PICOSECOND FLUORESCENCE STUDIES OF P700-ENRICHED PARTICLES OF SPINACH CHLOROPLASTS

Dynamic properties of the picosecond fluorescence of highly enriched reaction-center particles of photosystem I (8 - 10 chlorophylls/P700) prepared from spinach have been investigated. The number (N) of photons used to excite chlorophyll molecules per reaction center was controlled between 0.06 and 80. The 1/e lifetime was ca. 25 ps for N 1. which is much shorter than previously measured lifetimes of photosystem I particles. The initial fluorescence intensity saturated at higher excitation intensities (N ≲ 1). This was interpreted in terms of interaction and annihilation among excited chlorophyll molecules which occur almost entirely within the duration of a laser flash. The spectrum-resolved fluorescence decay was faster at 690 than at 680 nm. This implies that two kinds of antenna chlorophylls, apart from and in close proximity to P700, have different lifetimes. Upon heat treatment a component with a much longer fluorescence decay time was observed. The growth of this component upon heat treatment at increasing temperatures showed a correlation with a decrease in the amount of P700 that could be photooxidized.     

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.     

PHYSICAL AND CHEMICAL PROPERTIES OF CHLOROPHYLL RCI EXTRACTED FROM PHOTOSYSTEM I OF SPINACH LEAVES AND FROM GREEN ALGAE

Abstract— A new chlorophyll, designated chlorophyll RCI (Chi RCI), with absorption and fluorescence properties different to other known chlorophylls, has been extracted from photosystem I (PSI) sub-chloroplast particles of the green alga Scenedesmus obliquus; it was suggested that this chlorophyll is either the chromophore ofP–700 or the chromophore of another holochrome associated in a 1:1 molar ratio withP–700. We now report the extraction and isolation of a chlorophyll from PSI particle preparations from spinach leaves with properties identical to those of Chi RCI from Scenedesmus. Its molar ratio toP–700 measured in vivo is again approximately 1:1. Chlorophyll RCI is further characterized by its fluorescence characteristics and redox behaviour. Molecular weight determinations show that Chi RCI has a mol wt 35 units higher than that of chlorophyll a (Chi a).     

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.     

SPECTROSCOPY OF CHLOROPHYLL IN BIOLOGICAL AND SYNTHETIC SYSTEMS*

Abstract. –This review discusses recent spectroscopic studies aimed at discovering the structure, orientation, and function of chlorophyll in vivo. In plant membranes there appear to be at least two distinct types of chlorophyll a. The greater part, over 99%, is antenna chlorophyll which absorbs and transfers radiant energy to a few specialized chlorophyll molecules in a reaction center where the actual charge separation occurs. A dimer-oligomer model for antenna chlorophyll has been proposed on the basis of comparative studies of the absorption spectra of chlorophyll in various dry solvents and in vivo. Unfortunately a similarity between essentially structureless broad spectra is very weak evidence for their original identity. Also the requirement of an anhydrous environment for most of the chlorophyll in biological material is an unlikely postulate. A cross-linked, linear polymer model of chlorophyll in vivo has also been proposed. Recent Resonance Raman spectroscopic results appear to rule out, in large part, either polymer model and once again suggest that it is the various attachments of chlorophyll to proteins which determine its function as antenna pigment in vivo. Circular dichroism measurements of chlorophyll in various plant materials have also led to the conclusion that antenna chlorophyll has strong interaction with protein. However, some doubt still exists as to the interpretation of these CD results. New studies of fluorescence, polarized fluorescence and Resonance Raman spectroscopy of various plant species corroborate the original proposition, based upon deconvolution of absorption spectra, that antenna chlorophyll occurs in vivo in at least five discrete pools, and that each pool is likely to be located in the same environment in different plants. A new model-systems approach to simulating chlorophyll in vivo has come through the use of lipid bilayers and liposomes. Charge transfer has been observed between chlorophyll in a lipid phase and phycobiliproteins or cytochrome c. The most promising, newly synthesized model for the reaction center, P700, is a covalently bound dimeric derivative of pyrochlorophyllide a. Its properties are similar to P700 in several respects except for reversible photooxidation which has not yet been observed. By detergent treatments chlorophyll-protein complexes having about 20–40 chlorophyll a molecules for every P700 have been isolated from different plants, and their spectroscopic properties are under investigation in several laboratories. The several hypotheses to explain the shape of the oxidized minus reduced absorption difference spectrum of P700 have not yet been reconciled. The nature of the photosystem II reaction center chlorophyll, P680, is also a subject of active investigation. Its absorption difference spectrum appears to have two kinetic components.     

