CHLOROPHYLL ONE-ELECTRON PHOTOCHEMISTRY-II. PHOTOSENSITIZATION BY CHLOROPHYLL,PHEOPHYTIN AND BACTERIOCHLOROPHYLL OF A ONE-ELECTRON TRANSFER REACTION BETWEEN ETHANOL AND QUINONE*

Abstract— Chlorophyll, pheophytin and bacteriochlorophyll sensitize a one-electron transfer in the presence of quinones in ethanol, to produce a ternary complex of ground state porphyrin analog, alcohol cation radical and semiquinone anion radical as the primary photo-product at low temperature. A similar photo-oxidation of alcohol to produce a binary complex is caused by direct excitation of quin-one in the absence of chlorophyll. The mechanisms of these reactions and their implications for photosynthesis are discussed.     

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.     

TRANSFER OF ENERGY FROM REACTION CENTER TO LIGHT HARVESTING CHLOROPHYLL IN A PHOTOSYNTHETIC BACTERIUM*

Abstract— Previous evidence indicates that energy transfer in photosynthetic bacteria can occur from reaction center to light harvesting chlorophyll (the reverse of the usually considered flow) and that the amount of this flow depends on the strain of bacteria. The present report demonstrates that the action spectrum for fluorescence of Rhodopseudomonas spheroides, strain R26, is changed by adding the strong reductant dithionite. This change indicates that the amount of reverse flow can be altered chemically. The amount of reverse flow inferred from these measurements is consistent with the amount predicted from the absorption and fluorescence spectra of chromatophores and isolated reaction centers, and from the relative fluorescence yields of these two. The measurements permit an estimate of the transfer rates describing the energy flow from light harvesting to reaction center chlorophyll as well as the reverse flow. The spectrum for delayed fluorescence of Rps. spheroides, strain Ga, was found to be similar to that of the variable part of the fluorescence. This is a necessary, but not sufficient, condition that the energy for delayed fluorescence originates in the reaction centers.     

FAR-INFRARED SPECTRA OF LANGMUIR-BLODGETT FILMS OF CHLOROPHYLL a,CHLOROPHYLL b,PHEOPHYTIN a and THEIR ADDUCTS WITH WATER and DIOXANE*

Fourier transform infrared spectra in the low frequency region (500–150cm^?1) of Langmuir-Blodgett films of chlorophyll a (Chi a), chlorophyll b (Chi b) and pheophytin a have been studied. Correlations between spectral changes in monolayer and multilayers of Chi a and Chi b and their adducts with water and dioxane have been established. Spectroscopic evidence has indicated that, although there are no individual absorption bands that can be assigned to pure Mg-nitrogen and/or Mg-oxygen stretching or bending modes, there are several bands in the400–200 cm^?1 region of the spectra containing considerable contributions from metal-nitrogen and metal-oxygen vibrational modes. These specific vibrations exhibit marked intensity changes and shifts upon water and dioxane interaction. The different states of chlorophyll aggregation in Langmuir-Blodgett mono- and multilayers films resulted in noticeable changes in their far-IR spectra.     

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.     

REACTION OF EXCITED CHLOROPHYLL MOLECULES AT ELECTRODES AND IN PHOTOSYNTHESIS*

Abstract— Excited molecules can exchange electrons with suitable electrodes in an electrochemical cell. Excitation energy of molecules can therefore be converted into electrochemical energy. Electrochemical reactions of excited chlorophyll molecules have been investigated with the help of semiconductor electrodes. It is suggested that this type of reaction also occurs during the primary steps of photosynthesis. Consequently, concepts of electrochemical kinetics would have to be applied in order to elucidate chlorophyll-sensitized reactions in photosynthetic membranes. In order to provide evidence for this conclusion, electrochemical kinetics is applied to the calculation of decay of delayed light from photosynthetic membranes, and the result is compared with experimental data from Chlorella pyrenoidosa. The conversion of light into electrochemical energy via photoelectrochemical reactions of organic dyes in an electrochemical cell is demonstrated, and the postulated analogous electrochemical mechanism for photosynthesis is discussed.     

PHYTOCHROME IN ETIOPLASTS IN RELATION TO CHLOROPHYLL ACCUMULATION*

Abstract— De-etiolation of maize seedlings reduces their sensitivity for red light potentiation of rapid chlorophyll accumulation in white light. An earlier proposal (Raven and Spruit, 1973) attributes this to migration of the far-red absorbing form of phytochrome (P_fr) to receptors essential for chlorophyll synthesis, thereby increasing the local P_fr/total phytochrome (P_tot)ratio. We have studied etioplasts as possible loci for such P_(r receptors. The level of spectrophotometric phytochrome in purified etioplasts isolated from red preirradiated maize seedlings was higher than that of dark grown plants. The difference was marginally significant, however. We argue that migration of a fraction of cytoplasmic P_fr to the etioplasts, too small to be spectrophotometically demonstrable, could still meet the requirements of the model. Dark destruction of bulk spectrophotometric P_fr following saturating red irradiation of seedlings is not paralleled by a decrease of etioplast phytochrome. the latter remaining essentially constant over long periods. On the other hand, the potentiating effect of red light in intact seedlings is still partially reversible by far red light even after 24 h of darkness when destruction of bulk P_fr is complete. Since this demonstrates persistent presence of P_fr active in potentiation, we propose that at least part of this P_fr is associated with the etioplasts.     

