Photoreceptor pigments for photomovement of microorganisms: some spectroscopic and related studies

Optical spectroscopy of photoreceptor pigments can substantially contribute to our understanding of the molecular processes which are the basis of photoreception and sensory transduction in photomotile microorganisms. The main spectroscopic techniques are briefly illustrated, together with the most significant types of progress that can be achieved. A few "case examples" are discussed in some detail: Halobacterium, with particular attention to the contribution of flash photolysis studies to the identification and characterization of sensory rhodopsins; Euglena, and the role of in vivo microspectrofluorometry in confirming the flavin nature of its photoreceptor pigment; the first suggestions on the rhodopsin-like nature of the Chlamydomonas photosensing system; Stentor and Blepharisma and the contribution of static and time-resolved fluorescence studies to a molecular model of the primary events in their photoreceptor pigments (stentorin and blepharismin) and systems.     

SPECTROSCOPIC AND PHOTOACOUSTIC STUDIES OF HYPERICIN EMBEDDED IN LIPOSOMES AS A PHOTORECEPTOR MODEL*

Abstract— In photoresponsive ciliates, like Blepharisma japonicum and Stentor coeruleus, the photoreceptor pigments responsible for photomotile reactions are hypericin-type chromophores packed in highly osmiophilic subpellicular granules. Liposomes loaded with hypericin can constitute a simple model system, appropriate for understanding the primary light-induced molecular events triggering the sensory chain in these microorganisms. Optical absorption, steady-state and time-resolved fluorescence and pulsed photoacoustic calorimetry have been used to measure spectral distributions, fluorescence lifetimes, radiative and radiationless transition quantum yields of hypericin when assembled into egg L-a-phosphatidylcholine liposomes. With respect to hypericin ethanol solutions, both absorption and fluorescence maxima are 5 nm red shifted when the pigment is inserted into the lipidic microenvironment, regardless of the hypericin local concentration. Increasing by 100 times the hypericin local concentration decreases the relative fluorescence quantum yield by a factor of around 150 and the fraction of thermally released energy, conversely, increases from 0.6 to 0.9. From the analysis of fluorescence lifetimes and their relative amplitudes it appears that a subnanosecond living component is predominant at the highest hypericin local concentrations.     

In vivo absorption spectra of the two stable states of the Euglena photoreceptor photocycle

Euglena gracilis possesses a simple but sophisticated light detecting system, consisting of an eyespot formed by carotenoids globules and a photoreceptor. The photoreceptor of Euglena is characterized by optical bistability, with two stable states. In order to provide important and discriminating information on the series of structural changes that Euglena photoreceptive protein(s) undergoes inside the photoreceptor in response to light, we measured the in vivo absorption spectra of the two stable states A and B of photoreceptor photocycle. Data were collected using two different devices, i.e. a microspectrophotometer and a digital microscope. Our results show that the photocycle and the absorption spectra of the photoreceptor possess strong spectroscopic similarities with a rhodopsin-like protein. Moreover, the analysis of the absorption spectra of the two stable states of the photoreceptor and the absorption spectrum of the eyespot suggests an intriguing hypothesis for the orientation of microalgae toward light.     

CAFFEINE-ENHANCED PHOTOMOVEMENT IN THE CILIATE, STENTOR COERULEUS

The effects of caffeine, ionophores and calcium flux blockers on the step-up photophobic response, phototactic orientation and the intracellularly recorded, light-induced electrical action potential were studied in the ciliate, Stentor coeruleus . Caffeine alters the absorption and CD spectra and enhances the fluorescence of the photoreceptor pigment, stentorin. Independent of its effects on the spectroscopic properties of the photoreceptor pigment, caffeine shortens the photophobic response time by enhancing the Ca²⁺ conductivity of membranes, while Ca²⁺ flux blockers (LaCI₃ or ruthenium red) prolong it; both effects cancel each other. Evidence is presented that phototactic orientation is brought about by repetitive photophobic responses, since a change in the phobic response time results in a decreased accuracy of phototaxis.     

