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.     

ON THE TYROSINATE INVOLVEMENT IN THE SCHIFF BASE DEPROTONATION IN THE PROTON PUMP CYCLE OF BACTERIORHODOPSIN

Abstract— The ultraviolet transient absorption assigned to the tyrosinate species in bacteriorhodopsin is followed in time and as a function of pH. Both its rise time and titration curve closely resemble those observed for the production of the M₄₁₂ intermediate. These results may support a recently proposed mechanism that couples tyrosinate production to the Schiff base deprotonation in the proton pump of bacteriorhodopsin.     

A ROLE FOR MENADIONE IN THE PURPLE MEMBRANE PROTON PUMP?

Abstract— It has been assumed that proton pumps such as purple membrane lack redox loops. However, purple membrane does contain an electron carrier. Kates et al. (Meth. Enzymol. 88,98–1 111, 1982) reported the presence of 1 mole of vitaminMK–8 to 6 mol of bacteriorhodopsin among the nonpolar lipids. Is this quinone functionally important in the proton pump mechanism? Proton pumping rates were measured with lipid-free bacteriorhodopsin reconstituted in vesicles to which varying amounts of vitamin K₁ were added. With soybean lipids, in the presence of tetraphenyl boron, the pump quantum yield was 0.04H⁺/photon. This result was independent of the amount of vitamin K, added over a range of 0 to a 100-fold mole ratio to bacteriorhodopsin. A similar result was obtained with H. halobium lipids. The pump quantum yield in vesicles is much less than reported for membrane sheets and whole cells. The results support the conclusion that a vitamin K Q-cycle is not involved in the purple membrane proton pump.     

LIGHT-INDUCED PROTON DISSOCIATION AND ASSOCIATION IN BACTERIORHODOPSIN

Abstract— Light-induced proton release and uptake by acetylated and unmodified bacteriorhodopsin were measured. Bacteriorhodopsin, when illuminated, shows a net proton release at neutral and alkaline pH's, but in acidic pH, it shows an uptake of protons. In the presence of high concentrations of guanidine hydrochloride, light caused only proton release even in acidic pH and the maximum extent of the release was one proton per bacteriorhodopsin molecule around pH 8.
Acetylation of bacteriorhodopsin caused no alteration in the absorption spectrum of purple complex (bR₅₇₀) and M₄₁₂-intermediate, but decreased the decay rate of the M₄₁₂-intermediate. Light-induced release of protons was not observed even in neutral pH values, and only the proton uptake was noticed by acetylated purple membrane fragments. In high concentrations of guanidine hydrochloride, no proton uptake or release by illumination was observed. Vesicles were reconstituted from acetylated purple membrane. These vesicles had almost no ability for light-induced proton transport. The role of amino group(s) in light-induced proton release and transport through the purple membrane is discussed.     

ROLE OF MEROCYANINE DYE AS A PROTON PUMP IN PHOTOVOLTAGE GENERATION

A novel photoelectrochemical cell using a proton pump mechanism in the aggregated planar structure of oxidised cholesterol incorporating merocyanine dyes is reported. Lipid dye binding, as verified from spectral studies and photoisomerisation of the dye, is responsible for this photovoltage generation whose magnitude and storage duration are related to the equilibrium constant of dye-lipid binding through an empirical formula.     

ON THE ROLE OF TYROSINE IN THE PHOTOCYCLE OF BACTERIORHODOPSIN

Abstract -The rate of formation of the M intermediate ( k _M) in the photocycles of bacteriorhodopsin (bR₅₇₀) and of nitrated bacteriorhodopsin (bR₅₃₂ⁿ), is measured over the range between pH 6.5 and 11.5. In the case of bR₅₇₀, k _M is markedly pH dependent, exhibiting a titration-like curve with pK ∽ 10.3. The pH dependency is completely eliminated by nitration. On the basis of previous work by Lemke and Oesterhelt (1981), the effect is attributed to the specific modification of the Tyr 26 residue. The data are rationalized by a mechanism in which deprotonation of Tyr 26 at the stage of the L intermediate constitutes a prerequisite for deprotonation of the retinal-lysine SchifT base. Both reactions are intimately associated with the photo-induced proton pump mechanism.     

