KINETIC AND SPECTROSCOPIC EFFECTS OF PROTEIN-CHROMOPHORE ELECTROSTATIC INTERACTIONS IN BACTERIORHODOPSIN

Abstract— Available pH effects on the absorption spectrum and on the rates of dark adaptation (all- trans → 13 -cis interconversion) of bacteriorhodopsin are analyzed in terms of a model based on protein-chromophore electrostatic interactions. Plausible locations of three protein acid-base groups controlling the spectrum and the activation energy of the thermal trans → 13- cis process are suggested. The importance of the protein charges in controlling the cycles of bacteriorhodopsin is indicated.     

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.     

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.     

Theoretical Study of Halogen Effect in Isomerization of 2-Halo-[9]-annulen Anion at the DFT Level

Cis-trans isomerization of [9]-annulenanion(1) and its 2-fluoro-,2-chloro-and 2-bromo-derivatives(2,3 and 4,respectively) were investigated at the HF/6-31G* and B3LYP/6-311++G** levels of theory.Cis,cis,cis,cis structures appear more stable than their corresponding cis,cis,cis,trans-isomers.The relative height of energy barriers for cis-trans isomerization is:2cis > 1cis > 3cis > 4cis.This trend for the reverse trans-cis isomerization follows the electronegativity of the substituent at C-2(2trans > 3trans > 4trans > 1trans).     

POLARIZED ABSORPTION SPECTRA OF MONOCRYSTALLINE ALL-trans. AND 11-cis., 12-s-cis. RETINAL AT 4.2 K

Abstract Polarized absorption spectra of very thin crystal platelets of all- trans . retinal and both forms of 11- cis ., 12-s- cis . retinal were measured at liquid Helium temperature. Even at 4.2 K the spectra remain vibronically unresolved. They show, however, a distinct shoulder at 23.9 kK for all- trans . and at 22.7 kK for 11- cis ., 12-s- cis . retinal on the low energy side of the main band. An assignment of this transition to the lowest-lying ππ*-state ¹A_g-* is proposed.     

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.     

1H NMR study of protected and unprotected kainoid amino acids: facile assignment of C-4 stereochemistry

The kainoid amino acids exhibit potent neuroexcitatory activity in the mammalian central nervous system. Around their pyrrolidine ring, a trans disposition between the C-2 and C-3 substituents and a cis relationship between the C-3 and C-4 substituents are crucial for their potent biological activity. During synthetic studies into the kainoids, we have established a straightforward, empirical rule, which allows the facile assignment of C-4 stereochemistry to both protected and unprotected kainoids. When pairs of C-4 epimers are available, the rule indicates that, when their (1)H NMR spectra are compared, one of the methylene protons on the C-3 side chain appears at significantly lower chemical shift in the C-3, C-4 cis isomer than the corresponding signal for the proton in the spectrum for the C-3, C-4 trans isomer. In addition, the rule states that the difference in chemical shift between the two individual protons on the C-3 side chain of the C-3, C-4 cis isomer is significantly greater than the corresponding difference for the C-3, C-4 trans isomer. The rule is demonstrated for kainoids possessing an unsaturated substituent at C-4 and when comparing spectra in D(2)O for pairs of unprotected C-4 epimers, the spectra were recorded at approximately the same pD.     

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.     

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.     

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.     

Stereoelectronic effects in ring-chain tautomerism of 1,3-diarylnaphth[1,2-e][1,3]oxazines and 3-alkyl-1-arylnaphth[1,2-e][1,3]oxazines

The disubstitution effects of X and Y in 1-(Y-phenyl)-3-(X-phenyl)-2,3-dihydro-1H-naphth[1,2-e][1,3]oxazines on the ring-chain tautomerism, the delocalization of the nitrogen lone pair (anomeric effect), and the (13)C NMR chemical shifts were analyzed by using multiple linear regression analysis. Study of the three-component equilibrium B<==>A<==>C revealed that the chain<==>trans (A<==>B) equilibrium constants are significantly influenced by the inductive effect (sigma(F)) of substituent Y on the 1-phenyl ring. In contrast, no significant substituent dependence on Y was observed for the chain<==>cis (A<==>C) equilibrium. There was an analogous dependence for the epimerization (C<==>B) constants of 1-(Y-phenyl)-3-alkyl-2,3-dihydro-1H-naphth[1,2-e][1,3]oxazines. With these model compounds, significant overlapping energies of the nitrogen lone pair was observed by NBO analysis in the trans forms B (to sigma*(C1-C1'), sigma*(C1-C10b), and sigma*(C3-O4)) and in the cis forms C (to sigma*(C1-H), sigma*(C1-C10b), and sigma*(C3-O4)). The effects of disubstitution revealed some characteristic differences between the cis and trans isomers. However, the results do not suggest that the anomeric effect predominates in the preponderance of the trans over the cis isomer. When the (13)C chemical shift changes induced by substituents X and Y (SCS) were subjected to multiple linear regression analysis, negative rho(F)(Y) and rho(F)(X) values were observed at C-1 and C-3 for both the cis and trans isomers. In contrast, the positive rho(R)(Y) values at C-1 and the negative rho(R)(X) values at C-3 observed indicated the contribution of resonance structures f (rho(R) > 0) and g (rho(R) < 0), respectively. The classical double bond-no-bond resonance structures proved useful in explaining the substituent sensitivities of the donation energies and the behavior of the SCS values.     