EFFECTS OF PIGMENT HETEROGENEITY ON FLUORESCENCE IN PHOTOSYNTHETIC UNITS

A computer solution of the Pauli Master Equation is used to study the fluorescence quantum yield of a two-dimensional random host-trap-anti-trap system. Calculations predict that both the quantum yield and prompt fluorescence lifetime from photosynthetic units increase as pigment heterogeneity increases. From the temperature dependent action spectrum of fluorescence the pairwise Forster transfer rate is predicted to decrease with decreasing temperature.     

TIME-GATED FLUORESCENCE OF BLEPHARISMIN, THE PHOTORECEPTOR PIGMENT FOR PHOTOMOVEMENT OF Blepharisma

Abstract— A computer-controlled apparatus for time-resolved laser fluorescence spectroscopy has been used to measure fluorescence lifetimes, time-integrated and time-gated spectra of crude extracts of blepharismin, the photoreceptor pigment of the ciliated photoresponsive protozoan Blepharisma japonicum , in ethanol, aqueous solutions and detergent micelles. The effect of hydroxyl concentration has been investigated in both alcohol and water solutions. A short-living (0.2-0.4 ns) molecular species, emitting at 600 nm, is predominant in aqueous solutions at pH < 11.7, whereas in pure ethanol solutions an intermediate-living species (about 1 ns), still fluorescing at 600 nm, prevails. Upon increasing OFF concentration, a third, long-living (about4–6 ns) molecular species, emitting at 660 nm, is formed in all the examined media. This species has been tentatively identified as the negatively charged form of the photoreceptor pigment, whereas the short-living and the intermediate-living fluorescence emissions have been attributed respectively to the phenolic and the quinonic neutral forms of blepharismin. The phenolic form in its ground state is suggested to be the molecular species from which proton release occurs.     

THE USE OF A COMPUTERIZED SPECTRORADIOMETER TO PREDICT PHYTOCHROME PHOTOEQUILIBRIA UNDER POLYCHROMATIC IRRADIATION

Abstract A method is described for predicting the effect of polychromatic irradiation upon the photo-stationary equilibrium of the plant photoreceptor phytochrome. This method follows from the rate equations for phototransformation and utilizes the in vivo action spectra for phytochrome phototransformation (Pratt and Briggs, 1966). A scanning spectroradiometer interfaced with a microcomputer is used to determine a spectral photon distribution from 360 to 800 nm. The products of the photon fluence rate and the relative quantum efficiencies at 2-nm intervals are summed over the entire visible range to yield a predicted percentage of the pigment in the Pfr form. This value was determined under eight different polychromatic light sources and was generally within 7% Pfr of the value measured in etiolated corn coleoptiles under the same light sources.     

A theoretical and experimental study of two thiazole orange derivatives with single- and double-stranded oligonucleotides, polydeoxyribonucleotides and DNA

The effect of interaction with DNA and oligonucleotides on the photophysical properties of two thiazole orange (TO) derivatives, with different side chains (-(CH2)3-N+(CH3)3 and -(CH2)6-I)) linked to the nitrogen of the quinoline ring of the thiazole orange, is presented here. The first one called TO-PRO1 is a commercially available dye, whereas the second one called TO-MET has been specially synthesized for further covalent binding to oligonucleotides with the aim of being used for specific in situ detection of biomolecular interactions. Both photophysical measurements and molecular calculations have been done to assess their possible mode of interaction with DNA. When dissolved in buffered aqueous solutions both derivatives exhibit very low fluorescence quantum yields of 8 x 10(-5) and 2 x 10(-4), respectively. However, upon binding to double-stranded DNA, large spectroscopic changes result and the quantum yield of fluorescence is enhanced by four orders of magnitude, reaching values up to phi F = 0.2 and 0.3, respectively, as a result of an intercalation mechanism between DNA base pairs. A modulation of the quantum yield is observed as a function of the base sequence. The two derivatives also bind with single-stranded oligonucleotides, but the fluorescence quantum yield is not so great as that when bound to double-stranded samples. Typical fluorescence quantum yields of 7 x 10(-3) to 3 x 10(-2) are observed when the dyes interact with short oligonucleotides, whereas the fluorescence quantum yield remains below 10(-2) when interacting with single-stranded oligonucleotides. This slight but significant quantum-yield increase is interpreted as a folding of the single strand around the dye, which reduces the internal rotation of the two heterocycles around the central methine bridge that links the two moieties of the dye. From these properties, it is proposed to link monomer covalently to oligonucleotides for the subsequent detection of target sequences within cells.     

