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.     

TIME-RESOLVED ULTRAVIOLET ABSORPTION CHANGES IN THE PHOTOCYCLE OF BACTERIORHODOPSIN*

Abstract– The kinetics of the absorption changes associated with the perturbation of aromatic acids during the photocycle of bacteriorhodopsin (bR) were studied at room temperature with microsecond time-resolution. Flash experiments with nanosecond excitation at 532 nm were performed on the purple membrane suspension at a number of measuring wavelengths in the spectral range250–630 nm (to monitor both non-chromophore changes and the photocycle kinetics). The kinetic data collected at different wavelengths were simultaneously fitted with a sum of exponentials to obtain time-resolved UV-VIS difference spectra of photocycle intermediates. This approach allowed us to separate kinetically distinct contributions coupled with tryptophan(s) and tyrosine(s) perturbations. Contributions associated with a reversible perturbation of tryptophans appeared with complex (multistep) kinetics during the bRM transitions and relaxed in a single step during the M0 transition. A contribution associated with perturbation of the local environment of tyrosine appeared before the L and relaxed during the Ob̊ transition.     

EVIDENCE FOR BRANCHING IN THE PHOTOCYCLE OF BACTERIORHODOPSIN AND CONCENTRATION CHANGES OF LATE INTERMEDIATE FORMS

Abstract— Flash photolysis transients of bacteriorhodopsin were recorded with a spectrograph -multielement photodiode array combination and the recordings were analyzed to determine the concentrations of bacteriorhodopsin intermediates "M" and "O" relative to the amount of "bR" cycling (pH 7.1,10–40°C). Estimated concentration time courses were simulated with solutions to two kinetic decay models which could account for photocycle temperature dependence. A unidirectional unbranched decay model overpredicts our estimated levels of [O(r)], whereas a model branched at the "M" intermediate describes each of the later intermediate levels well (with no evidence for an independent "N" form). Our results are consistent with "M" decay regulating the level and rates of change of [bR (t)] and (bR(f)]- and also suggest that two temperature-dependent pathways form "bR" from "M", one directly, and the other indirectly through "O".     

菌紫质LB膜对闪光的响应

光敏生物大分子菌紫质(bR)是嗜盐菌(H.Halobium)紫膜中的唯一蛋白质,分子量为26,000 Da.视黄醛共价结合在蛋白上,当吸收了光子后它可在瞬间产生光化学反应,并与光电转换相联系.bR分子在结构与功能方面的这些特征,与动物光感受器中的视紫红质构为类似,从而引起了视觉研究者对它的兴趣.     

气体在固体表面吸附的热力学模型及吸附等温式

描述气体在固体表面吸附的两个著名吸附等温式Langmuir公式和Bronauer—Emmett—Teller(B·E·T)公式可以由动力学方法和统计热力学方法导出。但在导出过程中,都要引进一些基本假定之外的附加限制和近似. 根据经典热力学的唯象特点,能建立气体在固体表面吸附的吸附表面相热力学模型.从吸附平衡是一种热力学相平衡这一观点出发,可以由相平衡原理十分简洁地导出Langmuir吸附等温式和多层吸附的B·E·T吸附等温式.     

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.     

CORRELATION BETWEEN ABSORBANCE CHANGES AND A PHYSIOLOGICAL RESPONSE INDUCED BY BLUE LIGHT IN NEUROSPORA

Abstract— Absorbance changes due to the photoreduction of a b -type cytochrome are associated with many biological blue light-controlled processes. Evidence is presented for their causal relationship with the perception of light which induced conidia formation under conditions of starvation in Neurospora crassa mutant albino band, but not with light-induced phase shifts of conidiation bands.     

POWER- AND TIME-RESOLVED RESONANCE RAMAN STUDIES AND CONFORMATIONAL CHANGES IN BACTERIORHODOPSIN

Abstract— By comparing the resonance Raman spectra of the retinal of the intermediates of bacteriorho-dopsin (obtained by using fixed flow with residence of time of 10 ps. variable laser power and frequency as well as computer subtraction techniques) with those of model compounds and with each other, the following possible conclusions can be obtained: (1) There exists stronger interaction between the retinal and the opsin in bacteriorhodopsin than that present in rhodopsin. (2) Conformational changes seem to take place during the dark light adaptation process as well as during the photosynthetic cycle. (3) The appearance of the spectrum of the retinal in the fingerprint region for the bL₅₅₀ and bM₄₁₂ intermediates is similar despite large shifts in their optical absorption maxima. This might argue against the theory that proposes ground state retinal conformational changes to explain the observed red shift in the optical spectra of retinal upon combining with the opsin. (4) Contrary to bM ₄₁₂ , the bL₅₅₀ species seems to be protonated. The fact that loss of proton does not seem to change the retinal conformation greatly might suggest that the protein and its ionic environment might carry the larger share of the load in the deprotonation process.     

