Effect of quinones on the photoelectric properties of chlorophylla-containing lipid bilayers

Summary A Mueller-Rudin lipid bilayer formed from egg lecithin, cholesteroln-decane, and chlorophylla when separating asymmetric solutions of oxidant and reductant is capable of developing both a photoconductance and a photovoltage when exposed to continuous light focused on the bilayer region. The incorporation of plastoquinones (PQ-5 or PQ-9) increases the dark and photoconductances up to twenty times. Other quinones and hydroquinones also increased the conductance. The role of pH gradients on the electrical conductivity is discussed in terms of proton and electron transfer across the membrane solution interfaces.     

Pigment containing lipid vesicles

Summary Vesicles obtained by sonication of chlorophylla-lecithin mixtures dispersed in an aqueous medium closely resemble the well-characterized vesicles similarly prepared from pure lipids. They are bounded by one spherical lipid bilayer which contains the chlorophylla. Appropriate conditions for sonication prevent substantial degradation of the membrane constituents. Up to one chlorophylla molecule per 55 lecithins can be incorporated into the membranes. The average Stokes' radius of the vesicles determined by analytical sieve chromatography is 102±5 Å and independent of the chlorophylla content. The membrane is visible in the electron-microscope when the vesicles are treated with osmium tetroxide prior to negative staining. The osmium fixation is, however, not strong enough to allow for a preparation of the vesicles for thin sectioning (dehydration, embedding in epoxide).     

Low levels of irradiation modify lipid domains in model membranes: a laser Raman study

We have used Raman spectroscopy to study the effects of ionizing radiation on thermal transitions of dipalmitoyl lecithin + polyunsaturated fatty acid liposomes. Raman spectra in the CH (2800-3000 cm-1), C = C (1600-1680 cm-1), and C-C (1000-1150 cm-1) stretching regions are sensitive to ionizing radiation. The CH stretching of acyl chains yields three strong bands around 2850, 2880, and 2930 cm-1. The ratios of the relative intensities of 2880 and 2850 cm-1 bands, i.e., I2880/2850, when plotted against temperature show multiple infection points which correspond to multiple spectroscopic transitions. These are ascribed to a separate phase with distinctive proportions of lecithin and polyunsaturated fatty acids. We find these transitions sensitive to low levels of ionizing radiation. Doses as low as 5-15 rad after 48 h of 60Co gamma irradiation and 60 kVp X irradiation drastically broaden and shift the polyunsaturated rich phase which occurs at lower temperatures (-7 to +5 degrees C) than that of pure dipalmitoyl lecithin (39 degrees C). In addition a new transition around 46 degrees C also emerges upon irradiation (48 h postirradiation). These irradiation effects can be accelerated by the presence of catalytic amounts of Fe2+/EDTA +H2O2. The membrane transition modification is more sensitive to 60 kVp X rays in comparison to 60Co gamma rays owing to the high LET component of the former. The intensity of 1660 cm-1 band, assigned to C = C stretching in the cis-configuration, loses intensity upon irradiation. Concomitantly, a new band around 1675 cm-1, assigned to trans-configuration, emerges. Similarly the increase in the "order parameter" as calculated from the relative intensities of C--C stretching bands indicates rigidification of membrane. Various factors such as reduction in unsaturation, increase in trans-configuration, and the formation of multiple peroxidation products are invoked as lipid phase modifiers.     

A low temperature investigation of the intermediates of the photocycle of light-adapted bacteriorhodopsin. Optical absorption and fluorescence measurements.