Fluorescence and Time-Resolved Photoacoustics of Hypericin Inserted in Liposomes: Dependence on Pigment Concentration and Bilayer Phase

Abstract— Solvatochromic effects on the energy of visible absor-bance and fluorescence transitions in hypericin have been studied in organic solutions and in phosphatidylcholine liposomes, suggesting that the molecule in this last case is preferentially located in the polar aprotic zone close to the lipid-water interface. Nonradiative and radiative decays of the pigment have also been studied, combining photocalorimetric and fluorescence techniques, as a function of the pigment concentration in the liposomal phase and of the gel-to-sol thermotropic transition of the phos-pholipidic bilayer. The results show that hypericin can sense the phase transition by exhibiting a stepwise increasing of the fluorescence quantum yield; concomitantly photoacoustic data indicate that in the gel phase, above a certain concentration of hypericin, clustering of the pigment promotes the formation of nonradiative long-lived species, whereas when the bilayer is in the sol phase ultrafast nonradiative pathways become the main deactivation channels upon increasing concentration. As a consequence, as shown by photoacoustic results, the photosensitized formation of singlet oxygen is prevented when the local concentration of hypericin in the lipidic phase is greater than about 30 m M .     

Excited State Photophysics of Curcumin and its Modulation in Alkaline Non-Aqueous Medium

Curcumin, a well-known medicinal pigment, has seen limited applications in biology despite having great potential as a therapeutic drug. Deprotonation is one of the possible ways to enhance solubility of curcumin in polar solvent. Here, we have explored the effect of deprotonation on the ultrafast dynamics of this biomolecule with the help of the time-resolved fluorescence spectroscopic measurements using the femtosecond fluorescence upconversion technique. The excited state photophysics of fully deprotonated curcumin significantly differs from that of neutral curcumin. We have observed that the completely deprotonated curcumin not only has higher quantum yield, but also higher excited state lifetime and slower solvation dynamics in comparison to neutral curcumin. We propose solvation dynamics and intramolecular charge transfer as the excited state processes associated with the radiative decay of the completely deprotonated molecule, while ruling out the possibility of excited state proton exchange or proton transfer. Our results are well supported by time-dependent density-functional theory calculations. Lastly, we have also demonstrated the possibility of modulating the ultrafast dynamics of fully deprotonated curcumin using non-aqueous alkaline binary solvent mixtures. We believe our results will provide significant physical insight towards unveiling the excited state dynamics of this molecule.     

Alpha-retinals as rhodopsin chromophores--preference for the 9-Z configuration and partial agonist activity

The visual pigment rhodopsin, the photosensory element of the rod photoreceptor cell in the vertebrate retina, shows in combination with an endogenous ligand, 11-Z retinal, an astonishing photochemical performance. It exhibits an unprecedented quantum yield (0.67) in a highly defined and ultrafast photoisomerization process. This triggers the conformational changes leading to the active state Meta(rhodopsin) II. Retinal is covalently bound to Lys-296 of the protein opsin in a protonated Schiff base. The resulting positive charge delocalization over the terminal part of the polyene chain of retinal creates a conjugation defect that upon photoexcitation moves to the opposite end of the polyene. Shortening the polyene as in 4,5-dehydro,5,6-dihydro (alpha), 5,6-dihydro or 7,8-dihydro-analogs might facilitate photoisomerization of a 9-Z and a 11-Z bond. Here we describe pigment analogs generated with bovine opsin and 11-Z or 9-Z 4,5-dehydro,5,6-dihydro-retinal that were further characterized by UV-Vis and FTIR spectroscopy. The preference of opsin for native 11-Z retinal over the 9-Z isomer is reversed in 4,5-dehydro,5,6-dihydro-retinal. 9-Z 4,5-dehydro,5,6-dihydro-retinal readily generated a photosensitive pigment. This modification has no effect on the quantum yield, but affects the Batho<-->blueshifted intermediate (BSI) equilibrium and leads to a strong decrease in the G-protein activation rate because of a downshift of the pK(a) of the Meta I<-->Meta II equilibrium.     