BLUE LIGHT INDUCED PHOTOTRANSFORMATION OF PHYTOCHROME IN THE PRESENCE OF FLAVIN*

Abstract— Irradiation of the P_r form of phytochrome in the presence of flavin mononucleotide (FMN) which absorbs the actinic blue light yields P_fr at a rate greater than that in the absence of FMN. The actinic blue light absorbed by FMN enhances the phototransformation of P_r via the energy transfer from the former to the latter. On the other hand, the photoreversion of P_fr was inhibited by the presence of FMN when illuminated with blue light. The lack of photo-enhancement of the reversion of P_r, by blue light suggests that the P_fr chromophore (acceptor) transition dipole is virtually perpendicular to the FMN transition dipole, as the result of a chromophore reorientation in the P_r→P_fr phototransformation. The fact that blue light absorbed by flavin preferentially enhances the forward phototransformation of phytochrome while inhibiting the reversion may have an important implication in the high irradiance responses in plants in terms of a preferential accumulation of P_fr by blue light excitation.     

THE ROLE OF ARGININES 82 AND 227 IN THE BACTERIORHODOPSIN PROTON PUMP

Abstract
Arginine residues 82 and 227 in bacteriorhodopsin were replaced by glutamine residues, using the site-directed mutagenesis techniques. Mutant bacteriorhodopsins were found to be competent in formation and decomposition of the photocycle M412 intermediate as well as in generation of photoelectric potential provided that pH of the medium is sufficiently high. Lowering of pH results in transition of bacteriorhodopsin into a blue acidic form which cannot produce M412 and photo-potential. The p K values of these transitions for Arg-227 → Gln and Arg-82 → Gln mutants are shifted correspondently for 1 and 4 pH units to a higher pH region in comparison with native bacteriorhodopsin. The rate of the M412 formation in both mutants was similar to that in the native protein. As to M412 decay, it is much slower in Arg-227 → Gln mutant than in native and Arg-82 → Gln bacteriorhodopsins. In all cases, the decay depends only slightly upon pH. It is concluded that Arg-82 is involved in maintenance of a bacteriorhodopsin structure that is resistant to the pH decrease down to 4 whereas Arg-227 is required first of all for the process of Schiff base reprotonation.     

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.     

BACTERIORHODOPSIN AND THE SENSORY PIGMENT OF THE PHOTOSYSTEM 565 IN HALOBACTERIUM HALOBIUM

Abstract Blocking in vivo synthesis of retinal by addition of nicotine to the culture medium leads to the loss of photobehavior in Halobacterium halobium. Addition of rrans -retinol or frans-retinol₂ (3,4-dehy-droretinol) restores the responses to light decreases in the green-yellow spectral range. Action spectra of the reconstituted retinal- and retinal₂-photosystem show maximal sensitivity at 565 and 580 nm, respectively. Addition of retinol or retinol₂ also restores the formation of bacteriorhodopsin (BR) or bacteriorhodopsin₂ (BR₂= 3,4-dehydroretinal-bacterio-opsin complex). The absorption spectra of BR and BR₂, measured in isolated membranes, as well as in living bacteria, show maxima at 568 nm (BR) and at about 600 nm (BR₂), respectively. Comparison of the action spectrum of the retinal2-containing sensory photosystem with the absorption spectrum of BR₂ suggests that a retinal pigment different from BR is responsible for the photosensory behavior to green-yellow light.     

FLUORESCENCE OF BACTERIORHODOPSIN UNDER SUBPICOSECOND LIGHT EXCITATION

Abstract— A streak camera detection technique has been used to record the fluorescence of bacteriorhodopsin at room temperature induced by single subpicosecond light pulses. The fluorescence lifetime of bacteriorhodopsin has been found to be less than 2 ps.     

