Dipole-dipole transfer between acetone solvates of chlorophyll a and chlorophyll a dihydrate dimers in water/acetone mixtures. A model for P680 sensitized excitation

We describe a simple model for P680 sensitized excitation in photosynthesis. Chl a fluorescence quenching effects observed when water is added to Chl a solutions in acetone are shown to be the result of resonant transfer between acetone solvates of monomeric Chl a, Chl a·Ac, and dimers of Chl a dihydrate. The presence of (Chl a·2H₂O)₂ is evidenced by a 678 nm difference absorbance (ΔA band obtained on conversion of a 680 nm absorption shoulder to polycrystalline Chl a precipitate, (Chl a·H₂O)_n. The equilibration between (Chl a·2H₂O)₂ and Chl a·Ac as a principal mechanism for Chl a·Ac fluorescence quenching is supported by theoretical fits of the data.     

Chlorophyll a self-assembly in polar solvent-water mixtures

The conversion of chlorophyll a (Chl a) monomers into large aggregates in six polar solvents upon addition of water has been studied by means of absorption, fluorescence spectroscopy and fluorescence lifetime measurements for the purpose of elucidating the various environmental factors promoting Chl a self-assembly and determining the type of its organization. Two empirical solvent parameter scales were used for quantitative characterization of the different solvation properties of the solvents and their mixtures with water. The mole fractions of water f1/2 giving rise to the midpoint values of the relative fluorescence quantum yield were determined for each solvent, and then various solvent-water mixture parameters for the f1/2 values were compared. On the basis of their comparison, it is concluded that the hydrogen-bonding ability and the dipole-dipole interactions (function of the dielectric constant) of the solvent-water mixtures are those that promote Chl a self-assembly. The influence of the different nature of the non-aqueous solvents on the Chl aggregation is manifested by both the different water contents required to induce Chl monomer-->aggregate transition and the formation of two types of aggregates at the completion of the transition: species absorbing at 740-760 nm (in methanol, ethanol, acetonitrile, acetone) and at 667-670 nm (in pyridine and tetrahydrofuran). It is concluded that the type of Chl organization depends on the coordination ability and the polarizability (function of the index of refraction) of the organic solvent. The ordering of the solvents with respect to the f1/2 values--methanol < ethanol < acetonitrile < acetone < pyridine < tetrahydrofuran--yielded a typical lyotropic (Hofmeister) series. On the basis of this solvent ordering and the disparate effects of the two groups of solvents on the Chl a aggregate organization, it is pointed out that the mechanism of Chl a self-assembly in aqueous media can be considered a manifestation of the Hofmeister effect, as displayed in the lipid-phase behavior (Koynova et al., Eur. J. Biophys. 25, 261-274, 1997). It relates to the solvent ability to modify the bulk structure and to distribute unevenly between the Chl-water interface and bulk liquid.     

Examination of the photophysical processes of chlorophyll d leading to a clarification of proposed uphill energy transfer processes in cells of Acaryochloris marinas

A comprehensive study of the photophysical properties of chlorophyll (Chl) d in 1:40 acetonitrile-methanol solution is performed over the temperature range 170-295 K. From comparison of absorption and emission spectra, time-dependent density-functional calculations and homologies with those of Chl a, we assign the key features of the absorption and fluorescence spectra. Possible photophysical energy relaxation mechanisms are summarized, and thermal equilibration processes are studied in detail by monitoring the observed emission profiles and quantum yields as a function of excitation energy. In particular, we concentrate on emission subsequent to excitation in the extreme far-red tail of the Qy absorption spectrum, with this emission partitioned into contributions from hot-band absorptions as well as uphill energy transfer processes that occur subsequent to absorption. No unusual photophysical processes are detected for Chl d; it appears that all intramolecular relaxation processes reach thermal equilibration on shorter timescales than the fluorescence lifetime even at 170 K. The results from these studies are used to reinterpret a previous study of photochemical processes observed in intact cells and their acetone extracts of the photosynthetic system of Acaryochloris marina. In the study of Mimuro et al., light absorbed by Chl d at 736 nm is found to give rise to emission by another species, believed to also be Chl d, at 703 nm; this uphill energy transfer process is easily rationalized in terms of the thermal equilibration processes that we deduced for Chl d. However, no evidence is found in the experimental results of Mimuro et al. to support claims that (nonequilibrium) uphill energy transfer is additionally observed to Chl a species that emit at 670-680 nm. This finding is relevant to broader issues concerning the nature of the special pair in photosystem II of A. marina because suggestions that it is comprised of Chl a can only be correct if nonthermal uphill energy transfer processes from Chl d are operative.     