Interactions between colloidal silver and photosynthetic pigments located in cyanobacteria fragments and in solution

Changes in the yield of the fluorescence emitted by pigments of photosynthetic organisms could be used for the establishment of the presence of some toxic substances. The presence of colloidal metals can be indicated by enhancement of pigments' emission as a result of plasmons generation. The spectra of the pigments of cyanobacterium Synechocystis located in the bacterium fragments and in solutions with and without colloidal silver additions have been measured. The quantum yield of the pigments' fluorescence in solution has been observed to increase at some wavelength of excitation, while the fluorescence of the pigments in the bacteria fragments has been only quenched as a consequence of interactions with colloidal silver particles. Close contact between pigment molecules located in bacteria fragments and silver particles is probably not possible. We plan in future to investigate the influence of other, more typical metal pollutants of water, using similar spectral methods and several other photosynthetic bacteria pigments, in solution, in cell fragments and in the whole bacteria organisms.     

Photophysics of benzazole derived push–pull butadienes: A highly sensitive fluorescence probes

Two new homologues of 1,4-diphenylbutadiene, namely, 1-(2-benzazolyl)-4-(p-dimethylaminophenyl)buta-1,3-diene have been synthesized and their absorption and fluorescence properties have been investigated in different organic solvents. The absorption spectra are less sensitive to the solvent polarity than the corresponding fluorescence spectra, which show dual emission and high solvatochromic effect in polar solvents. Using an efficient solvatochromic method, a large excited state dipole moment parallel to the smaller ground state dipole moment was calculated. Other properties of the lowest excited state such as the planar ICT B_u nature, fluorescence quantum yield and the basicity of the two nitrogen atoms (of the benzoxazole or benzothiazole ring as well as the amino group) were studied by spectroscopic techniques and semiempirical AM1 quantum chemical calculations. The findings have been presented and discussed along with the promising fluorescence probing and pH-sensing properties of these two dienes. The main spectroscopic properties of the two derivatives have been also compared.     

Photophysics and photochemistry of naphthoxazinone derivatives

The photophysics and photochemistry of a series of naphthoxazinones have been studied using a combination of methods ranging from steady-state and time-resolved spectroscopic techniques to product analysis. The photophysics of naphthoxazinone derivatives is very dependent on the structure: phenanthrene-like compounds exhibit higher fluorescence quantum yield than the less aromatic anthracene-like homologous. The latter, exhibit a substantial degree of charge transfer in the excited singlet state. These compounds are fairly photostable in the absence of additives, yielding a single photoproduct arising from the triplet state. The presence of electron donors such as amines increases the photoconsumption quantum yield and changes the product distribution, the primary photoproduct being a dihydronaphthoxazinone that photoreacts further yielding ultimately an oxazoline derivative.     

FLUORESCENCE PROPERTIES OF C-PHYCOCYANIN ISOLATED FROM A THERMOPHILIC CYANOBACTERIUM

Abstract The fluorescence lifetime of purified C-phycocyanin from the thermophilic cyanobacterium, Phormidium laminosum (strain OH-1-p. Cl 1), was measured as 1.48 ± 0.06 ns using the technique of time-correlated single-photon counting under very weak excitation pulses. The natural radiative lifetime (∼6.1 ns) of the pigment was calculated by integrating the absorption spectrum using the Strickler–Berg equation. From these two lifetimes we calculate a fluorescence quantum yield of ∼0.24 which is very close to the value ∼0.22 which we measure relative to the known value of cresyl violet in methanol. Both the fluorescence lifetime and the quantum yield of the pigment from this organism are lower than most previous values reported in the literature. We conclude that our lower values are not due to high light intensity, pH, buffer, concentration, instrumentation artifacts, aggregation effects or the thermophilic nature of the organism. Instead, we suggest that the photophysical properties of C-phycocyanin are species dependent, perhaps due to the specific molecular environment of the tetrapyrrole.     