FLUORESCENCE SPECTROSCOPY OF A P700-CHLOROPHYLL-PROTEIN COMPLEX*

Abstract. Chlorophyll-protein complexes enriched in the Photosystem I reaction center chlorophyll (P700) exhibit a fluorescence emission maximum at 696 nm at - 196°C The height of this 696 nm emission relative to the emission at 683 nm from antenna chlorophyll a increases proportionally with the P700 concentration while the total fluorescence yield of the complex decreases. The 696 nm emission could possibly be from an absorbing form of antenna chlorophyll a that may be somewhat enriched along with P700 in Photosystem I fractions. However, evidence resulting from glycerol treatment which appears to decrease the rate of resonance energy transfer between antenna chlorophyll and P700 favors the hypothesis that the emission comes from a photooxidized P700 dimer (Chl⁺-Chl) absorbing near 690 nm. In turn, this fluorescence evidence provides additional support for the model of a P700 dimer involving exciton interaction. Absorption in the wavelength region of 450 nm specifically excites emission at 696 nm from the P700-chlorophyll complex.     

CHLOROPHYLL b: AN INTEGRAL COMPONENT OF PHOTOSYSTEM I OF HIGHER PLANT CHLOROPLASTS

Abstract— An undissociated photosystem I complex may be isolated from spinach thylakoids by mild gel electrophoresis (CP1a) or Triton X-100. CP1a has a Chl a / b ratio of 11 and a Chl/P700 ratio of 120. while the Triton X-100 PS I complex (Chl a / b ratio of 5.9) has a larger antenna unit size (Chl/P700 ratio of 180). None of the Chl a / b -proteins of the main light-harvesting complex (apoproteins of 30–27 kD) are present in CP1a, and they account for less than 10% of the total chlorophyll in the Triton X-100 PS I complex. Instead, these PS I complexes have specific, but as yet little characterized, Chi a / b -proteins (apoproteins in the 26–21 kD range). With both PS I complexes, Chi b transfers light excitation to the 735 nm low temperature fluorescence band characteristic of photosystem I. We suggest that Chi b is an integral but minor component of photosystem I.     

STRUCTURE OF PHOTOSYSTEM I AND PHOTOSYSTEM II OF PLANT CHLOROPLASTS*,†,§

Abstract— The action of Triton X-100 upon photosynthetic membranes which are devoid of carotenoids produces a small Photosystem I particle (HP700 particle) which is active in N ADP photoreduction and has a [Chl]/[P700] ratio of 30. The properties of the HP700 particle indicate that it is a reaction center complex which is served by an accessory complex containing the additional light-harvesting chlorophyll of Photosystem I as well as the cytochromes and plastoquinone. When Photosystem II particles obtained by the action of Triton X-100 are further washed with a solution 0.5 M in sucrose and 0.05 M in Tris buffer (pH 8.0), chlorophyll-containing material is released. After centrifugation, the supernatant contains about 1 per cent of the chlorophyll and contains three types of particles which can be separated by sucrose density gradient centrifugation. One of these particles, designated TSF-2b, has the same pigment composition as the original Photosystem II fragment, contains cytochrome 559, and shows Photosystem II activity (DCMU-sensitive diphenylcarbazide-supported photoreduction of 2,6-dichlorophenolindophenol). The other two particles (TSF-2a and TSF-2a′) have a [Chl a]/[Chl b] ratio of 8, have a low concentration of xanthophylls, and show a [Chl]/[Cyt 5591 ratio of about 20. Only the TSF-2a particle is active in the Photosystem II reaction described above. On the basis of these data, it is proposed that the Photosystem II unit consists of a reaction center complex which contains Chl a, Cyt 559, and an acceptor for the photochemical reaction. The reaction center complex would be served by an accessory complex which contains the light-harvesting pigments, Chl a. Chi b, and xanthophyils.     