Optical absorption and emission measurements have been made on samples of light-adapted purple membrane of Halobacterium halobium at temperatures ranging from 77 K to room temperature. As a result of these experiments a set of equations is given which described thermal and photochemical reactions interrelating various intermediates of the reaction cycle of the chromophore of light-adapted bacteriorhodopsin (BR). Further some specific problems connected to these intermediates have been investigated. Thus the room temperature emission spectrum of bacteriorhodopsin has been found to exhibit a Stokes shift of 3430 cm-1 only, if low excitation intensities are used. The recently detected intermiediate P-BR can be shown to convert thermally into bacteriorhodopsin following a first-order decay with the activation energy delta E = 2.4 +/- 0.2 kcal/mol. The thermal decay of K-BR consists of two exponentials if measured on purple membrane suspensions in a mixture of H2O and glycerol (1 : 1, v/v). A simple procedure is given for trapping the intermediate L-BR at 170 K in a very pure form. M-BR is shown to consist of two species, MI-BR and MII-BR. They are characterized by similar optical absorption spectra but different thermal stability. Further the oscillator strengths corresponding to the long wavelength absorption bands of the intermediates bacteriorhodopsin, K-, L, MI- and MII-BR have been calculated. They have been discussed with respect to the question which of the corresponding absorption spectra show the characteristics of isomerism of the chromophore or simply solvatochromism.     

Enhancement factor for oxygen absorption into fermentation broth

Values of the enhancement factor for oxygen absorption into fermentation broth, i.e., the ratio of the liquid phase mass transfer coefficients for oxygen absorption for both cases with and without respiration of microorganisms were predicted theoretically on the assumption of various cell concentration distributions. Calculations indicate that in the usual case the enhancement factor is only slightly or negligibly larger than unity, even when accumulation of microorganisms at or near the gas-liquid interface is assumed. Results of experiments with sparged-stirred fermentors on oxygen absorption into fermentation broths containing resting and growing cells of Candida tropicalis confirmed the theoretical prediction. Except for extreme cases, the effect of respiration of microorganisms on k_La, values can practically be ignored.     

Short-term variability of chlorophyll a concentrations in Lake Ontario

Research on aquatic ecosystems has shown the presence of diel (24 hour) variations of chlorophylla. In order to investigate this phenomenon in the Great Lakes, an IFYGL program was set up in which 8 cruises were made roughly monthly between April and October 1972. Two Lake Ontario Stations were chosen, one inshore near Oshawa, Ontario and the other mid-lake. Chlorophylla samples were taken approximately every two hours at depths of 1, 5 and 1o m at the inshore station and 1, 10 and 20 m at the mid-lake station.Chlorophylla concentrations at the 1 m depth were reduced during periods of high light intensities, but this phenomenon was observed only during the late June and July cruises at the near-shore station. Apparently, during most cruises, light intensities were too low to produce this effect. Coefficients of variation of chlorophylla concentrations as great as too% occurred at the deeper sampling depth during periods when the thermocline was well developed. This variability was associated with a thermocline motion in which the fixed depths sampled moved from the epilimnion into the thermocline or the hypolimnion and back again over the sampling period. When the lake was not thermally stratified, variability of chlorophylla concentrations was also observed with coefficients of variations between 20–3o%. Horizontal advection by currents, vertical turbulence, and presence of Langmuir cells were possible mechanisms causing such chlorophylla variability.In light of these observations, caution must be used in interpreting spatial and temporal distributions of chlorophylla in lakes. Special care must be taken when sampling at depths near the thermocline due to movements that take place. For this reason, water samples taken from the epilimnion during periods of thermal stratification are recommended as opposed to fixed depth or integrated samples which could be affected by thermocline movements.This study was undertaken as part of the International Field Year for the Great Lakes, a joint Canadian-U.S. contribution to the International Hydrological Decade Program     

Redshift of the purple membrane absorption band and the deprotonation of tyrosine residues at high pH: Origin of the parallel photocycles of trans-bacteriorhodopsin

At high pH (> 8) the 570 nm absorption band of all-trans bacteriorhodopsin (bR) in purple membrane undergoes a small (1.5 nm) shift to longer wavelengths, which causes a maximal increase in absorption at 615 nm. The pK of the shift is 9.0 in the presence of 167 mM KCl, and its intrinsic pK is ~8.3. The red shift of the trans-bR absorption spectrum correlates with the appearance of the fast component in the light-induced L to M transition, and absorption increases at 238 and 297 nm which are apparently caused by the deprotonation of a tyrosine residue and red shift of the absorption of tryptophan residues. This suggests that the deprotonation of a tyrosine residue with an exceptionally low pK (pK_a ≈ 8.3) is responsible for the absorption shift of the chromophore band and fast M formation. The pH and salt dependent equilibrium between the two forms of bR, “neutral” and “alkaline,” bR ↔ bR_a, results in two parallel photocycles of trans-bR at high pH, differing in the rate of the L to M transition. In the pH range 10-11.8 deprotonation of two more tyrosine residues is observed with pK's ~ 10.3 and 11.3 (in 167 mM KCL). Two simple models discussing the role of the pH induced tyrosine deprotonation in the photocycle and proton pumping are presented.

