhodopsin, in which the local thermalization of the chromophore is achieved. Then cooling down of the chromophore to the surrounding protein temperature takes place with 20 ± 10 ps along with blue-shifting of a spectrum of 10 ± 5 nm. In addition to these observations, a prominent gain in the region of > 850 nm was observed and decayed with 2–3 ps in H₂O. A similar time constant was estimated for a partial decay of an induced absorption around 600 nm. This process may be related with two forms of bathorhodopsin reported previously. In this scheme, two forms of bathorhodopsin are formed with time constants of about 400 fs and 2 ps. In the sample in D₂O, time constant of 3–4 ps was obtained for the slower process.     

LOW-TEMPERATURE TRAPPING OF EARLY PHOTOINTERMEDIATES OF α-ISORHODOPSIN

Abstract— a-Isorhodopsin, an artificial visual pigment with a 9- cis -4,5-dehydro-5,6-dihydro(a)retinal chromophore, was photolyzed at low temperatures and absorption difference spectra were collected as the sample was warmed. A bathorhodopsin (Batho)-like intermediate absorbing at ca 495 nm was detected below 55 K, a blue-shifted intermediate (BSI)-like intermediate absorbing at ca 453 nm was observed when the temperature was raised to 60 K and a lumirhodopsin (Lumi)-like intermediate absorbing at ca 470 nm was found when the sample was warmed to 115 K. Photointermediates from this pigment were compared to those of native rhodopsin and 5,6-dihydroisorhodopsin. As in native rho-dopsin, Batho is the first intermediate detected in a-isorhodopsin, though unlike native rhodopsin at low temperatures BSI is observed prior to Lumi formation. a-Isorhodopsin behaves similarly to 5,6-dihydroisorhodopsin, with the same early intermediates observed in both artificial visual pigments lacking the C₅-C₆ double bond. The transition temperature for BSI formation is higher in a-isorhodopsin, suggesting an interaction involving the chromophore ring in BSI formation. The transition temperature for Lumi formation is similar for these two pigments as well as for native rhodopsin, suggesting comparable changes in the protein environment in that transition.     

PHOTOEXCITATION OF RHODOPSIN: CONFORMATION CHANGES IN THE CHROMOPHORE, PROTEIN AND ASSOCIATED LIPIDS AS DETERMINED BY FTIR DIFFERENCE SPECTROSCOPY

Abstract— The visual pigment rhodopsin is the major membrane protein in the rod photoreceptor membrane. Rhodopsin's function is to transduce the light induced isomerization (ll-cis to all-trans) of its internally located retinylidene chromophore into transient expression of signal sites at the surface of the protein. Fourier transform infrared (FTIR) difference spectroscopy has been used to study all of the steps in the photobleaching sequence of rhodopsin. Early protein alterations involving the peptide backbone and aspartic and/or glutamic carboxyl groups were detected which increase upon lumirhodopsin formation and spread to water exposed carboxyl groups by metarhodopsin II. The intensified and frequency shifted hydrogen-out-of-plane vibrations of the chromophore that are present in bathorhodopsin are absent in lumirhodopsin. This indicates that by lumirhodopsin, the chromophore has relaxed relative to its more strained all-frans form in bathorhodopsin. Finally, the transition to metarhodopsin II is found to involve perturbation of the acyl tail region of unsaturated phospholipid molecules possibly in response to small changes in the shape of the rhodopsin.     

ON THE STATE OF CHROMOPHORE PROTONATION IN RHODOPSIN: IMPLICATION FOR PRIMARY PHOTOCHEMISTRY IN VISUAL PIGMENTS

Resonance Raman multicomponent spectra of bovine rhodopsin, isorhodopsin, and bathorhodopsin are obtained at low temperature. Application of the double beam, 'pump-probe' technique allows an extraction of the rhodopsin and bathorhodopsin spectra in both protonated and deuterated media. Our results show that the Schiff bases of both rhodopsin and bathorhodopsin are fully protonated and the degree of protonation is unaffected by the rhodopsin-bathorhodopsin transformation. Further, the data support the concept or cis-trans isomerization as occurring in this transition. The effect of these results on various models for the primary photochemical event in vision is discussed.     

A resonance Raman study of octopus bathorhodopsin with deuterium labeled retinal chromophores.

The resonance Raman spectrum of octopus bathorhodopsin in the fingerprint region and in the ethylenic-Schiff base region have been obtained at 80 K using the "pump-probe" technique as have its deuterated chromophore analogues at the C7D; C8D; C8,C7D2; C10D; C11D; C11, C12D2; C14D; C15D; C14, C15D2; and N16D positions. While these data are not sufficient to make definitive band assignments, many tentative assignments can be made. Because of the close spectral similarity between the octopus bathorhodopsin spectrum and that of bovine bathorhodopsin, we conclude that the essential configuration of octopus bathorhodopsin's chromophore is all-trans like. The data suggest that the Schiff base, C = N, configuration is trans (anti). The observed conformationally sensitive fingerprint bands show pronounced isotope shifts upon chromophore deuteration. The size of the shifts differ, in certain cases, from those found for bovine bathorhodopsin. Thus, the internal mode composition of the fingerprint bands differs somewhat from bovine bathorhodopsin, suggesting a somewhat different in situ chromophore conformation. An analysis of the NH bend frequency, the Schiff base C = N stretch frequency, and its shift upon Schiff base deuteration suggests that the hydrogen bonding between the protonated Schiff base with its protein binding pocket is weaker in octopus bathorhodopsin than in bovine bathorhodopsin but stronger than that found in bacteriorhodopsin's bR568 pigment.     