A PROTON RELEASE SITE ON THE C-TERMINAL SIDE OF BACTERIORHODOPSIN

We have studied the pH dependence of the light-induced proton release and uptake by bacteriorhodopsin. The quantum efficiency of proton release in cell envelopes and proton uptake in phospholipid vesicles is high in the low pH range and begins to decline between pH 6 and 7 in cell envelopes and between pH 7–8 in phospholipid vesicles. In the cell envelope vesicles the proton release increases again above pH 8–8.5; in phospholipid vesicles a proton release is observed before proton uptake at pHs greater than 9. We suggest that the light-induced proton release observed at high pHs are due to protons released and rebound on the carboxyl terminal side of bacteriorhodopsin.     

CONVERSION OF LIGHT ENERGY TO ELECTROSTATIC ENERGY IN THE PROTON PUMP OF HALOBACTERIUM HALOBIUM

Abstract— The energetics of the proton pump in Halobacterium halobium is considered on a molecular level. It is proposed that absorption of light by bacteriorhodopsin leads to charge separation and sequential changes in the proton affinities some of the protein groups resulting in proton pumping against the pH gradient. Two alternative models of 'through-space' and 'through-bond' charge separation are proposed. Novel diagrams that describe the energetics of the pumping process are presented. These diagrams can be used both to examine different models of bacteriorhodopsin and for considering other photobiological processes that involve conversion of light energy to electrostatic energy.     

古紫质等光驱质子泵以及该类活性蛋白质与聚合物的功能复合材料

将具有生物活性的蛋白质与具有良好加工和力学性能的合成高分子相结合制备高性能的杂化材料是材料科学一个新的生长点.本专论聚焦于对具有光驱质子泵功能的两类活性蛋白质——细菌视紫红质(BR)与古紫质4(AR4)的研究,其中BR作为一个著名的膜蛋白已有四十余年的研究历史,而AR4则为中国科研人员十余年前所发现.综述了AR4的研究,并对AR4和BR进行了对比,进一步介绍了光敏蛋白质(BR和AR4)与聚合物基质复合制备新型功能高分子材料的工作,还介绍了该类光敏蛋白质的基因工程改造以及蛋白质/聚合物复合膜用于信息材料方面的探索工作.论文总结了一系列创新成果,如(1)在光敏蛋白质的质子泵机理方面,提出了"弱偶联模型"并解释了AR4具有与BR相反的质子释放和提取时间顺序的内在机理;(2)光敏蛋白质与聚合物复合膜相关的高分子水凝胶和蛋白质聚集状态的研究,并发现与均聚物和两亲性小分子不同,两亲性嵌段共聚物可导致光敏蛋白质中间体的寿命有数量级的延长;(3)发展了光敏蛋白质与聚合物复合膜的制备技术,所得到的材料不仅保持了光学活性,其蛋白质的光学响应性能还得到进一步改善;(4)发现了含光敏蛋白质的紫膜强烈抗拒哺乳动物细胞黏附的新功能;(5)尝试将该蛋白质和聚合物的复合膜作为信息材料,实现了全光宽带图像显示.进一步展望了此类光敏蛋白质的后续研究和潜在的应用前景.     

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.     