Substituted 2-azabicyclo[2.1.1]hexanes as constrained proline analogues: implications for collagen stability

Among the proteinogenic amino acids, only proline is a secondary amine and only proline has a saturated ring. Electronegative substituents on C-4 (that is, C(gamma)) have a substantial effect on the trans/cis ratio of the prolyl peptide bond and the pucker of the pyrrolidine ring. 2-Azabicyclo[2.1.1]hexane is, in essence, a proline analogue with two C(gamma) atoms, one in each of the two prevalent ring puckers of proline. Here, 2-azabicyclo[2.1.1]hexane analogues of 2S-proline, (2S,4S)-4-hydroxyproline, and (2S,4S)-4-fluoroproline residues were synthesized, and their trans/cis ratios were shown to be invariant in a particular solvent. Thus, the substitution of a proline residue on C-4 affects the trans/cis ratio by altering the pucker of its pyrrolidine ring. This finding has implications for the conformation of collagen, which has an abundance of 2S-proline and (2S,4R)-4-hydroxyproline residues, and can be stabilized by (2S,4R)-4-fluoroproline and (2S,4S)-4-fluoroproline residues.     

PURPLE MEMBRANE ANALOGS SYNTHESIZED FROM C17 ALDEHYDE

Abstract— All- trans , 11- cis and 9- cis isomers of the C₁₇ aldehyde analogs of retinal bound with purple membrane apoprotein, probably through a Schiff base linkage at the normal retinal binding site. The complex formed from C₁₇ aldehyde and purple membrane apoprotein was slowly decomposed by 10m M hydroxylamine. The C₁₇ aldehyde competitively inhibited the regeneration of purple membrane from all- trans -retinal and purple membrane apoprotein. The differential ability of the different isomers to inhibit the regeneration suggests that purple membrane has a binding site for the side chain of retinal in addition to the Schiff base binding site.     

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.     

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.     

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.     

THE TRANS → CIS PHOTOISOMERIZATION OF PROTONATED RETINYL SCHIFF BASES

Schiff bases were prepared from all- trans -retinal (I) and pyrrolidine perchlorate (II) and from I and n-butyl amine, protonated with anhydrous hydrogen chloride gas, Ill. Initial quantum yields of trans → cis photoisomerization (Φ°PI) were determined and primary photoproducts and product ratios were measured in aerated methanol. Φ°PI of all- trans -III is independent of excitation energy. All analyses were made using high-pressure liquid chromatographic (LC) methods. It was necessary to hydrolize the Schiff bases to corresponding retinals prior to LC analysis.     

Direct Measurements for Isomer Ratio of Retinal in Bacteriorhodopsin by an Electrochemical Method

Abstract— The composition of retinal isomers in bacteriorhodopsin (bR) in purple membrane (PM) was determined by photoelectric response measurements using a sandwich-type electrochemical cell. The measured amplitude of the photocurrent obtained from a dark-adapted sample was 55% lower than that from a light-adapted sample. This ratio, 55:45, would correspond to the 13- cis /aU- trans isomer ratio of retinal in the dark if the 13- cis form of the pigment did not give a response. This amplitude change correlated with the visible spectral shift of bR. The isomer ratio in the dark depended only weakly on the temperature of the electrolyte, whereas the retinal isomerization rate strongly depended on the temperature and the pH of the electrolyte in the cell. Our results indicate that photoelectric response is elicited only by a species originating from bR containing all- trans retinal and that the behavior of the response in the dark is associated with the pKa of the proton release kinetics of Asp-85.     

ISOMERIZATION OF THE RETINYLIDENE CHROMOPHORE OF BACTERIORHODOPSIN IN LIGHT ADAPTATION: INTRINSIC ISOMERIZATION OF THE CHROMOPHORE AND ITS CONTROL BY THE APO-PROTEIN

Abstract— The dependence of the isomeric configuration of the retinylidene chromophore of bacteriorhodopsin on the pH value and on the wavelength of irradiation (in a photostationary state) were examined by high performance liquid chromatographic analyses of extracted retinal. The process of isomerization of the chromophore during light adaptation was also traced. More than 93% of all- trans and less than 5% of 13- cis retinal were extracted in the photostationary state for irradiation at 560 nm in the pH region of5–9 as well as for irradiation in the wavelength region of 400–650 nm at pH 7. Comparison of the above photostationary state composition with that of protonated n -butylamine Schiff base of retinal indicates that strong constraint is applied to the chromophore by the apo-protein. The constraint can be changed at low or high pH by a partial denaturation or transition of the apo-protein, which results in the generation of 11- cis retinal in the extract. At higher photon density, the isomerization process of the chromophore during light adaptation at pH 7 was characterized, as extracted isomeric retinal, by (1) the initial decrease in 13- cis and increase in all- trans , (2) a subsequent, transient toward the above photostationary state composition. The results are discussed in terms of both the photoisomerization pattern inherent in the retinylidene chromophore and the control by the apo-protein.     

TRIPLET STATES OF ISOMERS OF THE C15-ALDEHYDE AND THE C18-KETONE: LOWER HOMOLOGUES OF RETINAL

Abstract— Triplet extinction coefficients, singlettriplet intersystem crossing yields and triplet state kinetic data of all- trans , 7- cis , 9- cis , 7, 9- dicis isomers of the C₁₅-aldehyde and the C₁₈-ketone. lower homologues of retinal, have been determined in hexane using the methods of laser flash photolysis and pulse radiolysis.