THE CHLOROPHYLL-SENSITIZED REACTION BETWEEN BENZOQUINONE AND ETHANOL*

The electron-transfer reaction between triplet excited chlorophyll and quinones has been extensively studied as a model of the primary reaction in photosystem II. There has also been reported a minor reaction in which the chlorophyll cation radical ostensibly oxidizes the alcohol solvent or even water, leading to a gradual net reduction of quinone, but the exact mechanism and even the existence of this reaction has been uncertain. We have examined the consequences of prolonged irradation of ethyl chlorophyllide and benzoquinone in acidulated ethanol, and find a chlorophyllide-sensitized reaction which is not analogous to the better-known autosensitized reduction of quinones in blue or UV light. In the chlorophyllide-sensitized reaction, benzoquinone is apparently converted to ethoxy-substituted quinones and quinols, and polymeric material. Ethyl chlorophyllide (or chlorophyll) is simultaneously oxidized to more polar products which themselves continue to photosensitize the reaction of quinones. The production of acetaldehyde could not be demonstrated in the sensitized reaction. Chlorophyllide-sensitized reaction of (l-hydroxyethyl)benzoquinone, ethoxybenzoquinone and 2.5-diethoxybenzo-quinone were examined for additional information. A reaction sequence, tentatively proposed to accommodate the known facts, starts with oxidative attack by quinone on an oxidized chlorophyllide radical formed by loss of a hydroxyl proton from alcohol bound as a ligand to Mg²⁺. It is not likely that this reaction is closely related to events at the oxidizing side of photosystem II.     

PHOTOCHEMICAL REACTION BETWEEN PROTEIN AND NUCLEIC ACIDS: PHOTOADDITION OF TRYPTOPHAN TO PYRIMIDINE BASES*1

Abstract— The photochemical interactions between tryptophan and nucleic acid bases were studied. When aqueous solutions of tryptophan were irradiated (Λ > 260 nm) at neutral pH in the presence of each of the nucleic acid bases, pyrimidine bases but not purine bases were altered. Air was found to decrease the rate of reaction. Two classes of photoproducts were isolated by thin layer and ion-exchange chromatography and partially characterized. One was the dihydro-pyrimidine forms of the base (see Reeve and Hopkins, 1979) and the other consisted of tryptophan-pyrimidine photoadducts. Four tryptophan-uracil and two tryptophan-thymine adducts each with a 1:1 molecular stochiometry were found. Spectroscopic measurements and a positive reaction with Ehrlich's reagent indicate that the indole nucleus remained intact, but that the pyrimidine base was reduced at the 5, 6 double bond. The absence of a positive ninhydrin reaction and the effect of pH on the quantum yield of the photoadduct formation indicated that the ionized a-amino acid group of tryptophan was involved in photoadduct formation. Indole derivatives lacking an a-amino group were also found to form photoadducts with pyrimidine bases. The experimental results are consistent with a reaction mechanism involving tryptophan excitation to the first excited singlet state as the initial event. Radical scavenging experiments indicate that tryptophan free radicals, formed by electron dissociation from the excited state, react with the ground state pyrimidine.     

REVERSIBLE LIGHT-TNDUCED SINGLE ELECTRON TRANSFER REACTIONS BETWEEN CHLOROPHYLL AND HYDROQUINONE IN SOLUTION*

Abstract— EPR and optical studies demonstrate the occurrence of reversible temperature-independent light-induced single electron transfer reactions between chlorophyll (and pheophytin) and hydroquinone in degassed ethanol solutions. Oxygen is shown to quench this process, presumably by interacting with chlorophyll excited states. Pyridine and isoquino-line increase the effectiveness of the hydroquinone as an electron donor to chlorophyll, probably by acting as bases to form hydroquinone anions (or perhaps ion-pairs) which are more easily oxidized. Hydroquinone is found to be more effective in electron transfer than is benzoquinone, suggesting that the chlorophyll excited state is a better oxidizing agent than it is a reducing agent.     