Potassium persulphate as volumetric reagent

Summary Potassium persulphate has been used as volumetric reagent for the direct determination of hydrazine sulphate, phenyl hydrazine hydrochloride, semicarbazide hydrochloride, thiosemicarbazide, acetone semicarbazone, benzal semicarbazone, benzalazine, aminoguanidine hydrochloride, 4-phenyl semicarbazide hydrochloride, ethylmethyl ketone semicarbazone and 4-hydroxy-3-methoxy benzal semicarbazone in hydrochlorid acid medium at room temperature, using iodine monochloride as a catalyst and preoxidiser. Chloroform was used as an indicator. It was coloured violet owing to the liberation of iodine during the titration and became very pale yellow at the end-point due to the formation of iodine monochloride. Normality of the solution with respect to hydrochloric acid was kept between 6.0 and 7.5 N in these redox titrations.     

A Synthetic Chlorophyll Dimer Appending Fullerene: Effect of Chlorophyll Pairing on (Photo)redox Properties

Accurately mimicking structure and function of natural chlorophyll (Chl) assemblies is very challenging. Herein, we report the synthesis of a fullerene-appended Chl dimer being capable of intramolecular photoinduced charge separation (CS) with a unique structure reminiscent of reaction centers (RCs) in phototrophs. Structural analyses revealed that the Chl dimer adopts a bird-like structure in which two Chl components overlapped partially with one of the four pyrrole rings in a Chl ring similar to in a Chl pair in the natural RC complexes. A comparative study including voltammetry and spectrometric analyses using the Chl dimer and its corresponding monomer with and without a fullerene moiety was performed to gain insight into the effect of Chl pairing on (photo)redox properties. Our results suggest that the present dimer motif that closely resemble the Chl pair in natural RCs lead to more facile oxidation and lower energy of the CS state of the Chl dimer than those of the Chl monomer, resulting in its photoredox behavior different from that of the monomer Chl.     

Investigation of chlorophylls as a scavenger of manganese(II) and zinc(II) using a radiotracer technique

Uptake of manganese(II) and zinc(II) by chlorophylla (Chl) was investigated using a radiotracer technique in order to elucidate its ability of scavenging metals released into the soil environment. Two possibilities were considered for the metal uptake: (1) substitutional complexation with Chl and (2) inclusion of the metals into Chl aggregates. It was found that manganese(II) was not trapped appreciably by both Chl itself and its aggregates. Magnesium of Chl was exchanged with zinc(II) in aqueous phase giving Zn−Chl at pH values higher than 7. Zinc(II) was found to be trapped by Chl aggregates depending on the pH of the aqueous phase.     

THE RELATIONSHIP OF ACCESSORY PIGMENTS TO CHLOROPHYLL a CONTENT IN CHLOROPHYLL-DEFICIENT PEANUT AND SOYBEAN VARIETIES

Abstract— A relationship between Chl a and each of its accessory pigments, neoxanthin, violaxanthin, lutein, Chl b and carotene, is demonstrated in soybean and peanut genotypes. These genotypes include normal and Chl-deficient types. Plots of Chl a content against accessory pigment content generate straight-line relationships that are extrapolated to intersect the Chl a axis. The points of intersection correctly predict the order of origin of certain accessory pigments (lutein, carotene and Chl b ), but incorrectly predict that neoxanthin and violaxanthin arise after the introduction of Chl a .     