BRANCHING PATHWAYS IN THE PHOTOCYCLE OF BACTERIORHODOPSIN

Abstract— The pulsed laser photolysis of light-adapted bacteriorhodopsin (BR₅₇₀) is carried out over the temperature range between 25°C and—92°C in neutral and alkaline water-glycerol solutions. The results arc indicative of considerable complexity, introduced by two temperature dependent branching reactions associated with the intermediates K₆₁₀, L₅₅₀ and M₄₁₂, of the BR₅₇₀ photocycle. (a) At relatively low temperatures the primary photoproduct K-₆₁₀ equilibrates with a blue-shifted species, K_p. Both K₆₁₀ and the new intermediate subsequently decay into another species, K'_r, in a process which competes with the formation of L₅₅₀. Finally, K'_p converts very slowly to L₅₅₀. This branched pathway delays the formation of L₅₅₀ and thus of M₄₁₂, without affecting the final yield of either species, (b) A thermal back-reaction regenerating BR₅₇₀ takes place at the stage of L₅₅₀, inhibiting the formation of M₄₁₂. The reaction which also predominates at low temperatures, is relatively inefficient at high pH when the forward L₅₅₀→ M₄₁₂ step is highly catalyzed. It is the superposition of both branching mechanisms, (a) and (b), which accounts for the complex effects of temperature and pH on the photo-cycle of BR₅₇₀. Mechanism (b) is accounted for by a molecular scheme in which deprotonation of a tyrosine moiety at the stage of L₅₅₀ constitutes a prerequisite for deprotonation of the retinal-lysine schiff-base as required for forming M₄₁₂. This scheme appears to be directly related to the proton pump. Mechanism (a) introduces additional complexity in the photocycle at low temperatures but its molecular aspects are still unclear.     

LIGHT AND pH INDUCED CHANGES IN THE SURFACE PRESSURE-AREA ISOTHERMS OF BACTERIORHODOPSIN

Abstract— Pressure-area isotherms of monolayers of purple membrane fragments were measured at an air-water interface. The pH of the aqueous phase was varied from 2 to 10. Over the pH range 5 to 9 the area/molecule of bacteriorhodopsin was constant and illumination of the film had no effect on the isotherms.
At pH's more acid than 5 and more alkaline than 9, there were large decreases in area/molecule. Under these conditions of extreme pH the monolayer was very sensitive to light. Illumination resulted in irreversible increases in area/molecule.     

THE RADIOLYTIC REDUCTION OF THE SCHIFF BASE IN BACTERIORHODOPSIN

Abstract —Nanosecond electron beam pulses cause radiolytic reduction of the retinal Schiff base in bacteriorhodopsin. The effects of different scavengers show that both H atoms and the formate radical anion can act as the reducing species. Model system studies on the radiolysis of retinal indicate transient formation of a retinal radical anion and suggest an analogous two-step reaction in bR. Delocalization of the radical in the polyene system and reaction with surrounding groups would account for the relatively low yield of reduced Schiff base and the appearance of intra- and intermolecular crosslinks in irradiated bacteriorhodopsin.     

IDENTIFICATION OF THE PROTON ACCEPTOR OF SCHIFF BASE DEPROTONATION IN BACTERIORHODOPSIN: A FOURIER-TRANSFORM-INFRARED STUDY OF THE MUTANT ASP85 → GLU IN ITS NATURAL LIPID ENVIRONMENT

Abstract— In order to assign the proton acceptor for Schiff base deprotonation in bacteriorhodopsin to a specific Asp residue, the photoreaction of the Asp85 → Glu mutant, as expressed in Halobacterium sp . GRB, was investigated by static low-temperature and time-resolved infrared difference spec-troscopy. Measurements were also performed on the mutant protein labeled with [4-¹³C]Asp which allowed discrimination between Asp and Glu residues. 14,15-di¹³C-retinal was incorporated to distinguish amide-II absorbance changes from changes of the ethylenic mode of the chromophore. In agreement with earlier UV-VIS measurements, our data show that from both the 540 and 610 nm species present in a pH-dependent equilibrium, intermediates similar to K and L can be formed. The 14 ms time-resolved spectrum of the 540 nm species shows that a glutamic acid becomes protonated in the M-like intermediate, whereas the comparable difference spectrum of the 610 nm species demonstrates that in the initial state a glutamic acid is already protonated. In conjunction with earlier observations of protonation of an Asp residue in wild-type M, the data provide direct evidence that the proton acceptor in the deprotonation reaction of the Schiff base is Asp85.     