CHLOROPHYLL ONE-ELECTRON PHOTOCHEMISTRY: FLASH PHOTOLYSIS STUDIES OF THE CHLOROPHYLL-QUINONE REACTION IN ALCOHOL SOLVENTS*

Abstract— Flash photolysis of chlorophyll a alone in CBE (cyclohexanol-t-butanol-ethanol) yields a difference spectrum similar to those obtained upon steady illumination of chlorophyll a-quinone mixtures in this solvent. Decay kinetics in CBE and dimethylsulfoxide are faster at the Soret band than at 460–580 nm and red band regions. This difference is not obtained in other solvents (CHCI₃, CCI₄, t-butanol, ethanol), implying that two or more species are obtained in CBE and DMSO. β-Carotene in CBE increases the rate of decay of the flash-induced chlorophyll transients at 430 and 660 nm but only decreases the magnitude of the signal at 470 nm. This implies that the 470 nm absorbance is due to a product formed from the triplet state. This effect is not observed in ethanol. Adding quinone to chlorophyll solutions results in slowly decaying species being generated by flash excitation in CBE. Three components can be distinguished: the first (t_1/2? 0.2 msec) corresponds to the triplet state; the second (t_1/2= 5–10 msec) is quinone concentration and species independent; the third (t_1/2= several seconds) is dependent upon quinone concentration and species (rate is faster for higher concentrations and lower potential quinones). The ESR signal decay rate is approximately equal to the third component flash decay rate when the chlorophyll and quinone concentrations are equal. With excess quinone, the flash decay rate becomes faster, and the ESR decay rate decreases slightly. These slowly-decaying species are not produced when quinone is added to chlorophyll a in ethanol or t-butanol, or to pheophytin in CBE. One observes merely a decrease in signal height with no accompanying increase in decay rate. Mechanisms to account for all of these phenomena are presented which involve an initial chlorophyll triplet-solvent reaction with the subsequent formation of several species of chloro-phyll-quinone radical complexes.     

CORRELATION BETWEEN DELAYED LIGHT EMISSION AND FLUORESCENCE OF CHLOROPHYLL a IN SYSTEM II PARTICLES DERIVED FROM SPINACH CHLOROPLASTS

Abstract— The induction transient of delayed light of chlorophyll a, excited by repetitive flashes (0.5 ms in duration) and emitted 0.1 - 1.2 ms after the flashes, was measured in system II particles derived from spinach chloroplasts. An uncoupler, gramicidin S, was always added to the particles in order to eliminate the influence of the phosphorylation system on the delayed light and to isolate a direct relationship between the delayed light emission and the primary photochemical reaction, except for the experiments described in the next paragraph. The yield of delayed light emission from the system II particles was found to be about three–times higher than that of chloroplasts on a chlorophyll content basis. System I particles, on the other hand, emitted much weaker delayed light than chloroplasts. Upon intermittent illumination, induction of delayed light in system II particles showed a decrease from the initial rise level to the steady-state level. The initial rise level was the maximum. The fluorescence induction, on the other hand, exhibited an increase from the initial rise level to the maximum steady-state level. The induction of both delayed light emission and fluorescence arrived at their final steady-state levels after the same period of illumination. Induction of delayed light emission was measured under various conditions that changed the oxidation-reduction state of the primary electron acceptor, X, of photoreaction II: by adding an electron acceptor and an inhibitor of electron transport, and by changing the light intensity. The state of A'was monitored by measuring the fluorescence yield. The yield of delayed light emission excited by each flash was found to depend on the amount of oxidized form of X present before the flash. To examine the role of the primary electron donor Y of photoreaction II in delayed light emission, effects of electron donors of photoreaction II such as Mn²⁺, hydroquinone and p-phenylenediamine were investigated. These agents were found to markedly decrease the yield of delayed light emission without altering the pattern of its induction. They had little effect on the induction of fluorescence. These findings are interpreted by a mechanism in which transformation of the reaction center from the form (X^-Y⁺) into (X Y) produces a singlet excitation of chlorophyll a that is the source of millisecond delayed light emission. This reaction is probably non–physiological and must be very slow if compared to the transformation of (X^-Y⁺) into (X^-Y). Since the form (X^-Y⁺) is produced when the excitation is transferred to the reaction center in the form (XY), it is expected in this scheme that the yield of delayed light emission should depend on the amount of the form (X Y) present before the excitation flashes. Electron donors stimulate transformation of the reaction center from (X^-Y⁺) into (X^-Y). Since this reaction competes with the process of delayed light emission, electron donors are expected to suppress delayed light emission.     

SPECTROFLUOROMETRIC DETERMINATION OF CHLOROPHYLL(IDE) a AND b AND PHEOPHYTIN (OR PHEOPHORBIDE) a AND b IN UNSEGREGATED PIGMENT MIXTURES*

Abstract— Simultaneous equations were derived for the determination of chlorophyllide a and b and pheophorbide a and b in unsegregated mixtures of the four tetrapyrroles. The same equations can also be used for the determination of chlorophyll a and b and pheophytin a and b in unsegregated mixtures, after separation of the phytylated tetrapyrroles from their unphytylated analogs by solvent extraction. The equations are capable of calculating the fluorescence Soret excitation amplitude contributed solely by any one of the four tetrapyrroles in the mixture, from two Soret excitation spectra recorded at emission wavelengths of 674 and 660 nm respectively. The Soret excitation amplitudes are then converted into concentrations by reference to standard calibration curves. The equations are usable with any excitation corrected spectrofluorometer. They are particularly suited for the study of Chl‡catabo-lism with minimum generation of chemical artifacts.     