ON THE MULTIPLE CYCLES OF BACTERIORHODOPSIN AT HIGH pH

Bacteriorhodopsin (bR) shows at least two parallel photocycles at pH 10.5, suggesting that more than one form of bR exists in alkaline bR sample. Upon the absorption of visible light, the different forms of bR at high pH yield different parallel intermediates: M₄₁₂ with two rise and two decay components; and R, an extremely fast rising and extremely slow decaying intermediate with an absorption peak at 350 nm. The kinetics and spectra do not agree with the proposal of Kouyama el al. (1988, Biochemistry 27,5855–5863) that the 350 nm-absorbing species is the N intermediate which follows M₄₁₂ and that the slow decaying M₄₁₂ is an M-like photoproduct of N. Our results basically agree with the proposal of Dacshazy et al. (1988, Proc. Natl. Acad. Sci. USA 85,6358–6361) that the fast and slow decaying M₄₁₂ intermediates and R are in independent photocycles arising from different forms of bR. The different forms of bR are probably in dynamic equilibrium with their ratios controlled by pH and ionic strength.     

光生电子和由还原剂提供的电子在卤化银颗粒中性能的比较

本文研究了光生电子和还原剂提供的电子在卤化银晶体中的行为。光灰化和还原剂灰化生成的灰雾中心的位置不同。光灰化时灰雾中心优先在卤化银颗粒内部形成,表明光生电子可以直接进入卤化银晶体的导带。还原灰化时内部灰雾中心只有在灰化剂量足够多时才形成,而且滞后于表面灰雾中心的形成,表明还原剂提供的电子虽然可以进入卤化银的导带,但它们进入导带的方式与光生电子不同。     

GENETIC VARIATIONS AND LINOLENIC ACID INDUCED CHANGES IN THE ORIENTATION PATTERN OF CHLOROPHYLL a IN THYLAKOID MEMBRANES

Abstract Orientation pattern of the Q_y absorption and emission dipoles of chlorophyll a were studied in wild type Scenedesmus obliquus and in mutants deficient in chlorophyll b and carotenoids. Fluorescence polarization ratio at –140°C and linear dichroism at 25°C were measured in whole cells and thylakoids aligned in polyacrylamide gel. Unlike normal thylakoids, mutants displayed fluorescence polarization ratios significantly lower than 1.0 and showed a negative LD signal around 672 nm, indicating the tendency of the Q_y dipoles to tilt out from the membrane plane. Such an orientation pattern can also be artificially induced by treating normal thylakoids with linolenic acid.     

THE ROLE OF ARGININES 82 AND 227 IN THE BACTERIORHODOPSIN PROTON PUMP*

Abstract— Arginine residues 82 and 227 in bacteriorhodopsin were replaced by glutamine residues, using the site-directed mutagenesis techniques. Mutant bacteriorhodopsins were found to be competent in formation and decomposition of the photocycle M412 intermediate as well as in generation of photoelectric potential provided that pH of the medium is sufficiently high. Lowering of pH results in transition of bacteriorhodopsin into a blue acidic form which cannot produce M412 and photo-potential. The pK values of these transitions for Arg-227 → Gln and Arg-82 → Gln mutants are shifted correspondently for 1 and 4 pH units to a higher pH region in comparison with native bacteriorhodopsin. The rate of the M412 formation in both mutants was similar to that in the native protein. As to M412 decay, it is much slower in Arg-227 → Gln mutant than in native and Arg-82 → Gln bacteriorhodopsins. In all cases, the decay depends only slightly upon pH. It is concluded that Arg-82 is involved in maintenance of a bacteriorhodopsin structure that is resistant to the pH decrease down to 4 whereas Arg-227 is required first of all for the process of Schiff base reprotonation.     

AN EXPERIMENTAL STUDY OF THE CHANGES IN PIGMENTATION IN HUMAN SKIN IN VIVO WITH VISIBLE AND NEAR INFRARED LIGHT

Abstract— The skin of the lower inner arm of volunteers was irradiated, with a 390–1700 nm light source, through a fiber optic bundle for times of up to 1.2 × 10⁴ s and with powers of up to 0.35 W/cm². Simultaneously with the irradiation, spectra (390–720 nm) of the remitted intensity were measured, while a 5.0 cm in diameter area of the skin around the fiber bundle was maintained at constant temperature, within 0.2°C. The generation of a photoproduct was observed and measured as changes in the remitted intensity within 600 s (10 min) of the start of irradiation.
The photoproduct formed was characterized by a weak absorption in the blue part of the spectrum (400–450 nm), leading to a bluish appearance in the irradiated area only. The color change appears as a two step process. It starts with a "soluble" photoproduct, which disappears, within 24 h after irradiation, and an "insoluble" photoproduct which appears with irradiation greater than 3 ×10³ s (50 min). No spectral differences were detected between the two photoproducts. The "insoluble" photoproduct persists for periods of up to 8 weeks. The color change in the skin is immediate and there is no erythema associated with this color change.