ROOM TEMPERATURE TIME-RESOLVED IN μs RANGE DELAYED LUMINESCENCE OF CHLOROPHYLL a AND CHLOROPHYLL-LUTEIN MIXTURE SOLUTIONS

Using time-resolved in μS range luminescence spectroscopy, we observed at 20°C the emission of chlorophyll a, pheophytin a and chlorophyll a-lutein mixture solutions. This delayed emission exhibits several maxima in the650–750 nm region. The positions and kinetics of decay of delayed emission bands depend on chlorophyll concentration, and vary as a result of pheophytinization and addition of lutein. Our results can be explained by supposition that upon excitation, charge transfer species are formed in various pigment complexes. The back electron transfer reactions yield chlorophyll excited singlet states contributing to observed delayed emission. Delay in emission seems to be due also to the trapping of excitation on the triplet states of various forms of pigment and its detrapping with the participation of thermal energy followed by energy transfer to the forms of pigment characterized by different decay times.     

SPECTROSCOPIC CHARACTERIZATION OF TWO FORMS OF THE D1-D2-CYTOCHROME b559 COMPLEX FROM SUGAR BEET

Two D1-D2-cytochrome b559 complex forms, called RCIIa and RCIIb, with different pigment stoichi-ometry were characterized using absorption and surface-enhanced resonance Raman scattering spectroscopy and spectral gaussian deconvolution. Electronic absorption spectra of the RCIIb at 277 K showed significant differences compared to RCIIa, i.e . a strong decrease in the absorbance due to carotenoid and chlorophyll for the same amount of pheophytin. A reduced carotenoid and chlorophyll content in RCIIb was also observed in the surface-enhanced resonance Raman scattering spectra. Spectral deconvolution elicited three main absorption bands at 680, 672 and 669–670 nm, which were ascribed to P680, pheophytin and accessory chlorophyll, respectively. In addition, a minor component around 667 nm was observed in the RCIIb, most probably due to some reaction center inactivation. Calculation of the relative area under the gaussians together with pigment stoichiometry data suggest that the 680, 672 and 669–670 nm components contain, respectively, two chlorophylls, two pheophytins and four chlorophylls for the RCIIa, and two chlorophylls, two pheophytins and two chlorophylls for the RCIIb.     

Chromatographic determination of changes in pigments in spinach (Spinacia oleracea L.) during processing

The content of individual chlorophyll and carotenoid pigments is determined in three spinach varieties (Lorelei, Springfield, and Ballet) after processing. Raw spinach and spinach that is steam-blanched for 3, 9, or 15 min is stored frozen at -24 degrees C for 6 months. In addition, spinach is air-dried at 75 degrees C, packed in atmospheric air or nitrogen, and stored at ambient temperature for 6 months. Processing has a significant effect on the content of individual chlorophyll and carotenoid pigments; however, there are no differences between varieties in their content of total and individual pigments in raw, frozen spinach. Increasing blanching time resulted in decreased contents of chlorophyll a and b and increased contents of chlorophyll a' and b' and pheophytin a and b because of pheophytinization. Changes in the color because of pheophytinization are only detected after 15 min blanching. The carotenoid pigments are more stable than the chlorophyll pigments during blanching. (all-E)-Violaxanthin is significantly reduced, caused by degradation to other xanthophylls, such as neochrome, during blanching. There are no significant differences in the content of chlorophyll a and b of dried spinach and blanched, frozen spinach. Formation of chlorophyll a' and b', pheophytin a and b, and chlorophyll a-1 and b-1 is observed after drying. The content of pheophytin a and b is significantly lower in dried versus blanched frozen samples. In dried spinach that is stored in atmospheric air, the content of beta-carotene [599 mg/kg dry matter (DM)] is significantly lower compared with nitrogen (766 mg/kg DM), and the content of (all-E)-lutein is lower than in blanched frozen spinach. Neochrome is not detected in raw spinach but in steam-blanched and dried spinach. No differences are observed in the content of (all-E)-neoxanthin, (9'Z)-neoxanthin, (all-E)-violaxanthin, (all-E)-lutein epoxide, or neolutein A and B between spinach that is stored frozen after 3 min blanching and dried spinach.     