PHOTOELECTRON TRANSFER BETWEEN A CHARGED DERIVATIVE OF CHLOROPHYLL AND FERRICYANIDE AT THE LIPID BILAYER-WATER INTERFACE

Abstract— Flash illumination of a lipid bilayer containing a positively charge pigment: chlorophyll b cholyl hydrazone and separating two salt solutions, one of which contained ferricyanide, resulted in a photovoltage of ∼20mV, acceptor side negative. The positive charge on the pigment resulted in several novel effects. (1) The photo-emf is twice that of chlorophyll a and five times that of chlorophyll b at a given concentration. A higher surface concentration of the charged derivative is the likely cause of this effect. (2) The pheophytin of chlorophyll b cholyl hydrazone produces about one-half the photo emf of the magnesium derivative whereas pheophytin a or b produced only one-tenth the signal. This may be a reflection of the changed redox potential of the cation chlorophyll b cholyl hydrazone. (3) A voltage drop of 100 mV across the membrane, the acceptor side negatively biased, causes a 3–4-fold increase in the charge recombination rate. Biasing the acceptor side 100 mV positive has no effect. Chlorophyll a or b do not show this field effect. This asymmetric effect is explained as a movement of the more polar chlorophyll dication towards the water interface, leading to more rapid reaction with donor. Thus the kinetics of the charge reversal are a sensitive and specific probe of the polar interfacial region of the lipid bilayer-water interface.     

THE USE OF LIQUID CHROMATOGRAPHY-MASS SPECTROMETRY TO MONITOR THE ALLOMERIZATION REACTIONS OF CHLOROPHYLL a and PHEOPHYTIN a:m IDENTIFICATION OF THE ALLOMERS OF PHEOPHYTIN a

Abstract— This paper reports a new method for monitoring the allomerization reactions of chlorophyll a and pheo-phytin a. Complex mixtures are generated from illuminating pure compounds and monitored using both diode array high-performance liquid chromatography (DAD-HPLC) and liquid chromatography-mass spectrometry (LC-MS). LC-MS allows molecular weight and fragment ion analysis of significant HPLC peaks. Five products of the degradation of chlorophyll a can be simultaneously detected in a mixture, namely the monohydroxy allomer, the methoxylactone allomer, pheophytin a and the two corresponding allomers of the pheophytin. It is demonstrated that more than one pathway must be involved in the in vitro photodegradation of chlorophyll a as shown by the simultaneous existence of several intermediates.     

A REACTION BETWEEN PHEOPHYTIN and CHLOROPHYLL ADSORBED TO POLYETHYLENE-TETRADECANE PARTICLES*

Abstract— When chlorophyll is adsorbed to polyethylene-tetradecane particles along with ligating amphiphiles, and pheophytin is present also, a slow but irreversible photobleaching of the chlorophyll is observed in suspensions of the particles in aqueous media. It is suggested that the reaction starts by transfer of an electron from singlet excited chlorophyll to pheophytin and concludes by hydration and reduction of the chlorophyll radical cation.     

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.     

CHLOROPHYLL-PHOTOSENSITIZED OXIDATION OF HYDROQUINONE AND p-PHENYLENEDIAMINE DERIVATIVES

Abstract— ESR and photovoltaic studies on light-induced one-electron transfer between chlorophyll a and electron donors in the absence of oxygen show (1) the possible conversion of photo-reduced chlorophyll a and p-benzosemiquinone ion radicals to their non-ionic radicals in methanol solutions of low pH, (2) the production of ESR absorption of tetrachloro-p-benzosemi-quinone even in benzene, enhanced by the addition of triethylamine or methanol, and (3) the transfer of one electron from tetramethyl-p-phenylenediamine to either excited chlorophyll a or pheophytin a in methanol at pH above 3.6 but not to pheophytin a at pH below 1 0 where its radical cation appears to accept an electron from excited pheophytin a . Bacteteriochloro-phyll is also shown to be capable of photooxidizing hydroquinones and tetramethyl-p-phenyl-enediamine.
The presence of oxygen enhances chlorophyll a -photosensitized oxidation of hydroquinone and tetrachloro-hydroquinone by one-electron transfer to oxygen and of trimethylhydro-quinone probably by two-electron trnasfer to oxygen. A free radical from excited chlorophyll a-oxygen interaction is formed in these reactions, but rapidly quenched in the case of trimethyl-hydroquinone. This, kind of free radical is not formed in pheophytin a . Tetramethyl- p -phenyl-enediamine readily undergoes chlorophyll a-photosensitized oxidation by oxygen in any pH region.     

