COMPARISON OF THE PATHWAYS AND QUANTUM YIELDS OF DIRECT PHOTOISOMERIZATION OF UNPROTONATED AND PROTONATED N-BUTYLAMINE SCHIFF BASES OF ISOMERIC RETINYLIDENEACETALDEHYDE WITH THOSE OF SCHIFF BASES OF ISOMERIC RETINAL: RATIONALIZATION OF THE SELECTION OF THE RETINYLIDENE CHROMOPHORE BY RETINOCHROME

Abstract— The pathways and quantum yields of direct photoisomerization of unprotonated and pro-tonated n-butylamine Schiff bases (SB and PSB) of isomeric retinylideneacetaldehyde (C22 aldehyde) were determined in n-hexane, acetonitrile and methanol for the former and in acetonitrile and methanol for the latter. The results are compared with those of the Schiff bases of isomeric retinal (C20 SB and C20 PSB) reported previously (Koyama et al., Photochem. Photobiol. 54 , 433–443, 1991). The isomerization pathways and quantum yields of C22 SB are more or less similar to those of C20 SB, but conspicuous differences in the isomerization pathways are found between C22 PSB and C20 PSB. The homogeneous (exclusive) isomerization of the retinylidene chromophore from all-trum to 11-cis in retinochrome is rationalized not by C22 PSB but by C20 PSB.
Almost complete one-way isomerization from cis to trans of C22 SB (in n-hexane) is ascribed to isomerization via the T₁ state, while mutual isomeritation between cis and tram of C22 PSB is ascribed to isomerization via the S₁ (B_u) state. The T_I potential of C22 SB and the S₁ potential of C22 PSB are discussed based on photostationary state compositions.     

PHOTOREACTION OF THE ACIDIFIED FORM OF BACTERIORHODOPSIN AND ITS 9-CIS DERIVATIVE IN PURPLE MEMBRANE AT LOW TEMPERATURES

Abstract— The photoreaction of the acidified form of bacteriorhodopsin and its 9-cis derivative was studied by low temperature spectroscopy.
A short exposure of the acidified form of bacteriorhodopsin, which was prepared by adding 2 m M HC1 to purple membrane suspension in 67% glycerol at 0°C, to red light at – 72°C resulted in the blue-shift of the spectrum. The feature of the shift was very similar to that accompanied by the formation of stable 9- cis acidified form of bacteriorhodopsin at 0°C, but only 13- cis - and all- trans -retinals were found in the extract from this product. No blue-shifted product was found on irradiation at – 190°C.
Irradiation of the 9- cis form of acidified bacteriorhodopsin at -72°C with blue light caused the isomerization of its 9- cis -retinylidene chromophore to 13- cis and all- trans forms without a significant spectral change. It became greater only after the sample was warmed above – 24°C. These results indicate the presence of the light-induced product which has trans configuration on the 9-10 double bond and exhibits the 9- cis type spectrum.     

AN IMPROVED HPLC METHOD FOR THE SEPARATION OF RETINALDEHYDE ISOMERS FROM VISUAL PIGMENTS

Abstract— A method for the analytical separation of retinal isomers such as 13- cis , 11- cis , 9- cis and all- trans retinal, dissolved in aqueous solutions of detergents, is described. The retinals are extracted by means of a non-isomerizing procedure and separated by HPLC on an octadecyl silane column used in normal phase. This column retains detergents without deteriorating and gives a satisfactory separation of retinal isomers with a resolution comparable with that obtained with silica gel column. The reliability of the method is verified by analysing the chromophore of visual pigment rhodopsin in digitonin solution, before and after irradiation with white light.     

WAVELENGTH DEPENDENCE OF THE INTERSYSTEM CROSSING EFFICIENCY FOR RETINAL ISOMERS

Abstract— The quantum efficiencies of intersystem crossing (_ISC) fur four isomers of retinal, the all- trans , 9- cis , 11- cis and 13- cis , have been measured using both 265 nm and 353 nm excitation. The values for the all- trans and 9- cis isomers are independent of the excitation wavelength but the values for the 11- cis and 13- cis isomers show a marked increase in the efficiency of ISC for 353 nm excitation compared with the 265 nm excitation.     