Cyclic Tetramers of Zinc Chlorophylls as a Coupled Light‐Harvesting Antenna–Charge‐Separation System

A coupled light‐harvesting antenna–charge‐separation system, consisting of self‐assembled zinc chlorophyll derivatives that incorporate an electron‐accepting unit, is reported. The cyclic tetramers that incorporated an electron acceptor were constructed by the co‐assembly of a pyridine‐appended zinc chlorophyll derivative, ZnPy , and a zinc chlorophyll derivative further decorated with a fullerene unit, ZnPyC₆₀ . Comprehensive steady‐state and time‐resolved spectroscopic studies were conducted for the individual tetramers of ZnPy and ZnPyC₆₀ as well as their co‐tetramers. Intra‐assembly singlet energy transfer was confirmed by singlet–singlet annihilation in the ZnPy tetramer. Electron transfer from the singlet chlorin unit to the fullerene unit was clearly demonstrated by the transient absorption of the fullerene radical anion in the ZnPyC₆₀ tetramer. Finally, with the co‐tetramer, a coupled light‐harvesting and charge‐separation system with practically 100 % quantum efficiency was demonstrated.     

Modulation of redox potential in electron transfer proteins: effects of complex formation on the active site microenvironment of cytochrome b5

The reduction potential of cytochrome b5 is modulated via the formation of a complex with polylysine at the electrode surface (Rivera et al., Biochemistry, 1998, 37, 1485). This modulation is thought to originate from the neutralization of a solvent exposed heme propionate and from dehydration of the complex interface. Although direct evidence demonstrating that neutralization of the charge on the heme propionate contributes to the modulation of the redox potential of cytochrome b5 has been obtained, evidence demonstrating that water exclusion from the complex interface plays a similar role has not been conclusive. Herein we report the preparation of the V45I/V61I double mutant of rat liver outer mitochondrial membrane (OM) cytochrome b5. This mutant has been engineered with the aim of restricting water accessibility to the exposed heme edge of cytochrome b5. The X-ray crystal structure of the V45I/V61I mutant revealed that the side chain of Ile at positions 45 and 61 restricts water accessibility to the interior of the heme cavity and protects a large section of the heme edge from the aqueous environment. Electrochemical studies performed with the V45I/V61I mutant of cytochrome b5, and with a derivative in which the heme propionates have been converted into the corresponding dimethyl ester groups, clearly demonstrate that dehydration of the heme edge contributes to the modulation of the reduction potential of cytochrome b5. In fact, these studies showed that exclusion of water from the complex interface exerts an effect (approximately 40 mV shift) that is comparable, if not larger, than the one originating from neutralization of the charge on the solvent exposed heme propionate (approximately 30 mV shift).     

A comparative study of dimerization of chlorophylls and pheophytins by fluorescence and odmir

Optical and ODMR data for dimers of pheophytin a and b and chlorophyll a and b are presented. It is proposed that pheophytin a forms a parallel dimer arising from π-π interaction, binding being essentially different from that in the corresponding chlorophyll dimer. The dimer of phcophytin a is less stable (K ≈ 10⁴ ol/mol) than that of chlorophyll a(K ≈ 10⁶ ol/mol).     

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.     

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.     

Effect of Side Chains on Charge Transfer in Quaterthiophene-Naphthalene Diimide Based Donor-Bridge-Acceptor Dyads

We have probed the effect of side chains on the charge transfer dynamics in dyads containing quaterthiophene (QT) donor and naphthalene diimide (NDI) acceptor. The donor and the acceptor are covalently linked using a flexible linker. Four dyads (1–4) were synthesized with the quaterthiophene bearing hexyl side chain and the naphthalene diimide bearing hydrocarbon, fluorocarbon, branched or polar side chains. The UV-Vis spectra for these dyads showed the existence of a donor-acceptor complex. The time-resolved fluorescence (TRF) decay studies show a rapid quenching of fluorescence in all the dyads upon excitation of the donor. We found that the side chains on the NDI did not alter the quenching rates in solution.     