Excited state dynamics in recombinant water-soluble chlorophyll proteins (WSCP) from cauliflower investigated by transient fluorescence spectroscopy

The present study describes the fluorescence emission properties of recombinant water-soluble chlorophyll (Chl) protein (WSCP) complexes reconstituted with either Chl a or Chl b alone (Chl a only or Chl b only WSCP, respectively) or mixtures of both pigments at different stoichiometrical ratios. Detailed investigations were performed with time and space correlated ps fluorescence spectroscopy within the temperature range from 10 to 295 K. The following points were found: (a) The emission spectra at room temperature (295 K) are well characterized by bands with a dominating Lorentzian profile broadened due to phonon scattering and peak positions located at 677, 684 and 693 nm in the case of Chl a only WSCP and at 665, 675 and 689 nm for Chl b only WSCP. In addition, all spectra contain minor bands in the longer wavelength region. (b) The emission spectra at 10 K of samples suspended in buffer containing 50% glycerol are dominated by bands peaking at 668 nm for Chl b only WSCP and at 685 nm for Chl a only WSCP and samples reconstituted with mixtures of Chl a and Chl b. (c) At 10 K and in buffer with 50% glycerol the decay kinetics of WSCP samples with Chl a only are dominated by a component with a time constant of 6.2 (+/-0.2) ns at 685 nm while those of WSCP containing mixtures of Chl a and Chl b are characterized by a slightly shorter value of 6.0 (+/-0.2) ns. WSCP containing Chl b only exhibits a distinctly longer value of 7.0 (+/-0.3) ns at an emission wavelength of 668 nm. (d) The decay associated emission spectra at 10 K of all samples exhibit at least 3 decay components with time constants of 80-120 ps, 2-4 ns and 6-7 ns in 50% glycerol. These results are consistently described within the framework of our previously presented model (J. Phys. Chem. B 2007, 111, No. 46, 13325; J. Phys. Chem. B 2007, 111, No. 35, 10487) , for the structural motifs of chlorophyll binding to the tetrameric protein matrix of WSCP. It is shown that formation of strongly coupled open sandwich dimers does not lead to quenching of 1Chl a* or 1Chl b*.     

Separation and determination of minor photosynthetic pigments by reversed-phase HPLC with minimal alteration of chlorophylls.

Reversed-phase HPLC conditions for separation of chlorophyll (Chl) a, Chl a' (the C132-epimer of Chl a), pheophytin (Pheo) a (the primary electron acceptor of photosystem (PS) II), and phylloquinone (PhQ) (the secondary electron acceptor of PS 1), have been developed. Pigment extraction conditions were optimized in terms of pigment alteration and extraction efficiency. Pigment composition analysis of light-harvesting complex II, which would not contain Chl a' nor Pheo a, showed the Chl a'/Chl a ratio of 3-4 x 10(-4) and the Pheo a/Chl a ratio of 4-5 x 10(-4), showing that the conditions developed here were sufficiently inert for Chl analysis. Preliminary analysis of thylakoid membranes with this analytical system gave the PhQ/Chl a' ratio of 0.58 +/- 0.03 (n = 4), in line with the stoichiometry of one molecule of Chl a' per PS I.     

ELECTRON TRANSFER FROM CHLOROPHYLL a TO QUINONE IN MONO- AND MULTILAYER ARRAYS

Abstract— Mono- and multilayers of chlorophyll a (Chl a )– lecithin have been prepared on quartz slides, by means of the Blodgett-Langmuir technique, for fluorescence studies. Self-quenching of the Chl a fluorescence has been observed in Chl a -lecithin single layer excited with a laser light at 632.8 nm. The fluorescence yield is reduced by 50% at a concentration of 7 ± 10¹² Chl a molecules cm⁻². Chl a fluorescence quenching, by adding N,N -distearoyl-1,4-diaminoanthraquinone (SAQ), has been studied. in a single layer, in pure Chl a and also at various dilutions of Chl a in lecithin. The results are explained in terms of a dynamic quenching rather than in terms of a permanent complex formation, at the ground state, between Chl a and SAQ. The fluorescence quenching has been interpreted as the result of an electron transfer from excited Chl a to SAQ, and rate constants of 8.3 ± 10⁻⁵ cm² molecule⁻¹ S⁻¹ and 2.4 ± 10⁻⁴ cm² molecule⁻¹ s⁻¹ have been found for pure diluted Chl a , respectively. Ten per cent of the diluted Chl a fluorescence always remains unquenchable and independent of the quinone concentration. In multilayers, where SAQ and Chl a are in different layers, there is no fluorescence quenching for pure or diluted Chl a even when the chromophores are in two adjacent layers. This happens only if SAQ is not able to diffuse from one layer to another. A minimum value of 22.4 nm has been found for the singlet exciton diffusion length in pure Chl a multilayers.     

