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.     

STOCHASTIC MODELING OF LIGHT ENERGY CONVERSION IN PHOTOSYNTHESIS

Abstract— Photosynthetic quantum conversion and early electron transport is modeled as a stochastic process on a digital computer to determine what free-energy losses are a necessary consequence of specific assumptions about the reaction structure, kinetics, and thermodynamics of the participating molecules. Maximal free-energy yield requires that all dark reactions be near equilibrium, so the potentials of all half-cells on each side of the light act are nearly the same. This near equilibrium requires that all forward rate constants be at least 10² times the rate of light absorption, and that all reverse rate constants be at least the rate of light absorption. The behavior of model systems with one primary donor and one primary acceptor is comparatively independent whether there is one or an infinite number of secondary electron donors and acceptors. A system having no metastable (e.g. triplet) state of the light-activated donor can convert light energy with nearly ideal efficiency, provided that the standard electrode potentials of the primary donor and primary acceptor half-cells are precisely located with respect to one another and to the potentials of the ultimate donor and acceptor. While not necessary for near maximal free-energy yield, a metastable intermediate allows a flexibility in the choice of half-cell potentials which is not possible in the absence of such an intermediate.     

BIOSYNTHESIS AND ASSEMBLY OF PURPLE MEMBRANE OF HALOBACTERIUM HALOBIUM S9: LIGHT STIMULATION OF BACTERIORHODOPSIN FORMATION

Abstract— The effect of light on purple membrane biogenesis in Halobacterium halobium S9 strain was investigated. When bacteria were grown in the dark, the 570nm absorption due to bacteriorhodopsin increased more slowly than under illumination, but eventually after longer incubation, reached the same level as that seen in the illuminated culture.
Analysis of membrane fractions by sucrose density gradient centrifugation revealed that two different membrane fractions, containing purple and brown membrane could be detected in the exponential growth phase. Another fraction whose density was higher than that of purple membrane, disappeared concomitantly with the increase in purple membrane and brown membrane, indicating that it may be related to purple membrane formation.
HPLC analysis of membrane proteins showed that there was no significant difference in de novo synthesis of bacterio–opsin between dark and illuminated cultures. This led us to conclude that light stimulated retinal binding to bacterio–opsin and/or retinal biosynthesis rather than bacterio–opsin synthesis. Bacteriorhodopsin seemed to form the brown membrane fraction first, which then spontaneously reorganized into purple membrane.
When incorporated in liposomes, bacteriorhodopsin in brown membrane was found to have rather higher proton pump activity than that in purple membrane. The H⁺ pumping activity was quite heat labile. This and the CD spectra indicated that bacteriorhodopsin in brown membrane might exist without forming normal timer unit.     

INTERPRETATIONS OF THE SOLUTION AND ORIENTED FILM SPECTRA OF BROWN MEMBRANE OF HALOBACTERIUM HALOBIUM

Abstract— The absorption and circular dichroic spectra of the brown holo-membrane (retinal present) and apo-membrane (retinal absent) of Halobacterium halobium in solution and oriented as a film have been studied over the accessible wavelength region, 800–183nm. Since the structure of the well-studied purple membrane can be considered to be a modification of the structure of the brown membrane and much is known about the structure of the purple membrane, interpretations of the brown membrane spectra are based on our previous interpretations of similar studies of the purple membrane. The brown membrane contains two membrane proteins, bacteriorhodopsin and cytochrome b-561 in a 3:1 molar ratio in contrast to the purple membrane which contains only bacteriorhodopsin. Main findings are (a) degenerate oscillator coupling (exciton) among the retinyl chromophores of the bacteriorhodopsins, (b) a relatively strong in-plane interaction between the retinal and the bacteriorhodopsin apoprotein environment, possibly due to a dissymmetric static charge distribution, (c) the planes of the aromatic rings of some of the tryptophans must be nearly parallel to the plane of the membrane, (d) the helical axes of the bacterio-opsin polypeptide segments are significantly tilted in respect to the normal to the membrane plane in contrast to the helical axes of the bacteriorhodopsin polypeptide segments which are nearly parallel to the normal, (e) no detectable interaction between the two membrane proteins, (f) the plane of the heme of the cytochrome cannot be parallel to the membrane plane and is most likely perpendicular to it. (g) the dipole moments of the two mutually perpendicular Soret porphyrin transitions of the heme are most likely oriented at an angle to the membrane plane, (h) there seems to be a significant reduction in the symmetry of the heme group in the environment of the apoprotein, (i) the possibility of a unique geometrical arrangement and resonance interaction between the Soret porphyrin and nearby cytochrome aromatic amino acid π–π* transitions, (j) the secondary structure of the cytochrome is significantly α-helical, and (k) the helical axes of the cytochrome polypeptide segments are randomly oriented in respect to the normal to the membrane plane. A consequence of these findings is that the fine structures of the bacteriorhodopsins of the brown and purple membranes are very similar in spite of differences in the composition and the structure of the two membranes. In addition, the orientation of the helical segments of the bacteriorhodopsin polypeptides relative to the membrane plane in the brown and purple membranes can be regulated by the retinal–apoprotein interactions. Significance of this possible regulation in respect to the proton-pumping function of these membranes is discussed.     