CHROMOPHORE OF A LONG-LIVED PHOTOPRODUCT FORMED WITH METARHODOPSIN III IN THE ISOLATED FROG RETINA

Abstract Long-lived photoproducts of frog rhodopsin in isolated retina and digitonin solution have been investigated by spectrophotometry and their chromophores have been analyzed by high-pressure liquid chromatography (HPLC). By irradiation (> 560 nm) at 3°C and pH 8.6, a product analogous to metarhodopsin III (MIII) is formed, whose absorption maximum is at about 450 nm. This product decays more slowly than MIII does. The results of HPLC analysis indicate that the chromophore of this photoproduct is 7- cis retinal and that of MIII is all-trans retinal. The product possessing 7- cis retinal is called 7- cis photoproduct. The amount of 7- cis isomer in rhodopsin solution irradiated at various temperatures between 15°C and –82°C, has been determined. The results suggest that the 7- cis photoproduct can be formed by the photoconversion of lumirhodopsin and metarhodopsin I.     

LIGHT INDUCED ISOMERISATION, AT ACIDIC pH, INITIATES HYDROLYSIS OF BACTERIORHODOPSIN TO BACTERIO-OPSIN AND 9-CIS-RETINAL

Abstract— Bacteriorhodopsin (BR) from the purple membrane of Haiobacterium halobium contains covalently bound retinal in the 13- cis and all- trans configurations. Several forms of bacteriorhodopsin are known, with different absorption maxima which are designated as BRλ_max (nm). At acidic pH, BR605 is formed from BR560. The following sequence of reactions was found, which is initiated by irradiation of BR605 with red light:

An all- trans /13- cis to 9- cis isomerisation occurs in the light induced reaction BR605 ∼ BR500. BR500 seems to contain covalently bound retinal, whereas BR390 contains free retinal. By irradiation with light, BR500, BR450 and BR390 can be reconverted to BR560.     

DEPENDENCE OF THE TRIPLET YIELD ON EXCITATION ENERGY IN ALL-TRANS AND 11-CIS RETINAL*

Abstract. The triplet-triplet absorption of all-trans and 11- cis retinal was measured as a function of the exciting radiation from 423 nm to 365 nm in a glass of 3-methylpentane at 77 K. This experiment was also accomplished with all-trans retinal in hexane at ambient temperature. The relative triplet formation quantum yields of all-trans and 11-cis retinal at 77 K were found to be independent (±10%) of the frequency of the exciting radiation. At room temperature we measured an increase in this relative quantum yield for all- trans retinal from 1.0 at 365 nm to 1.82 at 423 nm [Bensasson et al. (1975) measured an absolute quantum yield of 0.45 at 353 nm]. These results are used to evaluate previous interpretations for photophysical decay processes in all-trans retinal, and previous suggestions for wavelength dependent radiationless transitions are shown to be unacceptable. High energy excitation of 300 K solutions of all- trans retinal produce excited states that result in less efficient intersystem crossing. These states appear to be inaccessible in the 77 K matrices. We suggest that steric restrictions introduced by the retinal matrix interaction at 77 K are able to block this new internal conversion pathway back to the ground state.     

TRIPLET STATES AND cis-trans PHOTOISOMERIZATION PROCESSES IN THE SCHIFF BASES OF RETINAL ISOMERS

Abstract —The triplet states of the n -butyl-amine Schiff bases of 11- cis , 9- cis , 13- cis and all- trans retinal are produced via triplet-triplet energy transfer. Their absorption spectra, peaking around 435 nm, and their decay kinetics are recorded using pulsed-laser photolysis. Direct-excitation (φ^D_ISO) and triplet-sensitized (φ^T_ISO) photoisomerization yields, determined using steady irradiation methods, are found to be: φ^T_ISO (9- cis ) = 0.06, φ^T_ISO (11- cis ) = 045, φ^T_ISO (13- cis ) = 008, φ^T_ISO (all- trans ) = 0.02-0.05, φ^D_ISO (11- cis , = (4 ± 1) × 10^-3, φ^D_ISO (all- trans ) = (2 ± 1) × 10^-3. The possible role of the triplet state in the isomerization of rhodospin is discussed.     

BLEACHING OF BACTERIORHODOPSIN IN PURPLE MEMBRANE, BROWN HOLO-MEMBRANE ANDL–1690-SOLUBILIZED MONOMERS WITH HYDROXYLAMINE IN LIGHT AND THE DARK

Abstract— The reactions of hydroxylamine with bacteriorhodopsin in the states of the purple membrane, the brown holo-membrane andL–1690-solubilized monomers were studied in light and the dark. The bleaching rate was strongly dependent on the state of the bacteriorhodopsin and largely enhanced by light. Analysis of the isomeric composition of the resultant retinaloximes by high performance liquid chromatography (HPLC) showed that more all- trans retinaloximes were present in the dark than 13- cis retinaloximes. However, under illumination, the relative amounts of 13- cis retinaloximes increased greatly, indicating the occurrence of trans to cis isomerization during the photochemical cycle of bacteriorhodopsin. Molar extinction coefficients of 13- cis retinaloximes were determined to estimate the isomeric composition of retinaloximes by HPLC analysis.     