Copper, iron, and zinc interactions with chlorophyll in extracts of photosynthetic pigments studied by VIS spectroscopy

Interactions of copper, iron and zinc with chlorophyll, the major photosynthesis pigment, were studied by VIS spectrophotometry in extracts of photosynthetic pigments (in vitro). Copper predominantly forms Cu-Chl complexes in all studied systems (Cu-, Cu/Fe-, Cu/Zn- and Cu/Fe/Zn-incubated pigment solutions). It is not clear whether iron forms Fe-Chl complexes or produces pheophytin. It’s effect is dominant over zinc (Fe/Zn-system) but highly minor compared to Cu-effect when all three metals are present (Cu/Fe/Zn-Chl system) in the same concentrations. The article is published in the original.     

Light energy conversion with pheophytina monolayer at the SnO2 optically transparent electrode

The photoelectrochemical, absorption and fluorescence properties of pheophytin a mono- and multilayers, deposited on optically transparent tin oxide electrodes and quartz slides were investigated. Spectra of photocurrents coincided with the absorption spectra of photosynthetic pigment in monolayers at the SnO₂/solution interfaces. The anodic and cathodic photocurrents were measured at various electrode potentials. Effects of pH, electrode potentials, and concentration of redox reagents on the conversion of solar energy in monolayers on optically transparent electrodes are discussed. The absorption and fluorescence spectral characteristics, and fluorescence lifetime measurements of pheophytina in monolayers and thin films are also discussed in view of the aggregation properties of the photosynthetic pigment. The thermodynamics of adsorption of large amphiphilic compounds at the interface between two immiscible liquids is considered. The adsorption behavior of pheophytin a dissolved in different solvents is investigated. The thermodynamic parameters of pheophytin a adsorption at octane/water and benzene/water interfaces were determined.Presented at the Symposium, 76^th CSC Congress, Sherbrooke, Quebec, May 30–June 3, 1993, honoring Professor Donald Patterson on the occasion of his 65^th birthday.     

The heliobacteria, a new group of photosynthetic bacteria

A review is given of the photosynthetic properties of the heliobacteria, a new group of photosynthetic bacteria, discovered only 14 years ago. These bacteria contain a “new” pigment, bacteriochlorophyll g, and they have a relatively simple pigment system, consisting of a core-reaction center complex only. Like the green sulfur bacteria, they have a Photosystem I-type reaction center, with a chlorophyll a derivative as primary electron acceptor. Because of the absence of an extensive peripheral antenna system, the reaction center processes in these bacteria are much easier to study than those in the green sulfur bacteria.     

Expanded separation technique for chlorophyll metabolites in Oriental tobacco leaf using non aqueous reversed phase chromatography

An improved separation method for chlorophyll metabolites in Oriental tobacco leaf was developed. While Oriental leaf still gives the green color even after the curing process, little attention has been paid to the detailed composition of the remaining green pigments. This study aimed to identify the green pigments using non aqueous reversed phase chromatography (NARPC). To this end, liquid chromatograph (LC) equipped with a photo diode array detector (DAD) and an atmospheric pressure chemical ionization/mass spectrometer (APCI/MSD) was selected, because it is useful for detecting low polar non-volatile compounds giving green color such as pheophytin a. Identification was based on the wavelength spectrum, mass spectrum and retention time, comparing the analytes in Oriental leaf with the commercially available and synthesized components. Consequently, several chlorophyll metabolites such as hydroxypheophytin a, solanesyl pheophorbide a and solanesyl hydroxypheophorbide a were newly identified, in addition to typical green pigments such as chlorophyll a and pheophytin a. Chlorophyll metabolites bound to solanesol were considered the tobacco specific components. NARPC expanded the number of detectable low polar chlorophyll metabolites in Oriental tobacco leaf.     