PHOTOPHYSICAL BEHAVIOUR OF NEW PYRROLOCOUMARIN DERIVATIVES

Abstract— The photophysical behaviour of new pyrrolocoumarins with different substituents on the nitrogen are reported. The photophysical properties of these pyrrolocoumarins are generally in agreement with those of the psoralens: a strong absorption (240–400 nm), a weak fluorescence (400–680 nm) characterized by a short singlet lifetime, and a rather strong phosphorescence at 77 K (480–600 nm). The absorption and fluorescence properties were investigated in several solvents. The shift of the fluorescence maximum is interpreted on the basis of the solvatochromic parameters π*, α and β. The triplet-triplet absorption spectra also depend on the nature of the solvent, while the triplet excited state has a lifetime of a few microseconds at room temperature (concentration 2.5 × 10⁻⁴ M ). Some absorption and fluorescence characteristics of the 4',5'-dihydropyrrolocoumarins, which are suitable models for the 4',5'-monoadducts to pyrrolocoumarins are reported.     

ON THE ORIGIN OF 628 nm FLUORESCENCE IN CERTAIN CHLOROPHYLL PREPARATIONS

Abstract— A fluorescence band at 628 nm seen in certain preparations of chlorophyll on polyethylene particles swollen with tetradecane comes not from an impurity of protochlorophyll in the original chlorophyll, but from a substance like protochlorophyll formed upon adsorption of chlorophyll to the particles. Much of the chlorophyll is grafted to polyethylene in the same particles by a process that does not affect the chromophore. These and other properties peculiar to the particles are tentatively ascribed to a prior free radical oxidation process in the swollen particles.     

USE OF A PULSED LASER DIODE TO MEASURE PICOSECOND FLUORESCENCE LIFETIMES

Abstract— The use of an inexpensive pulsed laser diode (Hamamatsu picosecond light pulser PLP-01) as the excitation source for a single photon timing spectrolluorimeter with microchannel plate photomultiplier detection was dem-onstrated. The performance of the instrument was tested with two very short-lived fluorescent dyes and two pho-tosynthetic systems with wcll-defined decay characteristics. Individual fluorescence decays were analyzed by modeling with a convolution of the instrument response function to a sum of exponential decay components. Accurate fluorcscence lifetimcs of the dyes cryptocyanine (55 ps in acetone and 83 ps in ethanol) and 1,1'-diethyl-2,2'-dicarbocyanine iodide (13 ps in acetone and 26 ps in ethanol) were obtained by analysis of the decay kinetics with a single exponential component. Fits to the fluorescence decay kinetics of isolated photosystem I particles and intact cyanobacterial cells required three and four decay components. respectively. The decay kinetics of the isolated photosystem I preparation were dominated (99%) by a very fast 9 ps lifetime, reflecting the preparation's small antenna size of approximately 30 chlorophyll a . The cyanobackria showed decay components of 35 ps, 160 ps, 400 ps and 1.95 ns similar to those described previously by Mullincaux and Holzwarth ( Rinchim. Biophys. Acfa 1098 , 68–78, 1991). The performance of the pulsed laser diode as an excitation source for single photon timing is discussed in comparison with conventional sources of picosecond light pulses.     

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.     