SPECTROSCOPIC BEHAVIOUR OF RETINYL POLYENES IN SOLID FILMS

Abstract— The absorption spectroscopic study of retinyl polyenes, i.e. all- trans retinal, 9- cis retinal, 13- cis retinal, all- trans retinol, 13- cis retinol and all- trans retinyl acetate in solid films is discussed. The spectra of the films with low surface coverages obtained from dilute solutions are red shifted relative to their solution spectra. This shift is interpreted as due to the weak interaction between the surface and polyene molecules. With increased surface coverages, i.e. the films obtained from concentrated solutions, the retinals show a large red shift whereas a blue shift is observed with the retinols. Chromophore–chromophore interactions in addition to the surface effects are believed to be responsible for such observations. Effect of surface causing permanent changes in molecule is also discussed.     

Trans-cis isomerization is responsible for the red-shifted fluorescence in variants of the red fluorescent protein eqFP611

An important class of red fluorescent proteins (RFPs) feature a 2-iminomethyl-5-(4-hydroxybenzylidene)imidazolinone chromophore. Among these proteins, eqFP611 has the chromophore in a coplanar trans orientation, whereas the cis isomer is preferred by other RFPs such as DsRed and its variants. In the photoactivatable protein asFP595, the chromophore can even be switched from the nonfluorescent trans to the fluorescent cis state by light. By using X-ray crystallography, we have determined the structure of dimeric eqFP611 at high resolution (up to 1.1 A). In the far-red emitting eqFP611 variant d2RFP630, which carries an additional Asn143Ser mutation, the chromophore resides predominantly (approximately 80%) in the cis isomeric state, and in RFP639, which has Asn143Ser and Ser158Cys mutations, the chromophore is found completely in the cis form. The pronounced red shift of excitation and emission maxima of RFP639 can thus unambiguously be assigned to trans-cis isomerization of the chromophore. Among RFPs, eqFP611 is thus unique because its chromophore is highly fluorescent in both the cis and trans isomeric forms.     

POLARIZED UV-ABSORPTION SPECTRA OF RETINAL ISOMERS-I. MEASUREMENTS IN EXTREMELY THIN MONOCRYSTAL PLATELETS

Abstract Crystals of all- trans retinal and both different forms of 11- cis , 12-s- cis retinal were grown on quartz slides with faces (101), (001) and (101), respectively, forming thin platelets of less than 0.2 μm thickness. Polarized UV absorption spectra at room temperature were measured in the range from 20 to 43 × 10³ cm⁻¹ with a microscope-spectrophotometer. In this spectral range three diffuse absorption bands were observed for all crystal types at similar wave numbers. A main absorption band was found at 25–28 × 10³ cm⁻¹, and two further bands at 32–34 and 38–40 × 10³ cm⁻¹. In case of all- trans retinal the latter band is by far the weakest in this spectral range. Additionally, the crystal spectrum of all- trans retinal shows a shoulder at the low wavenumber side of the main band which cannot be resolved in the corresponding solution spectrum. In the crystal spectra of 11- cis , 12-s- cis retinal, however, only a strong dissymmetry is observed at this side of the main band.     

PHOTOCHEMISTRY OF POLYENES—XI. FLASH PHOTOLYTIC STUDIES OF HINDERED ISOMERS OF RETINAL

Abstract— The μs transient absorption spectra of benzene solutions of 7- cis , 7,9- dicis , 11- cis and all- trans isomers of retinal were recorded both by direct and sensitized excitation. The spectra are indistinguishable, as are the transient decay rates. The results are consistent with the notion of rapid isomerization of the 7- cis triplets and possibly equilibration of all retinal triplets.     