THE WHITE MEMBRANE OF CRYSTALLINE BACTERIOOPSIN IN HALOBACTERIUM HALOBIUM STRAIN R1mW AND ITS CONVERSION INTO PURPLE MEMBRANE BY EXOGENOUS RETINAL

Abstract— A new strain isolated from Halobacterium halobium designated R₁mW, contained negligible amounts of isoprenoid pigments, had a yellowish white color due to respiratory pigments and showed no proton movement in response to light. However, addition of all-trans-retinal converted R₁mW into purple cells. Formation of both halorhodopsin and bacteriorhodopsin was indicated by induction of light-dependent proton uptake and release, respectively. Both haloopsin and bacterioopsin were thus postulated to be present in R₁mW. Electron micrographs of freeze-fractured cytoplasmic membranes revealed patches in a hexagonal array of trimeric particles, comparable to the purple membrane structure. These white membrane patches were isolated by procedures similar to those for the purple membrane. The white membrane's buoyant density was about 1.18 g/m/, and its main component migrated on sodium dodecylsulfate polyacrylamide gels at the same rate as bacteriorhodopsin. The white membrane showed only a small absorption peak at ～410nm due to contaminating respiratory pigments and a strong absorption at around 275 nm and shorter wavelengths. The white membrane was thus considered to be mainly composed of bacterioopsin, which was readily converted into bacteriorhodopsin by an addition of all-trans-retinal. The absorption and CD spectra of the white membrane were measured before and after addition of retinal. The molar extinction coefficient of dark-adapted bacteriorhodopsin formed was determined to be 53000M^?1 cm^?1 at 560 nm from retinal binding studies. The CD spectrum of the white membrane was negligible in the visible region but showed several bands assigned to aromatic and backbone structures in the UV region. Retinal addition caused considerable changes in the spectrum, yielding the CD spectrum of crystalline purple membrane bacteriorhodopsin. The white membrane thus appears to be a preparation suitable for structure-function studies of bacteriorhodopsin.     

SUBSTITUTION OF RETINAL BY ANALOGUES IN RETINAL PIGMENTS OF HALOBACTERIUM HALOBIUM. CONTRIBUTION OF BACTERIORHODOPSIN AND HALORHODOPSIN TO PHOTOSENSORY ACTIVITY

Abstract— In Halobacrerium hnlobium. retinal is the chromophore of the light-energy converting pigments bacteriorhodopsin (BR) and halorhodopsin (HR) and of the sensory photosystems. PS 370 and PS 565. In both photosystems as well as in BR and HR. retinal was substituted by retinal analogues. Retinal₂ ( 3,4-dehydro-retinal ) . shifts the main sensitivity maximum of PS 370 and of PS 565 by about 1.5 nm to longer wavelengths. The absorption maxima of BR and HR are both shifted in the same direction, but by 37 nm. 13-Ethylretinal and 13-propylretirnal shift the main sensitivity maximum of each sensory photosystem to shorter wavclengths; the absorption maxima of BR and HR are shifted in the same direction but to a smaller extent. Both sensory photosystems are equally active with retinal and with each of the three analogues as the chromophore. After substitution of retinal by the analogues, the action spectra of PS 565 of the BR-containing strain R₁L₃ show a secondary bensitivity peak in addition to the main peak. This secondary peak matches the absorption maximum of the corresponding BR. In the action spectra of the BR-deficient strainET–15 this secondary peak is missing. Action spectra of PS 565 of the BR-deficient strainL–33, which synthesizes increased amounts of HR. with all retinals show a secondary peak which matches the absorption maximum of the corresponding HR.
The results show that the analogues can substitute retinal in both sensory pigments as well as in BR and HR. Moreover, the data support the previous assumption that both BRand HR, although not required for photosensory activity can contribute to photosensing through PS 565.     