Magneto-optic spectroscopy of a protein tetramer binding two exciton-coupled chlorophylls

In vitro chlorophyll (Chl) aggregates have often served as models for in vivo forms of long-wavelength Chl. However, the interaction of protein-bound Chl molecules is typically different than that occurring in solvent-based self-aggregates. We have chosen a water-soluble Chl-binding protein (WSCP) from cauliflower in order to help characterize the spectroscopic properties of Chl in a single well-defined native environment and also to study the pigment-pigment (exciton) interactions present in assemblies of this protein. WSCP forms tetrameric units upon binding two Chl molecules. We present the absorption, circular dichroism (CD), magnetic circular dichroism (MCD), and emission spectra at 1.7 K of recombinant WSCP tetramers containing either Chl a or Chl d. The spectroscopic characteristics provide evidence for significant exciton interaction between equivalent Chl molecules. Our simple exciton analysis allows an estimate of the molecular geometry of the dimer, which is predicted to have an "open sandwich"-type structure. We find that the ratio of the magnetic circular dichroism to absorption, deltaA/A, is substantially increased (approximately 60%) for Chl a in this system compared to its value in solution. This increase is in marked contrast to substantial reductions (>50%) of deltaA/A seen in solvent-based Chl aggregates and in photosynthetic reaction centers. Current theoretical models are unable to account for such large variations in the MCD to absorption ratio for Chl. We propose that spectroscopic studies of WSCP mutants will enable a fundamental understanding of Chl-Chl and Chl-protein interactions.     

Demetalation kinetics of chlorophyll derivatives possessing different substituents at the 7-position under acidic conditions

Conversion of the formyl group at the 7-position in chlorophyll (Chl) b to the methyl group via the hydroxymethyl group is biologically important in Chl b degradation. To clarify the effects of the 7-substituents on demetalation properties of chlorophyllous pigments in the early process of Chl b degradation, we report demetalation kinetics of the zinc Chl derivative possessing a 7-hydroxymethyl group, which is a good model compound of the intermediate molecule in the early process of Chl b degradation, under acidic conditions, and compare its properties with those of zinc Chl derivatives possessing a methyl and a formyl group, which are model compounds of Chls a and b, respectively. Demetalation rate constants of 7-hydroxymethyl zinc chlorin were much larger than those of 7-formyl zinc chlorin, but were slightly smaller than those of 7-methyl zinc chlorin. The activation energy for demetalation reaction of 7-formyl zinc chlorin was larger than those of other derivatives. Demetalation rate constants of 7-deformyl-7-hydroxymethyl Chl b were also larger than those of Chl b, and were similar to those of Chl a. These indicate that the 7-hydroxymethyl group in the chlorin macrocycle has a smaller effect on demetalation compared with the 7-formyl group.     

PROPERTIES OF CHLOROPH'YLL-LECITHIN VESICLES: ULTRACENTRIFUGATION, ABSORBANCE, EMISSION AND PHOTOBLEACHING

Abstract— Egg lecithin vesicles (liposomes) containing up to one Chl a per 11 lecithin molecules were prepared without sonication. Their absorbance and emission properties indicate that Chl is dispersed in the lecithin, that the vesicles are dispersed in the medium and that there is a strong quenching of fluorescence.
Their photobleaching and chemical bleaching show that there is a negligible amount of colloidal Chl present. In vesicles with a Chl:lecithin molar ratio higher than 0.05, there are probably some small aggregates of Chl a in the bilayer.
The ultracentrifugation of such vesicles shows that their sedimentation constant increases, though not regularly, with their relative Chl content     

Quantification of the effect of nonphotochemical quenching on the determination of in vivo chl a from phytoplankton along the water column of a freshwater reservoir