THEORY OF THE OPTICAL ABSORPTION OF LIGHT-ADAPTED BACTERIORHODOPSIN AND ITS ACIDIFIED FORMS

Abstract— We assume a model for bacteriorhodopsin chromophore such that the protonated retinal Schiff-base (PRSB) interacts with two anions in the case of light-adapted bacteriorhodopsin (bR^L), while it does with one anion in the case of the acidified form of bacteriorhodopsin (bR^acid₆₀₀). On the basis of this model, the π-electronic states of all- trans -PRSB are calculated according to our LCAO-ASMO-SCF-CI method, the anions being approximated by negative point-charges in the plane of PRSB π-system. A possible distribution of the negative point-charges around PRSB is proposed for the chromophores of bR^L, bR^acid₆₀₀, and the two irradiated forms of bR^acid₆₀₀ (the one at 3°C containing 9- cis -PRSB, and the other at — 72°C all- trans -PRSB). It is shown that the wavelength λ_max of absorption maximum observed for each form of bacteriorhodopsin can be explained reasonably well by the suggested charge distribution. Furthermore, a model for the structure of the active site of bR^L is proposed, considering that two COO⁻ groups form the anions that interact with PRSB. The calculated optical absorption of all- trans -PRSB at such a site is shown to be consistent with the observed absorption spectrum of bR^L.     

ALL-trans RETINAL PHOTOISOMERIZATION AND PHOTOOXIDATION FROM UV LASER RADIATION. VIBRATIONAL ASSIGNMENTS OF ALL-trans 5,8-PEROXYRETINAL

Abstract— Photoisomerization and photooxygenation of all- trans retinal in acetonitrile, illuminated by laser radiations (λ_act, = 333.6 or 350.7 nm), were investigated under various experimental conditions. In deoxygenated solutions, the major photoproducts are 13- cis and 9- cis retinal. All- trans and 13- cis 5,8-peroxyretinal are obtained in large amounts in oxygenated solutions. 11-cis derivatives have not been detected in any of these solutions. The photoproducts were identified by UV, NMR, mass and vibrational spectroscopies and HPLC chromatography. All- trans and 13- cis 5,8-peroxyretinal were isolated. Their vibrational spectra (IR and Raman) are analyzed. Most of the bands are assigned by comparison with previous studies on all- trans and 13- cis retinal, ( E,E,E )-3-methyl-2,4,6-octatrienal and peroxides.     

Photoswitchable multivalent sugar ligands: synthesis, isomerization, and lectin binding studies of azobenzene-glycopyranoside derivatives

Coating of azobenzene chromophore with multivalent sugar ligands has been accomplished. Such sugar coating allows the study of the isomerization properties of this chromophore in aqueous solutions. The predominantly cis-isomer-containing photostationary state (PS) mixture of these azobenzene derivatives is found to be stable for hours. The rate constants for their isomerization, as well as the Arrhenius activation energies, are determined experimentally. An assessment of the lectin binding properties of the lactoside bearing isomeric azobenzene derivatives, by isothermal calorimetric methods, reveals the existence of an unusual cooperativity in their binding to lectin peanut agglutinin. Thermodynamic parameters evaluated for the trans and the PS mixture are discussed, in detail, for the lactoside bearing bivalent azobenzene derivative.     

TRIPLET STATES OF CAROTENOIDS BOUND TO REACTION CENTERS OF PHOTOSYNTHETIC BACTERIA: TIME-RESOLVED RESONANCE RAMAN SPECTROSCOPY

Time-resolved, low-temperature resonance Raman spectra of triplet states of the carotenoids specifically present in bacterial reaction centers in a strained cis conformation have been obtained, thus demonstrating the possibility of studying intermediate transient states of these structures using resonance Raman spectroscopy. Resonance Raman spectra of triplet cis spheroidene and cis methoxyneurosporene present in reaction centers of Rhodopseudomonas spheroides, (strains 2.4.1. and Ga, respectively) exhibit marked differences with those of triplet, all- trans carotenoids previously studied in vitro. These differences, together with the frequency shifts measured for the v ₁ modes, indicate that triplet carotenoids bound to reaction centers retain a cis conformation, and that probably no isomerization occurs to all- trans carotenoids upon T ← S₀ excitation. P_i electron distributions along the polyene backbone are probably less regular in the triplet state than in the singlet ground state, although probably not to the extent suggested by previous theoretical calculations. The apparently anomalous behaviour of the v ₂ bands of all- trans carotenoids upon T ← S₀ excitation is shown to result largely from the actual complexity of this region of the Raman spectra, together with a weak participation of the v _c—–c internal coordinate in the corresponding modes. Finally, the Raman scattering efficiency of triplet spheroidene bound to reaction centers is lower than that of the singlet, ground state form, under equivalent excitation conditions.     