THE ORIENTATION OF THE TRANSITION DIPOLE MOMENTS OF CHLOROPHYLL a and PHEOPHYTIN a IN THEIR MOLECULAR FRAME

Abstract— In this paper we describe the determination of the orientation of the absorption and emission transition dipoles of chlorophyll a and pheophytin a in their molecular frame. For this purpose we have embedded the pigments in anhydrous nitrocellulose films with a concentration of 2 × 10^-7 mol/g. We have shown previously that under these conditions the pigments are in a purely monomeric state, are distributed uniformly both before and after stretching and that no intermolecular energy transfer among the molecules takes place.
Using a combination of steady-state anisotropy experiments on unstretched films and angle-resolved fluorescence depolarization measurements on stretched films, we obtain the orientation of the transition dipole moments of both pigments in their molecular frame and the orientational distribution function of the molecules relative to the stretching direction of the film.
The steady-state anisotropy measurements indicate that chlorophyll a has two distinct emission dipole moments and that excitation in the Soret-region results in simultaneous excitation of two or more absorption transition dipole moments. On the other hand, excitation in the Q_Y-band involves only a single dipole moment. The directions of the transition dipole moments in the molecular frame are obtained from the angle-resolved measurements. Pheophytin a also exhibits two emission dipole moments, but the angle between them is much smaller than that between the corresponding dipoles for chlorophyll a . As a consequence the dipole moments contributing to the Soret-region could not be resolved and only an effective absorption transition dipole moment in the Soret-region is extracted.     

A THEORETICAL STUDY OF EXCITONS IN CHLOROPHYLL a PHOTOSYSTEMS ON A PICOSECOND TIMESCALE

Abstract— The creation and decay of singlet excitons in structurally-explicit Chi a photosystems were investigated by numerical integration of the appropriate Master Equations. The antenna chlorophylls were excited by a simulated 6ps Gaussian light pulse. Positions and orientations for the antenna chlorophylls were randomly generated within regions whose size and shape were chosen to be appropriate for the particles observed in freeze-fractured photosynthetic membranes. Among the variables considered were chlorophyll concentration, depth of the trap (i.e. P680 or P700), and the R₀ parameter for Förster transfer. Among the properties considered were antenna fluorescence lifetime; detrapping rates; the instantaneous and integrated quantum yields for antenna fluorescence and intersystem crossing; and the instantaneous and integrated quantum yields for trap fluorescence, intersystem crossing, and photochemistry. A variety of experimental evidence was used as input to the computational model.     

PHOTOCHEMICAL AND SPECTRAL PROPERTIES OF A PARTICULATE MODEL SYSTEM OF CHLOROPHYLL WITH AMPHIPHILES PREPARED FROM HISTAMINE

Abstract— In the photosynthesis model system described, chlorophyll a at an interface photosensitizes the transfer of hydrogen equivalents from a hydrocarbon phase to an aqueous phase. The hydrocarbon phase, to which chlorophyll is adsorbed, consists of polyethylene particles swollen with tetradecane. The particles are also charged positive by co-adsorption of dodecylpyridinium iodide. Furthermore, chlorophyll is ligated with the imidazole function of one of several amphiphiles derived from histamine, which may or may not contain a reducible nitroaromatic group that can serve as primary electron acceptor from photoexcited chlorophyll. The fluorescence quantum yield of chlorophyll on these particles is diminished by self-association of the pigment and by reaction with an oxidizing amphiphile; in the latter case, the quantum yield is correlated with the one-electron redox potential of the amphiphile. Fluorescence-lifetime analysis reveals that most excited singlet states of chlorophyll are quenched rather quickly, and that most of the fluorescence comes from a small fraction of chlorophyll with long lifetime. All preparations sensitize the photoreduction of 5,5′-dithiobis(2-nitrobenzoate) (DTNB) to the water-soluble thiolate by hydrazobenzene. When the amphiphile that ligates chlorophyll is not oxidizing, the quantum yield of photoreduction is unrelated to the fluorescence yield of the particles, but may be related to the degree of self-association of chlorophyll. When the amphiphile that ligates chlorophyll is oxidizing, the kinetics of photoreduction of DTNB require that the electron passes through the primary oxidant to DTNB. Quantum yields for photosensitized reducton of oxidizing amphiphiles in the absence of DTNB have a reversed correlation with redox potential, which can be rationalized in terms of the Marcus theory of electron transfer. All data are most consistently accounted for if the primary photoproduct is an ion pair of chlorophyll and primary oxidant when the latter is available, and a chlorophyll radical ion pair when it is not, both formed by electron transfer from the singlet excited state of chlorophyll.