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.     

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.     

轻烃在改性ZSM－5催化剂上转化成芳烃与低碳烯烃的选择性控制

研究了Ｃ３－Ｃ９烷烃在不同的改性ＺＳＭ－５沸石上的反应规律．过渡金属离子改性的ＺＳＭ－５沸石显示了较高的芳烃选择性，芳烃的选择性还取决于改性金属离子的脱氧能力并可通过预硫化得到改进．钾、钡改性的沸石显示了较高的烯烃选择性，增加钾含量，烯烃选择性显著提高，但也明显抑制了催化剂的活性；提高Ｂａ含量，同样可提高烯烃选择性，但对活性影响不大．环己烯的模型反应结果表明，Ｋ，Ｂａ的作用在于抑制了双分子氢转移反应和提高了脱氢能力．红外表征结果表明，沸石表面的羟基，因不同金属改性而发生不同的变化．此外，气相氧对提高烯烃和芳烃的收率有明显作用     

ENERGY CONVERSION IN THE DECAY OF TRIPLET LUMIFLAVIN IN THE PRESENCE OF FERRI- AND FERROCYANIDE

Abstract— Flash photolysis at 450 nm has been used to study the quenching of the excited triplet state of lumiflavin and the transient species formed in subsequent reactions in deaerated phosphate buffer (pH 6.9).
The effect of the presence of ferricyanide on the life time of triplet lumiflavin has been studied. The results suggest an energy transfer reaction without concurrent electron transfer reactions. The rate constant for the process was 2.8 times 10⁹ M ^-1 s^-1. The analogous reaction with ferrocyanide could not be observed because of the efficient electron transfer reaction (δG = -20.6 kcal mol^-1) leading to the formation of the semireduced lumiflavin and ferricyanide. The rate constant for this reaction was 3.3 times 10⁹ M ^-1 s^-1. The semireduced lumiflavin radical was found to disappear in a second order reaction with a rate constant of 1.7 times 10⁹ M ^-1 s^-1. It was found to react with ferricyanide with a rate constant of 0.7 times 10⁹ M ^-1 s^-1.
A model for the various photochemical and photophysical processes involved in the decay and quenching of the lumiflavin triplet state is suggested and discussed.     

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.     

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.     

P588, A SECOND RETINAL-CONTAINING PIGMENT IN HALOBACTERIUM HALOBIUM

Abstract— Halobacterium mutant strains with defects in the biosynthesis of various pigments have been isolated. One of these strains, mutant ET-15, is incapable of producing the light-driven proton pump bacteriorhodopsin and the carotenoid bacterioruberin. However, ET-15 synthesizes another photochemically active, retinal-containing pigment, P₅₈₈, which mediates light-induced proton uptake enhanced by uncouplers. P₅₈₈ and bacteriorhodopsin are simultaneously present in wild-type cells grown under normal conditions; however, they can be distinguished by the following criteria.

• 1 They can be separated by independent mutational events.
• 2 Proton ionophores such as FCCP diminish bacteriorhodopsin-driven proton translocation but enhance P₅₈₈-mediated proton flows. We define here proton translocations which can be diminished by the addition of uncouplers (e.g. FCCP) as ‘active’, others which can be enhanced by FCCP as ‘passive’.
• 3 The kinetic and spectral properties of the photocycle intermediates of bacteriorhodopsin and P₅₈₈ are different.
• 4 The action spectrum for photocycling of P₅₈₈ is red-shifted with respect to that for bacteriorhodopsin.

A comparison of action spectra for proton translocations with that for photocycling links pigment P₅₈₈ and light-driven, passive proton uptake by ET-15 envelope vesicles. When chemically bleached P₅₈₈ was regenerated with all-trans-retinal, both photochemical activity and light-induced proton inflow were restored to equal extents. This identifies P₅₈₈ with the energizer for the passive proton flows.     

BINDING OF ANTHRACENE TO CELLULAR MACROMOLECULES IN THE PRESENCE OF LIGHT

Ultraviolet radiation (δ > 295 nm) induced covalent binding of anthracene to DNA which increased with time and was not affected by oxygen. Irradiation in the presence of anthracene induced nicking of Col E, circular DNA and decreased the thermal denaturation temperature of calf thymus DNA. These effects were oxygen dependent, and were decreased by GMP. Irradiation of anthracene and human serum albumin resulted in covalent binding of the hydrocarbon to the protein accompanied by crosslinking of the protein. Protein crosslinking decreased under anaerobic conditions. Irradiation of anthracene bound to liposomes induced lipid peroxidation which was not affected by superoxide dismutase or catalase.     