POLARIZED UV-ABSORPTION SPECTRA OF RETINAL ISOMERS—II. ON THE ASSIGNMENT OF THE LOW AND HIGH ENERGY ABSORPTION BANDS

Abstract The polarized UV-absorption spectra of all- trans retinal and both crystal forms of 11- cis , 12-s- cis retinal (presented in the previous paper, Part I) are analyzed using Lowry-Hudson functions to describe the band profiles. The polarization ratios of the polarized bands is used to determine the direction of the corresponding transition moments. For all- trans retinal the polarization spectra show that the absorption between 23 and 36 X 10³ cm⁻¹ is caused by three overlapping bands labeled S, A and B. For 11- cis retinal the B-band is also clearly resolved whereas the S and A bands are separated with much less certainty than for all- trans retinal.
Comparing these bands with the excited state manifold resulting from semiempirical CI-calculations including double excitations, the S-band could be assigned to the ¹A_g⁻→¹A_g-* and the A-band to the ¹A_g⁻→¹B_u+* transition. However, no transition is found in this manifold which could positively be assigned to the B-band because the transitions predicted in this spectral region have negligible oscillator strengths. In all the crystal spectra a further band C is observed around 39 X 10³ cm⁻¹ which is particularly pronounced in the case of 11- cis retinal. For this band an assignment to the ¹A_g⁻→¹A_g+*-transition is proposed.     

TIME-RESOLVED RESONANCE RAMAN SPECTRA OF THE PHOTOCYCLE INTERMEDIATES OF ACID AND DEIONIZED BACTERIORHODOPSIN

Abstract— The photocycle of bacteriorhodopsin (bR) and its perturbed forms are investigated by a time-resolved resonance Raman study. These experiments were performed in the C=C stretching and in the fingerprint spectral regions for the acid blue, acid purple and deionized forms of bR.
The main observations are as follows: (1) isomerization of the retinal, from all- trans to 13- cis , occurs in native bR and in all of the acid and deionized perturbed bR species; (2) formation of the early intermediates (the K₆₁₀ and L₅₅₀ analogues) also occur in native bR and in all of the perturbed species; and (3) deprotonation of the protonated Schiff base (PSB), to give the M₄₁₂ type intermediate, occurs in native bR, but is inhibited in all of the perturbed bR species on the time-scale of the native bR photocycle.
The results show that isomerization alone is not a prerequisite for the PSB deprotonation process. The observed photocycle, initiated with retinal isomerization, is found to occur from all- trans to 13- cis in all of the perturbed forms of bR. In addition, the results imply that removal of the cations, of an increase in the hydrogen ion concentration, prevent only the PSB deprotonation process and not the formation of earlier cycle intermediates. Some attention is focused on the two blue forms of bR (acid and deionized) due to the fact that their ground-state absorption maximum, unphotolyzed Raman spectra, and Raman spectra changes during the photocycle are all very similar. The similarities between the acid blue and deionized blue forms in the fingerprint region support previous suggestions that both blue species have nearly the same retinal active site.     

Tetrathiafulvalenenaphthalenophanes: planar chirality and cis/trans photoisomerization

A cyclophane incorporating one 1,5-dioxynaphthalene ring system and one tetrathiafulvalene (TTF) unit bridged by [SCH(2)CH(2)O] linkages has been synthesized. In this cyclophane, the TTF unit can adopt either cis or trans configurations. In addition, the 1, 5-dioxynaphthalene ring system imposes one element of planar chirality on this cyclophane. A second element of planar chirality is introduced by the trans form of the TTF unit. Thus, the cyclophane exists in diastereoisomeric forms as three pairs of enantiomers. The enantiomeric pairs associated with the cis form of the TTF unit, as well as one of those associated with the trans form, have been isolated by crystallization, and their structures assigned in the solid state by single-crystal X-ray analyses. In solution, cis/trans isomerization occurs when either the cis or the trans form of the cyclophane is exposed to light. The photoisomerization reaction can be followed by (1)H NMR and UV-vis spectroscopies, as well as by HPLC. The photoisomerization quantum yield has been measured at two different excitation wavelengths (406 and 313 nm). In both cases, the trans --> cis process (Phi = 0.20 at 406 nm) is much more efficient than the reverse cis --> trans process (Phi = 0.030 at 406 nm). Since the absorption spectra of the trans and cis isomers are different and the quantum yield of the trans --> cis photoisomerization reaction depends on the excitation wavelength, the mole fraction of the two diastereoisomers present at the photostationary state depends on the wavelength of the exciting light. No isomerization occurs when the solutions, regardless of the mole fraction of the two diastereoisomers, are stored in the dark.