Nonphotochemical quenching (NPQ) is a well-known collection of different photoprotective mechanisms of plants and algae to avoid photodamage under an excess of light energy. In order to evaluate the overall effect of NPQ processes on the fluorometric determination of in vivo Chl a from a phytoplankton community dominated by diatoms, we compared the results obtained by two different fluorometric field devices with the total concentration of extracted Chl a measured by HPLC ( in vitro Chl a ). A different set of measurements were made to assess the performance of these fluorometers at high, moderate and low irradiance conditions. The Fbbe fluorometer, which is capable of distinguishing different algal groups according to their pigment content, allowed a better determination of in vivo Chl a under high irradiance conditions, with only a 10% mean difference from the in vitro Chl a concentration. In turn, the FMII fluorometer underestimated by as much as 50% the in vitro Chl a concentration under the same light conditions. As data from both fluorometers were in accordance with the in vitro Chl a values at moderate irradiance levels, the differences observed at high irradiances were attributed to the decrease in the yield of Chl a fluorescence caused by photoprotective NPQ processes. Accordingly, we estimated the effect of NPQ processes on the in vivo Chl a determination and the results allow us to provide an equation to correct this effect when in situ fluorometric measurements are carried out under high irradiance regimes. Our results demonstrate that under certain circumstances NPQ seriously compromises the results obtained by in situ fluorometric probes and highlight the need for a cautious interpretation of field data under such environmental conditions.     

Reversed-phase HPLC determination of chlorophyll a' and naphthoquinones in photosystem I of red algae: existence of two menaquinone-4 molecules in photosystem I of Cyanidium caldarium.

Chlorophyll (Chl) a', the C13(2)-epimer of Chl a, is one of the two Chl molecules constituting the primary electron donor (P700) of photosystem (PS) I of a thermophilic cyanobacterium Synechococcus elongatus. To examine whether PS I of other oxygenic photosynthetic organisms in general contain one Chl a' molecule in P700, the pigment composition of thylakoid membranes and PS I preparations isolated from red algae Porphyridium purpureum and Cyanidium caldarium was examined by reversed-phase HPLC with particular attention to Chl a' and phylloquinone (PhQ), the secondary electron acceptor of PS I. The two red algae contained one Chl a' molecule at the core part of PS I. In PS I of C. caldarium, two menaquinone-4 (MQ-4) molecules were detected in place of PhQ used by higher plants and cyanobacteria. The 1:2:1 stoichiometry among Chl a', PhQ (MQ-4) and P700 in PS I of the red algae indicates that one Chl a' molecule universally exists in PS I of oxygenic photosynthetic organisms, and two MQ-4 molecules are associated with PS I of C. caldarium.     

Origin of chlorophyll fluorescence in plants at 55-75 degrees C

The origin of heat-induced chlorophyll fluorescence rise that appears at about 55-60 degrees C during linear heating of leaves, chloroplasts or thylakoids (especially with a reduced content of grana thylakoids) was studied. This fluorescence rise was earlier attributed to photosystem I (PSI) emission. Our data show that the fluorescence rise originates from chlorophyll a (Chl a) molecules released from chlorophyll-containing protein complexes denaturing at 55-60 degrees C. This conclusion results mainly from Chl a fluorescence lifetime measurements with barley leaves of different Chl a content and absorption and emission spectra measurements with barley leaves preheated to selected temperatures. These data, supported by measurements of liposomes with different Chl a/lipid ratios, suggest that the released Chl a is dissolved in lipids of thylakoid membranes and that with increasing Chl a content in the lipid phase, the released Chl a tends to form low-fluorescing aggregates. This is probably the reason for the suppressed fluorescence rise at 55-60 degrees C and the decreasing fluorescence course at 60-75 degrees C, which are observable during linear heating of plant material with a high Chl a/lipid ratio (e.g. green leaves, grana thylakoids, isolated PSII particles).     