It is suggested that the shifts of the absorption bands at high pH are due to the appearance of a negatively charged group inside the protein (tyrosinate) which causes electrochromic shifts of the chromophore and protein absorption bands due to the interaction with the dipole moments in the ground and excited states of bR (Stark effect). This effect gives evidence for a significant change in the dipole moment of the chromophore of bR upon excitation.

Under illumination alkaline bR forms, besides the usual photocycle intermediates, a long-lived species with absorption maximum at 500 nm (P500). P500 slowly converts into bR_a in the dark. Upon illumination P500 is transformed into an intermediate having an absorption maximum at 380 nm (P380). P380 can be reconverted to P500 by blue light illumination or by incubation in the dark.

     

Conformational studies of proteins with aromatic side-chain effects

We present here a brief analysis of ultraviolet isotropic absorption and related circular dichroism of the n–π* and π–π* transitions for the peptide (amide) chromophore in the 185–240 mμ region. Investigations by ultraviolet absorption and circular dichroism techniques on natural amino acids with aromatic chromophores in their side chains are also reported. By taking into account both the peptide and aromatic transitions we discuss the conformational studies of proteins with aromatic side-chain effects. Our attention is largely focused on the optical rotatory dispersion and circular dichroism spectra of these proteins in the near ultraviolet region, where characteristic aromatic side-chain bands occur. The 185–240 mμ region is also discussed when evidence exists of overlapping Cotton effects of aromatic and peptide groups.     

Spectral transitions in purple membranes fromHalobacterium halobium. I. Effect of preliminary illumination on photochemical processes

The effect of preliminary illumination of purple membranes by yellow light on the difference spectra of short-lived intermediates has been studied. It has been found that changes of the optical density of two of these intermediates, which have the maxima of the difference spectrum at 412 nm and 650 nm, coincides well with the kinetics of the known reversible transitions of the main band of the purple membrane absorption (560 570), i.e., 13-cis-trans transitions. The changes at 412 nm and 650 nm are proportional to the concentration changes of the all-trans retinal and 13-cis retinal, respectively. It was concluded that the formation of the short-lived 412 and 650 intermediates occurs in different photochemical cycles. The pH is found to affect the formation of the 650 intermediate.     

Some properties of chlorophylla at hydrocarbon-water interfaces and in black lipid membranes

Summary Chlorophylla is very surface active in the system 2,2,4-trimethylpentanewater. The standard free energy of adsorption may be as high as 10.6 kcal/mole. However, chlorophyll adsorption at this interface is unable to stabilize black membranes. Black films formed from solutions of glyceryl monooleate and chlorophylla exhibit a weak fluorescence which indicates that a small amount of pigment, ca. 1 to 2% by area, may be contained in the membranes. Calculations based on adsorption data show that inclusion of somewhat more chlorophylla than this might be expected. However, interfacial tension data for mixed solutions do not support this expectation.Whether or not they are illuminated, black lipid membranes formed from mixed solutions of chlorophylla and glyceryl monooleate have electrical properties indistinguishable from those of films made in the absence of pigment.     

Studies on the absorption spectra of chlorophyll a in aqueous dispersions of lipids from the photosynthetic membranes