Stimulated fluorescence and fluorescence quenching in chlorophyll a and bacteriochlorophyll a

This communication deals with the photophysical processes that take place in chlorophyll solutions under intense nitrogen laser irradiation. The effect of the pump photon density on the fluorescence yield depends strongly on the geometry of the irradiation and the sampling set-up. If the fluorescence cell and sampling probe are placed close to the transverse arrangement used for obtaining laser output, line narrowing and gain, which are processes associated with high population inversions and stimulated fluorescence, are observed. A normal fluorescence spectrum and a decrease in fluorescence quantum yield with increasing pump power are observed in the fluorescence cells in oriented at an angle of 20–40° with respect to the transverse axis of the exciting beam. The decrease in quantum yield appears to result from absorption of the pump photons by the excited singlet of the chlorophylls, and it is suggested that an analogous mechanism may be responsible for the anomalous fluorescence quantum yield reported for in in vivo Chlorella vulgaris algae.     

New photochromic diarylethenes bearing a pyridine moiety

A new class of diarylethenes bearing a pyridine unit has been firstly developed and their properties such as photochromism, fatigue resistance, and fluorescence have been discussed. The pyridine unit was connected directly to the central cyclopentene ring as one aryl moiety and availably participated in the photoinduced cyclization reaction even in the crystalline phase. All of these diarylethenes exhibited excellent photochromism, remarkable fatigue resistance, and notable fluorescence photoswitches both in solution and in poly(methylmethacrylate) films. Moreover, the different substituents had a significant effect on their properties. The results indicated that the electron-donating substituent could significantly enhance the cyclization quantum yield and depress the cycloreversion quantum yield while the electron-withdrawing group had a notable contribution to the cycloreversion quantum yield and fluorescence quantum yield for these diarylethenes.     

反丁烯二酰基桥连的带不同取代基的卟啉二联体的合成与性质

合成了反丁烯二酰基桥连的3种带不同取代基的卟啉二联体, 通过红外光谱, 紫外-可见光谱, 核磁共振波谱和质谱对化合物的结构进行了确认, 并研究了二联体的表面光电压谱, 荧光光谱和激光拉曼光谱的变化. 结果表明, 取代基的类型对卟啉二联体分子的荧光量子产率有显著影响, 带供电子基团的甲氧基增强了荧光量子产率, 而带吸电子基团的氯则降低了荧光量子产率, 并且吸电子基团的氯比供电子基团的甲氧基对荧光的影响更大. 取代基的电子效应对卟啉二联体的荧光性和激光拉曼光谱有较大影响.     

Fluorescence studies on photosynthetic pigment development in Rhodopseudomonas spheroides

Abstract— When bleached, aerobically grown cells of Rhodopseudomonas spheroides are transferred to semi-aerobic conditions to induce bacteriochlorophyll synthesis, a new fluorescence band, with a maximum at 790 nm, is observed in addition to the 885 nm emission maximum normally seen in pigmented cells. The 790 nm fluorescence may be due to bacterio-chlorophyll which has not been bound into the chromatophore membrane. The quantum yield of the 885 nm fluorescence is at first relatively high and then, about 1 hour after transfer, drops to the level found in pigmented photosynthetic cells. The coupling to the rest of the photo-synthetic apparatus, as indicated by the effect of dithionite on the fluorescence, also seems to occur during the first hour of pigment development, which suggests that the onset of fluorescence quenching is due at least in part to the synthesis of photochemical reaction centers. Continuation of these studies should provide new information on the formation, structure and molecular interactions of the pigments and the photosynthetic membranes.     

Maristentorin, a novel pigment from the positively phototactic marine ciliate Maristentor dinoferus, is structurally related to hypericin and stentorin

The photoreceptor pigment of the heterotrich ciliate, Maristentor dinoferus, has been characterized. It is structurally similar to those of Stentor coeruleus and Blepharisma japonicum but differs significantly in that it bears no aromatic hydrogens. The structure of the pigment, maristentorin, is based upon the hypericin skeleton, and its spectra are nearly identical to those of hypericin but shifted toward the red. Within experimental error, its fluorescence lifetime is identical to that of hypericin, approximately 5.5 ns in dimethylsulfoxide. It is remarkable that while the pigments are structurally similar in S. coeruleus and M. dinoferus, in the former there is an abrupt photophobic response, whereas in the latter there is a slow response toward light. The roles of the hypericin-like pigments in the heterotrich ciliates are discussed as potentially analogous in Maristentor.