THE ULTRAVIOLET FLUORESCENCE OF BACTERIORHODOPSIN AND THE LOCATION OF TRYPTOPHANYL RESIDUES

Abstract— The ultraviolet fluorescence spectrum of bacteriorhodopsin is characterized by emission from an ensemble of internal, surface and exposed Trp residues. The temperature dependence of fluorescence yields exhibits a discontinuity at about 30°C coincident with previously observed transitions in membrane lipid microviscosity, photocycle lifetime and photoconductivity. Quenching at high pH coincides with ionization of Tyr and an emission red shift to a spectrum typical of that of tyrosinate. Guanidine hydrochloride produces only partial protein denaturation, increasing the number of exposed Trp by 50%. While exposed Trp in native bacteriorhodopsin are in the minority, they represent a higher proportion of total Trp than is found in rhodopsin of animal rod outer sections.     

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".     

PASSIVE AND ACTIVE ROLE OF FLUORESCERS IN THE CHEMILUMINESCENCE OF TETRAMETHOXYDIOXETANE

Abstract— Tests for the possible catalytic role of fluorescers in the chemiluminescent decomposition of dioxetanes are discussed and applied to the case of tetramethoxydioxetane. This dioxetane, which can undergo direct thermal decomposition yielding excited methyl carbonate with subsequent excitation of fluorescers (for example 9, 10-dibromoanthracene. DBA), is shown to be sensitive also to catalysis by fluorescers such as rubrene. 9, 10-diphenyl-anthracene (DPA) and 9, IOdicyanoanthracene (DCNA). The possibility of a charge-transfer mechanism of chemiluminescent catalysis is suggested.     

THE ROLE OF FRONTAL NEURONS IN PAIN AND ACUPUNCTURE ANALGESIA

To study the role of frontal neurons in pain and acupuncture analgesia, experiments were carried out on 4 monkeys (Macaca mulatta) during performing two tasks, the Heat Discrimination Response GO/NO-GO Task (Task Ⅰ) and the Heat Delayed Discrimination GO/NO-GO Task (Task Ⅱ). After a criterion of 90% of correct performances over three successive days had been reached, activity of single neurons was recorded from the frontal cortex. Of 276 Task Ⅰ-related neurons, 211 responded to noxious and/or innocuous heat stimuli. Of 73 Task Ⅱ-related neurons, 59 responded to noxious and/or innocuous heat stimuli. During acupuncture, the nociceptive reaction time for lever release to avoid painful stimuli was increased, the percentage of correct performances was lowered, and the neuronal responses to noxious and/or innocuous stimuli were suppressed. These neurons were mainly located in a circumscribed area medial to the superior ramus of the arcuate sulcus in frontal cortex, including the prefrontal and premotor a     

THE ROLE OF SUPEROXIDE AND SINGLET OXYGEN IN LIPID PEROXIDATION

Abstract— An investigation into the mechanism of lipid peroxidation catalyzed by xanthine oxidase showed a dependence upon superoxide, singlet oxygen and adenosine 5'-diphosphate chelated iron (ADP-Fe³⁺). In the absence of ADP-Fe³⁺ or in the presence of superoxide dismutase there is complete inhibition of enzymatic peroxidation. Initiation of peroxidation likely occurs through an ADP-perferryl ion complex formed by ADP-Fe³⁺ and superoxide. Use of the singlet oxygen trapping agent 2,5-diphenylfuran showed that singlet oxygen does not participate in the initiation of peroxidation but rather in the propagation of peroxidation. The mechanisms of NADPH-cytochrome P450 reductase-catalyzed and ADP-Fe²⁺ catalyzed lipid peroxidation parallel that of xanthine oxidase in that initiation occurs through a superoxide dismutase-sensitive reaction and that singlet oxygen is present during propagation of lipid peroxidation.