The absorption spectra of chlorophyll a were studied in aqueousdispersions of four major lipid components present in the thylakoidmembranes. Chlorophyll a in aqueous dispersions of uncharged galactolipidsrevealed two absorption bands, at 670 and 745 nm, when the molecularratio of chlorophyll to lipid was higher than 0.2. The latterband may be due to the formation of microcrystals of chlorophylla. Chlorophyll a in aqueous dispersions of negatively chargedlipids revealed a single absorption band at 670 nm. However,chlorophyll a was decomposed during measurement in these lipiddispersions. The absorption spectra of chlorophyll a in aqueous dispersionsof mixture of galactolipid and charged lipid were apparentlysimilar to those of chlorophyll a in the charged lipid dispersion.Chlorophyll a, however, was not decomposed in these aqueousdispersions of lipid mixtures. It is concluded that the presence of both galactolipid and chargedlipid are necessary to reconstruct the state of chlorophylla dissolved in the lipid phase in the thylakoid membranes. The red absorption band of chlorophyll a in the reconstructedsystem composed of chlorophyll a, charged and uncharged lipids,appeared at 670 nm with a half bandwidth of 22 nm. Analysisof the absorption spectrum in the fourth derivative and thecurve-fitting methods indicated that the red band was composedmainly of a single band with a peak at 670–671 nm. ¹ Present address: Department of Biology, College of GeneralEducation, University of Tokyo, Komaba, Meguro-ku, Tokyo 153,Japan. (Received October 13, 1977; )     

Chiroptical properties of lecithin reverse micelles and organogels

The ultraviolet absorbance and circular dichroism (CD) spectra of lecithin reverse micelles and gels were investigated in order to establish whether the formation of these noncovalent macromolecular aggregates, which was induced by the addition of water to solutions of lecithin in organic solvents, was accompanied by specific spectroscopic changes. Systems containing the synthetic short-chain lecithins, 1,2-hexanoyl-, 1,2-diheptanoyl-, 1,2-dioctanoyl-, and 1,2-dinonaoyl-sn-glycero-3-phosphatidylcholines were used as models for the long-chain lecithins, soybean phosphatidylcholine and palmitoyl-oleoyl-phosphatidylcholine. All the molecules studied had asymmetric centres, formed reverse micelles under appropriate conditions, and, while both the long-chain lecithins also formed gels, none of the short-chain molecules did. As well as having CD spectra that were simpler to interpret, spectroscopic observations on solutions of the short-chain lecithins could be carried out over a large water content range. The ester chromophore of these compounds was shown to be highly sensitive to variation in both the solvent environment and the temperature, and components of both direct solvent effects and conformational change upon the addition of water were detected in the spectra. The spectra of the longer chain lecithins were complicated by the presence of double bonds although, here again, it was found that significant changes occurred as the water content increased, as monitored by the ester chromophore. However, no specific effect that could be ascribed to gelation alone was detected. The overall picture that emerged was that the ester chromophore of anhydrous micelles gave rise to a specific negative band in the CD spectrum (λ_max ≈ 210 nm) whereas a positive CD signal (λ_max ≈ 233 nm) was associated with the same chromophore in filled (i.e., hydrated) micelles. The two signals correspond to two different conformational states of the lecithin molecule, the hydrated state being not only more conformationally restricted but also providing a less polar environment for the ester groups, while the addition of water to the system shifts the conformational equilibrium. These observations have been interpreted as showing that only a limited range of lecithin conformation is compatible with the formation of the micellar structure and that it is this constraint, together with those introduced by the overall geometry of the aggregated state, that gives rise to the changes observed in the CD spectrum.     

Extraction of chlorophyll and carotenoid pigments from Antarctic benthic mats for analysis by HPLC

The efficiency of 9:1 acetone-water, DMSO and boiling 9:1 ethanol-water in extracting chlorophyll and carotenoid pigments from benthic cyanobacterial mats from Antarctica for HPLC (high performance liquid chromatography) analysis was examined. Considerable breakdown of chlorophylla was observed after 5 min extraction in boiling ethanol and 2 h extraction in DMSO. Over 50% of the chlorophylla was degraded to chlorophyllidea and there was substantial loss of carotenoids after a 15 h exposure of ground cells to cold 9:1 acetone-water.Mild sonication of ground mat material in 9:1 acetone-water followed by a 4 h extraction at 4 ° C was found to minimise chlorophylla breakdown and dramatically improved the extraction efficiency of chlorophylla, myxoxanthophyll and -carotene.     