潜影形成过程中的空穴及其转换

最近几年,在研究卤化银体系的潜影形成效率时,在如何对待和处理光生空穴问题上出现了一些突破传统思路并具有开拓性的方法,我们把它称之为"空穴-电子转换"法.这种方法的关键是寻找或设计一种"化学转换器".这种转换器既能捕获空穴,消除或减少潜影形成过程中由于电子与空穴的复合造成的电子损失,又能经过它的转换产生并释放出额外电子或某种电子载体(如自由基)促进潜影的形成,起到一举两得显著提高卤化银乳剂感光度的作用.本文对当前已有的3种化学转换器的研究进展进行了综合评述,望引起国内科学家的密切关注.     

合成气转化为乙醇的反应机理

本文在助剂型Rh催化剂上采用了以CH_2OD、D_2~(18)O为捕获剂的原位化学捕获反应, 以及以D_2~(18)O为重氧源试剂的原位氧同位素交换反应, 对合成气转化为乙醇的反应机理进行了研究。在原位捕获反应中检测到CH_2DCOOCH_3、CH_3COOCH_3和CH_2DCOOD、CH_3COOD的生成, 表明合成乙醇反应过程中存在中间体乙烯酮和乙酰基, 当CH_3OD/H_2比值足够大时主要捕获到CH_2DCOOCH_3, 说明乙酰基主要由乙烯酮的部分氢化反应生成。原位氧同位素交换反应检测到含~(18)O的乙醇、乙醛、乙酸的生成, 表明乙烯酮等C_2-含氧化合物前驱怵与重氧水发生了氧同位素交换反应。籍此, 无须如Katzer等人那样假设乙烯酮互变异构为位能较商的环氧乙烯而后进行氧同位素交换, 就可以得到Katzer等人在~(13)C~(16)O/~(13)C~(18)O+H_2反应中观察到的产物乙醇的同位素组成结果。本文的实验结果进一步说明我们提出的“CO缔合—卡宾—乙烯酮—乙酰基—乙醇(醛)”机理是合理的。     

EFFECTS OF HEMATOPORPHYRIN DERIVATIVE ON THE CELLULAR ENERGY METABOLISM IN THE ABSENCE AND PRESENCE OF LIGHT

Effects of Photofrin II on energy metabolism and metabolic viability were studied in a mammalian transformed cell line (BHK-21) in dark and after photo-irradiation with visible light. Cells were allowed to accumulate Photofrin by incubating for 4 h in buffer containing Photofrin (5-60 micrograms/ml). The results show that Photofrin significantly affects the cellular energy metabolism even in the absence of light; activity of cytochrome c oxidase is decreased and glucose utilization and lactate production (glycolysis) are increased. Irradiation with light resulted in a significant decrease in the activity of cytochrome c oxidase, glycolysis, ATP content, energy charge, ratios of adenine nucleotides like ATP/ADP, ATP/AMP and cell viability (dye exclusion test). Presence of inhibitors of energy metabolism, potassium cyanide (respiration) and 2-deoxyglucose (glycolysis), further enhanced the cytotoxic effects induced by hematoporphyrin derivative and light.     

TRANSFER OF ENERGY FROM REACTION CENTER TO LIGHT HARVESTING CHLOROPHYLL IN A PHOTOSYNTHETIC BACTERIUM*

Abstract— Previous evidence indicates that energy transfer in photosynthetic bacteria can occur from reaction center to light harvesting chlorophyll (the reverse of the usually considered flow) and that the amount of this flow depends on the strain of bacteria. The present report demonstrates that the action spectrum for fluorescence of Rhodopseudomonas spheroides, strain R26, is changed by adding the strong reductant dithionite. This change indicates that the amount of reverse flow can be altered chemically. The amount of reverse flow inferred from these measurements is consistent with the amount predicted from the absorption and fluorescence spectra of chromatophores and isolated reaction centers, and from the relative fluorescence yields of these two. The measurements permit an estimate of the transfer rates describing the energy flow from light harvesting to reaction center chlorophyll as well as the reverse flow. The spectrum for delayed fluorescence of Rps. spheroides, strain Ga, was found to be similar to that of the variable part of the fluorescence. This is a necessary, but not sufficient, condition that the energy for delayed fluorescence originates in the reaction centers.