Der Einbau von Magnesium in Liganden der Chlorophyll-Reihe mit (2,6-Di-t-butyl-4-methylphenoxy)magnesiumjodid

Introduction of Magnesium into Ligands of the Chlorophyll Series by (2,6-Di-t-butyl-4-methylphenoxy)magnesium Iodide Experimental details are given for the new method of introducing magnesium into porphinoid ligands by (2,6-di-t-butyl-4-methylphenoxy)magnesium iodide (1) , previously published in preliminary form [1]. Besides magnesium octaethylporphyrinate (14) , methyl pyrochlorophyllide a (10) , methyl chlorophyllide a (8) , and methyl bacteriochlorophyllide a (12) , the complexation of pheophytin a (2) to chlorophyll a (3) and of pheophytin b (4) to chlorophyll b (5) are described.     

SPECTRAL PROPERTIES OF CHLOROPHYLL a MONOLAYERS: MONOLAYERS OF CHLOROPHYLL a AND PHEOPHYTIN AT A GAS-WATER INTERFACE*

Abstract— Surface and spectral properties of chlorophyll a monolayers were studied at a nitrogen-water interface. Direct spectral analysis of Chl monolayers indicated that compression results in a heterogenous mixture of Chl species. Fourth derivative and difference spectra showed the presence of minor bands at 692, 726 and 748 nm. The state of compression determines the quantity and type of spectral species formed. A Chl monolayer on an acid subphase results in the formation of a long wavelength absorbing species (705 nm) similar to that of pheophytin. The half-band width, optical density/monolayer, and extinction coefficients of Chl monolayers are given. It is concluded that in the monolayer the formation of various aggregated species of Chl can be induced.     

CHLOROPHYLLS IN POLYMERS. II. PHEOPHYTIN a IN POLYMERS and THE INFLUENCE OF STRETCHING ON THE STATE OF CHLOROPHYLLS IN ANHYDROUS POLYMER FILMS

Abstract— In the first part of this study the spectral properties of pheophytin a in rigid, unstretched anhydrous polyvinyl alcohol and nitrocellulose films have been studied in order to establish the influence of the central magnesium atom on the state of chlorophylls in polymer systems. The absorption, fluorescence, excitation spectra and fluorescence intensity decays in the polymer films and in the solutions from which they are cast are reported. It is shown that pheophytin a aggregate formation is influenced by the nature of the polymer system. An aggregate of pheophytin a is found in polyvinyl alcohol films over a wide concentration range. On the other hand, pheophytin a exists in the monomeric form in unstretched nitrocellulose films at concentrations below 6 × 10^-6 mol/g.
In the second part of this work, the influence of stretching of the films on the state and distribution of embedded chlorophyll pigments, is described. Here we show that the chlorophyll a molecules are found to undertake a heterogenous distribution in polyvinylalcohol matrices, since stretching partially disrupts the pocket-like structures present in unstretched films. In contrast, chlorophyll a and pheophytin a molecules can be embedded in a monomeric state in nitrocellulose matrices and moreover they remain homogeneously distributed upon stretching. The chlorophyll/nitrocellulose system is concluded to be a useful model system for studies of donor-donor energy transfer processes.     