A Monte Carlo–quantum mechanics study of a solvatochromic π* probe

The solvation and the solvatochromic behavior of 5-(dimethylamino)-5′-nitro-2,2′-bithiophene 1, the basis of a π* scale of solvent polarities, was investigated theoretically in toluene, dichloromethane, methanol and formamide with a Monte Carlo and quantum mechanics (QM/MM) iterative approach. The calculated transition energies of the solvatochromic band of 1, obtained as averages of statistically uncorrelated configurations, including the solute and explicit solvent molecules of the first solvation layer, besides showing good agreement with the experimental transitions, reproduced very well the positive solvatochromism of this probe in various solvents.     

Effect of ammonium load on the growth of attached microalgae in the northern Baltic Sea

The effect of ammonium discharge from a food factory on the growth of attached microalgae was monitored north of the Hanko peninsula, on the southwestern coast of Finland. The impact of the discharge was studied at twelve localities, at four stages of seasonal succession. The microalgae were sampled from glass slides exposed at 0.4 m depth for two weeks. The variables measured for the microalgal growth were chlorophylla, primary production and total organic carbon (TOC). These were compared with planktonic chlorophylla and nutrient concentrations. The growth of attached microalgae displayed a consistent pattern of spatial distribution. Depending on season, TOC and primary production values were 7 to 70 times higher and chlorophylla values up to 1000 times higher close to the effluent outlet than in undisturbed areas of the archipelago. The microalgal samples near the discharge were characterized by low TOC/chlorophylla and TOC/primary production ratios. The temporal consistency of microalgal distribution illustrates the advantages of using attached algal assemblages in monitoring programmes.     

Pigment organization and energy transfer in the green photosynthetic bacterium Chloroflexus aurantiacus

The transfer of excitation energy and the pigment arrangement in isolated chlorosomes of the thermophilic green bacterium Chloroflexus aurantiacus were studied by means of absorption, fluorescence and linear dichroism spectroscopy, both at room temperature and at 4 K. The low temperature absorption spectrum shows bands of the main antenna pigments BChl c and carotenoid, in addition to which bands of BChl a are present at 798 and 613 nm. Fluorescence measurements showed that excitation energy from BChl c and carotenoid is transferred to BChl a, which presumably functions as an intermediate in energy transfer from the chlorosome to the cytoplasmic membrane. Measurements of fluorescence polarization and the use of two different orientation techniques for linear dichroism experiments enabled us to determine the orientation of several transition dipole moments with respect to each other and to the three principal axes of the chlorosome. The Q_y transition of BChl a is oriented almost perfectly perpendicular to the long axis of the chlorosome. The Q_y transition of BChl c and the -carotene transition dipole are almost parallel to each other. They make an angle of about 40° with the long axis and of about 70° with the short axis of the chlorosome; the angle between these transitions and the BChl a Q_y transition is close to the magic angle (55°).Abbreviations BChl bacteriochlorophyll - CD circular dichroism - LD linear dichroism Dedicated to Prof. L.N.M. Duysens on the occasion of his retirement.     

A solvent partitioning procedure for the separation of chlorophylls from their degradation products and carotenoid pigments

A simple liquid/liquid partitioning procedure was developed which employed aqueous acetonitrile and hexane, for the isolation of chlorophyll and pheophytin. This procedure separated these pigments from other interfering pigmented compounds. The efficacy of this solvent separation method was evaluated using commercially available chlorophylla, b, pheophytina, b, carotenoids, and algal pigment extracts. The recovery efficiencies of this solvent partitioning process for chlorophyll a and pheophytina have been shown to be 95–98% and 93–96%, respectively, furthermore, the chlorophylla fraction was practically free of any contaminating pigments. It appears that a more accurate assessment of chlorophylla and pheophytina can be accomplished employing liquid/liquid partitioning than with the present standard method.     