Structure‐Dependent Demetalation Kinetics of Chlorophyll a Analogs under Acidic Conditions

Demetalation of chlorophyll (Chl) a and its analogs is an important reaction in oxygenic photosynthetic organisms, which produces the primary electron acceptors in photosystem II reaction centers and is crucial in the Chl degradation. From these viewpoints, demetalation reactions of four Chl a analogs, 3,8‐divinyl‐Chl a (DV‐Chl a), 3‐devinyl‐3‐ethyl‐Chl a (mesoChl a), 13²‐demethoxycarbonyl‐Chl a (pyroChl a) and protochlorophyll a (PChl a), were kinetically analyzed under weakly acidic conditions, and were compared with that of Chl a. DV‐Chl a exhibited slower demetalation kinetics than did Chl a, whereas demetalation of mesoChl a was faster than that of Chl a. The difference in demetalation kinetics of the three chlorophyllous pigments originates from the electron‐withdrawing ability of the vinyl group as the peripheral substituent compared with the ethyl group. Removal of the electron‐withdrawing and homoconjugating 13²‐methoxycarbonyl group in Chl a (Chl a → pyroChl a) accelerated demetalation kinetics by two‐fold. PChl a possessing the porphyrin‐type skeleton exhibited slower demetalation kinetics than Chl a. The structure‐dependent demetalation properties of Chl a analogs will be useful for understanding in vivo Chl demetalation reactions in oxygenic photosynthetic organisms.     

Effects of metal and the phytyl chain on chlorophyll derivatives: physicochemical evaluation for photodynamic inactivation of microorganisms

Chlorophyll compounds and their derivatives containing metal or phytyl chain can be used as photosensitizer in photodynamic inactivation of microorganisms (PDI). So, the physicochemical properties and antimicrobial effect of chlorophyll derivatives were investigated: Mg‐chlorophyll (Mg‐Chl), Zn‐chlorophyll (Zn‐Chl), Zn‐chlorophyllide (Zn‐Chlde), Cu‐chlorophyll (Cu‐Chl), pheophytin (Pheo) and pheophorbide (Pheid). The photobleaching experiments showed photostability according to Cu‐Chl > Pheo ∼ Pheid ≫ Zn‐Chl ∼ Zn‐Chlde > Mg‐Chl. This order was discussed in terms of metal and the phytyl chain presences. Pheid and Zn‐Chl in aqueous Tween 80 solution exhibited highest singlet oxygen yield compared with the other derivatives. Chlorophyll derivatives (CD) with phytyl chain was limited by the self‐aggregation phenomenon at high concentrations, even in micellar systems (Tween 80 and P‐123). The antimicrobial effect of CD derivatives was investigated against Staphylococcus aureus, Escherichia coli, Candida albicans and Artemia salina. Pheid showed the best results against all organisms tested, Zn‐Chlde was an excellent bactericide in the dark and Cu‐Chl had no PDI effect. No correlation with CD uptake by microorganisms and darkness cytotoxicity was found. The physicochemical properties allied to bioassays results indicate that Mg‐Chl, Pheo, Zn‐Chl and Pheid are good candidates for PDI.     

A simple AuNPs-based colorimetric aptasensor for chlorpyrifos detection

In this work, a simple gold nanoparticles(Au NPs) based colorimetric biosensor was developed for chlorpyrifos(Chl) detection using an aptamer as the capture probe. The Chl-aptamer with low dissociation constant(Kd) of 58.59 ± 6.08 nmol/L was selected by ss DNA library immobilized systematic evolution of ligands by enrichment(SELEX). In the absence of Chl, the Chl-aptamer acted as the stabilizer for Au NPs in salt solution. In the presence of Chl, the highly specific Chl-aptamer bound with Chl ta...     

Normal-phase HPLC separation of possible biosynthetic intermediates of pheophytin a and chlorophyll a'.

Normal-phase HPLC conditions have been developed for separating the C17(3) isoprenoid isomers, which are expected to be formed as biosynthetic intermediates of chlorophyll (Chl) a, Chl a' (C13(2)-epimer of Chl a), pheophytin (Pheo) a and protochlorophyll (PChl). The application of these conditions to pigment composition analysis of greening etiolated barley leaves allowed us to detect, for the first time, the C17(3) isomers of Chl a', a possible constituent of the primary electron donor of photosystem (PS) I, P700, and those of Pheo a, the primary electron acceptor of PS II, in the very early stage of greening. The C17(3) isomer distribution patterns were approximately the same between Chl a and Chl a', but significantly different between Pheo a and Chl a', probably reflecting the similarity and difference, respectively, in the biosynthetic pathways of these pigment pairs.