TEMPERATURE DEPENDENCE OF DELAYED LIGHT EMISSION IN THE 6 to 340 MICROSECOND RANGE AFTER A SINGLE FLASH IN CHLOROPLASTS

Abstract. The delayed light emission decay rate (up to 120 μs) and the rise in chlorophyll a fluorescence yield (from 3 to 35 μs) in isolated chloroplasts from several species, following a saturating 10 ns flash, are temperature independent in the 0–35°C range. However, delayed light in the 120–340 μs range is temperature dependent. Arrhenius plots of the exponential decay constants are: (a) linear for lettuce and pea chloroplasts but discontinuous for bush bean (12–17°C) and spinach (12–20°C) chloroplasts; (b) unaffected by 3-(3,4 dichlorophenyl)-1,1-dimethylurea (inhibitor of electron flow), gramicidin D (which eliminates light-induced membrane potential) and glutaraldehyde fixation (which stops gross structural changes).
The discontinuities, noted above for bush bean and spinach chloroplasts, are correlated with abrupt changes in (a) the thylakoid membrane lipid fluidity (monitored by EPR spectra of 12 nixtroxide stearate, 12NS) and (b) the fluidity of extracted lipids (monitored by differential calorimetry and EPR spectra of 12 NS). However, no such discontinuity was observed in (a) chlorophyll a fluorescence intensity of thylakoids and (b) fluorescence of tryptophan residues of delipidated chloroplasts.
Microsecond delayed light is linearly dependent on light intensity at flash intensities as low as one quantum per 2 times 10⁴ chlorophyll molecules. We suggest that this delayed light could originate from a one quantum process in agreement with the hypothesis that recombination of primary charges leads to this light emission. A working hypothesis for the energy levels of Photosystem II components is proposed involving a charge stabilization step on the primary acceptor side, which is in a lipid environment.
Finally, the redox potential of P680 (the reaction center for chlorophyll of system II) is calculated to be close to 1.0–1.3 V.     

REDUCTION OF THE EXCITED SINGLET STATE OF CHLOROPHYLL AND PHEOPHYTIN AT THE ORGANIC CRYSTAL/AQUEOUS SOLUTION INTERFACE

Abstract— Reduction of the excited singlet state of chlorophyll a , chlorophyll b , and pheophytin a at the surface of perylene, anthracene, and chrysene single crystals has been measured as hole injection current. The dependence of the quantum yield on the standard free-energy difference of the reaction was in accordance with theoretical expectations without correcting for an interaction energy. The maximum quantum yield of only about 10⁻² holes per absorbed photon is ascribed to a very small effective lifetime of the excited singlet state due to concentration quenching of the excited singlet state in the adsorbed dye layer.     

On the photodecomposition of chlorophyll in vitro. I. Reaction rates

Abstract— Chlorophyll solutions are irreversibly bleached by light in the presence of oxygen. The action spectra parallel the absorption spectra for both chlorophyll a and b. The reaction is of second order with a Q₁₀ of 1.26. The reaction rates for chlorophylls a and b are of the same order of magnitude. Depending upon the light source, the initial rate for chlorophyll a is slightly higher, by a factor of 1.15 to 1.30. The rate for pheophytin is lowe 3 by several orders of magnitude. No pheophytin has been detected in the reaction products of the irradiated chlorophyll solutions in the absence of water.     

FOURIER-TRANSFORM INFRARED SPECTROSCOPIC STUDY OF THE STRUCTURE OF CHLOROPHYLL a and CHLOROPHYLL b IN HIGHLY DILUTE WATER-SATURATED CARBON TETRACHLORIDE SOLUTIONS

Abstract— Fourier-transform (FT) infrared (IR) absorption spectra have been measured for chlorophyll a (Chi a ), chlorophyll b (Chi b ), pheophytin a (Pheo a ), and pheophytin b (Pheo b ) in highly dilute (10^-5-10^-6 M ) water-saturated carbon tetrachloride solutions. Frequencies of IR bands due to C=O stretching modes of the 9-keto group indicate that Chi a assumes largely a dimeric structure in the concentrated (10^-2-10^-3 M ) water-saturated carbon tetrachloride solutions but it remains mostly a monomer with one or two coordinated water molecules in dilute (10^-5-10^-6 M ) solutions. Although it seems that Chi b also assumes predominantly dimeric form in concentrated solutions and monomelic form in dilute solutions, the relative intensity change of two C=O stretching bands ascribed to the free and coordinated 3-aldehyde groups with decreasing concentration suggests that the aldehyde group is also involved in formation of the dimer. The relative intensity of two C=O stretching bands due to the free and coordinated aldehyde groups changes significantly for Pheo b in water-saturated carbon tetrachloride solutions. This observation suggests that some of Pheo b also assume dimeric form via the aldehyde group in concentrated solutions.