Assigning the resonance Raman spectral features of rhodopsin, isorhodopsin and bathorhodopsin in bovine photostationary state spectra.

Abstract—High resolution resonance Raman spectra of rhodopsin. isorhodopsin and photostationary state mixtures containing a high percentage of bathorhodopsin arc presented. New spectral features are detected which were not obsei-ved in lower resolution studies by other workers. All of the hands in the photostationary state spcctra arc assigned based on pure rhodopsin and isorhodopsin resonance Raman results and alterations in the photostationary state mixture. The spectral features in these spectra are invariant from 20 to 150K indicating that retinal and protein structural alteration, consistent with a model of excitation proposed by Lewis, occurs in steady-state spectra even at 20 K. In addition, the relative intensity of certain features in the photostationary state spectra are altered upon D₂O suspension. One explanation for these alterations is that the contributions of various intcrmediates to the photostationary state mixture are changed when membrane fragments are suspended in D₂O.     

LOW TEMPERATURE SPECTROPHOTOMETRIC STUDY OF CATTLE BATHORHODOPSINS PRODUCED FROM RHODOPSIN AND ISORHODOPSIN IN TRANSPARENT MEDIUM WITHOUT CRACKS

Abstract— Cattle rhodopsin (11-cis-Rh) and isorhodopsin (9-cis-Rh) were prepared from the same extract of cattle opsin by incubation in the dark with 114– and 9-cis-retinals, respectively. The preparations obtained were mixed with glycerol (67% in the final concentration) and then cooled to 77 K by a rapid cooling method for freezing the samples as a clear glass, without any cracks. The spectral changes during their photochemical reactions were measured. A computer analysis of the spectra obtained verified that bathorhodopsin from 11-cis-Rh was identical in spectrum with that from 9-cⁱs-Rh not only in photosteady state but also under a short time irradiation.     

LASER FLASH PHOTOLYSIS OF RHODOPSIN AT ROOM TEMPERATURE

Abstract. Nanosecond flash photolysis of rhodopsin with 530 or 353 nm light produces an initial transient absorption spectrum with peaks at ˜57O and ˜420nm, and a subsequent transient species with a maximum absorption at 480 nm. These results are interpreted as the initial formation of prelumi-rhodopsin (570 nm) followed by its conversion to lumirhodopsin (470 nm). The peak at 420 nm in the first transient may be due to either hypsorhodopsin or isorhodopsin.     

CONFORMATIONAL ANALYSIS OF THE RHODOPSIN CHROMOPHORE USING BICYCLIC RETINAL ANALOGUES

Abstract— For investigation of the chromophore conformation around the trimethyl cyclohexene ring and of the origin of the induced β-circular dichroism band in rhodopsin, two C₆-C₇ single bond-fixed retinal analogues, 6s-cb- and 6s-trans-locked bicyclic retinals (6 and 7, respectively) were synthesized and incorporated into bovine opsin in CHAPS-PC mixture. 6s-cb- and 6s-tram-Locked rhodopsin analogues (8 and 9 ) with A max at 539 and 545 nm, respectively, were formed. Interestingly, both 8 and 9 displayed α- and β-circular dichroism bands. The ellipticity of α-bands are similar in each other, while the β-band of 8 was about three times stronger than that of 9. Irradiation of 6s-trans-locked rhodopsin, 9, in the presence of hyroxylamine, resulted in the formation of only one of the enantiomers of 6s-rrans-locked retinal oxime showing a positive circular dichroism signal at around 390 nm. This fact strongly suggests that the retinal binding site of rhodopsin shows a chiral discrimination. From these experimental results, the interactions between the trimethyl cyclohexene ring portion in the chromophore and the neighbouring protein moiety in the rhodopsin molecule are discussed.     

PHOTODICHROISM OF RHODOPSIN SOLUTIONS AT -196°C

Abstract— The photodichroism of a system of randomly oriented, bleachable pigment molecules, rigidly fixed in an inert transparent matrix was investigated theoretically. A formula was derived for the photochemical equilibrium reaction between three pigments, N ₁⇆ N ₂⇆ N ₃ in the case that the absorption ellipsoids of the pigments are rotationally symmetric and their symmetry axes coincide. The formula was applied to the dichroism induced by plane polarized light of different wavelengths in an aqueous rhodopsin-glycerol mixture at –196°C. It was found that the absorption ellipsoids of this visual pigment, its analogue, isorhodopsin, and its first bleaching product, prelumirhodopsin, are elongated with an apparent axial ratio of about 5. It was concluded that the light absorbing properties of these pigments cannot be described by a single transition moment vector (linear absorber). The absolute value of the quantum efficiency of the conversion of rhodopsin to prelumirhodopsin was shown to be approximately equal to the quantum efficiency of bleaching rhodopsin at room temperature. Some evidence was obtained that indicates that the relative quantum efficiencies of the rhodopsin system at – 196°C may be wavelength dependent.