Composition and optical properties of reaction centre core complexes from the green sulfur bacteria Prosthecochloris aestuarii and Chlorobium tepidum

Photosynthetically active reaction centre core (RCC) complexes were isolated from two species of green sulfur bacteria, Prosthecochloris (Ptc.) aestuarii strain 2K and Chlorobium (Chl.) tepidum, using the same isolation procedure. Both complexes contained the main reaction centre protein PscA and the iron–sulfur protein PscB, but were devoid of Fenna–Matthews–Olson (FMO) protein. The Chl. tepidum RCC preparation contained in addition PscC (cytochrome c). In order to allow accurate determination of the pigment content of the RCC complexes, the extinction coefficients of bacteriochlorophyll (BChl) a in several solvents were redetermined with high precision. They varied between 54.8 mM⁻¹ cm⁻¹ for methanol and 97.0 mM⁻¹ cm⁻¹ for diethylether in the Q_Y maximum. Both preparations appeared to contain 16 BChls a of which two are probably the 13²-epimers, 4 chlorophylls (Chls) a 670 and 2 carotenoids per RCC. The latter were of at least two different types. Quinones were virtually absent. The absorption spectra were similar for the two species, but not identical. Eight bands were present at 6 K in the BChl a Q_Y region, with positions varying from 777 to 837 nm. The linear dichroism spectra showed that the orientation of the BChl a Q_Y transitions is roughly parallel to the membrane plane; most nearly parallel were transitions at 800 and 806 nm. For both species, the circular dichroism spectra were dominated by a strong band at 807–809 nm, indicating strong interactions between at least some of the BChls. The absorption, CD and LD spectra of the four Chls a 670 were virtually identical for both RCC complexes, indicating that their binding sites are highly conserved and that they are an essential part of the RCC complexes, possibly as components of the electron transfer chain. Low temperature absorption spectroscopy indicated that typical FMO–RCC complexes of Ptc. aestuarii and Chl. tepidum contain two FMO trimers per reaction centre. This revised version was published online in June 2006 with corrections to the Cover Date.     

Photophysics of the carotenoids associated with the xanthophyll cycle in photosynthesis

Green plants use the xanthophyll cycle to regulate the flow of energy to chlorophylla within photosynthetic proteins. Under conditions of low light intensity violaxanthin, a carotenoid possessing nine conjugated double bonds, functions as an antenna pigment by transferring energy from its lowest excited singlet state to that of chlorophylla within light-harvesting proteins. When the light intensity increases, violaxanthin is biochemically transformed into zeaxanthin, a carotenoid that possesses eleven conjugated double bonds. The results presented here show that extension of the conjugation of the polyene lowers the energy of the lowest excited singlet state of the carotenoid below that of chlorophylla. As a consequence zeaxanthin can act as a trap for the excess excitation energy on chlorophylla pigments within the protein, thus regulating the flow of energy within photosynthetic light-harvesting proteins.     

Development of a push–π–pull phenothiazine-vinyl-isophorone fluorophore: a novel solvatochromic and pH indicator

Knoevenagel condensation of phenothiazine-3,7-dicarbaldehyde with an isophorone yielded a new phenothiazine derivative ( PTZ-c ) fluorophore. The solvatochromic and pH-sensing abilities of PTZ-c , an asymmetric fluorophore with a single isophorone molecule, were shown to be exceptional. PTZ-c produced very delicate absorbance and emission spectra. When the polarity of the solvent was increased, the PTZ-c emission spectra showed greater sensitivity than the absorption spectra. Multiple spectroscopic techniques, including Fourier transform infrared spectroscopy, nuclear magnetic resonance, and mass spectrometry, were used to characterize the manufactured PTZ-c sensor. To demonstrate the beneficial solvatochromic behaviour associated with intramolecular charge transfer, the absorption spectra of the synthesized DA PTZ-c dye were analyzed in different solvents of varying polarity. Band intensity and the wavelength of PTZ-c emission were also found to be highly solvent dependent. It was observed that when solvent polarity was increased to a maximum of 4122 cm⁻¹, Stokes’ shift also increased. To analyze the Stokes' shift that depended on the solvent, a linear correlation between solvation and energy was used. An investigation of PTZ-c quantum yield (ф) was also conducted. Both the absorbance and fluorescence spectra of the sensor in dimethylformamide as a function of pH were studied. A fluorescence peak was seen at 562 nm, whereas the greatest absorption wavelengths were found at 403 and 317 nm. It was shown that the pH-sensing mechanism depended on protons removed from the PTZ-c chromophore, which caused a colour shift and variation in both emission and colorimetric properties.