ENERGY TRANSFER FROM CHLOROPHYLL b TO CHLOROPHYLL a IN A HYDROPHOBIC MODEL SYSTEM

Abstract— Chlorophyll a and chlorophyll b purified by high-performance liquid chromatography (HPLC) were subsequently adsorbed on the surface of a pellicular reverse phase packing normally used in HPLC. The granule surface is reacted with octadecyl groups and furnishes an hydrophobic substrate for pigment adsorption. Reflectance spectra of chlorophyll a and chlorophyll b , each adsorbed at average spacings of about 11 nm² per molecule, had red region maxima at 664 and 643nm respectively. Fluorescence excitation spectra for 740nm emission from these surfaces peaked at about 420nm for chlorophyll a and 460nm for chlorophyll b. Adsorbed pigments excited at either of the two wave lengths had a single fluorescence emission peak at 683nm for chlorophyll a and at 664nm for chlorophyll b. A surface having both pigments adsorbed in approximately equal amounts with an overall average spacing of about 5.6nm² per molecule also had peaks at 420 and 460nm in the excitation spectrum. However, excitation of adsorbed molecules on this (latter) surface, at either 420 or 460nm, produced emission with the single chlorophyll a peak at 683nm. It is concluded that, under the conditions of our experiment, exciting adsorbed chlorophyll b contributes strongly to emission from adsorbed chlorophyll a.     

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.     

HOMOGENEOUS PHOTOBLEACHING OF CHLOROPHYLL HOLOCHROMES IN A PHOTOSYSTEM I REACTION CENTER COMPLEX

Chlorophyll (Chl) photobleaching and P700 photodegradation were followed simultaneously in a P700 Chi a-protein complex (Chl a /P700 < 35). During strong illumination, the photobleaching kinetics of the bulk Chl corresponded with that of P700 photodegradation. The absence of a blue shift of the absorption maximum at 678 nm after photobleaching indicated the simultaneous degradation of all Chl holochromes and the absence of long wavelength-absorbing energy traps that would protect P700 against excess light energy. It was deduced that in the P700 reaction center core complex, the excitons are uniformly distributed amongst the Chl holochromes, which decreases the deleterious effects of excess light energy on P700 and protects the photosystem against photoinhibition.     

BIMANES—26. AN ELECTRON TRANSFER REACTION BETWEEN PHOTOSYSTEM I1 AND MONOBROMOBIMANE INDUCES STATIC CHLOROPHYLL a QUENCHING IN SPINACH CHLOROPLASTS

Abstract— Monobromobimane in chloroplasts lowers both the quantum yield of system II photochemistry and the yield of chlorophyll a fluorescence. Illumination of the chloroplasts in the presence of monohromobimane is an absolute prerequisite to the manifestation of this phenomenon, which proceeds via the Photosystem II intermediate, the semiquinone radical anion, Q_A-. The latter transfers an electron to monobromobimane to yield an anion radical (mBBr^·), which may either lose bromide ion to yield a reactive radical (mB^·), or acquire a proton and undergo further reduction, eventually forming syn-(methyl, methyl) bimane. In turn, mB reacts with the protein of the light-harvesting complex, to form a product which acts as static excitation energy quencher in the chlorophyll pigment bed of photosystem 11. Polarographic reduction of monobromobimane shows an adsorption wave at O V and two reduction waves. Prolonged reduction in water at -0.5 V yields syn-(methyl, methyl) bimane (which is further reduced at more negative potentials) and bromide ion. Thus, both electrochemical and chloroplast-induced reduction produce syn-(methyl, methyl) bimane. Monobromobimane may then serve as a Photosystem II activated probe in elucidating the conformation of intrinsic thylakoid membrane polypeptides.     

A REVERSIBLE PHOTOCHEMICAL REACTION OF CHLOROPHYLL a WITH I2

Abstract— Chlorophyll a and I₂ form a 1:1 addition compound, which exhibits a strong absorption maximum at about 360 nm. When dissolved in anaerobic methanol, this compound can be reversibly bleached by illumination with red light. Prolonged illumination at low temperatures (˜– 75°C) or intense illumination at room temperature reduces the height of the red absorp tion maximum to about half of its normal value. Since the thermal back reaction is negligibly slow at – 75°C, presumably the chlorophyll-I₂ complex is converted completely to its bleached form, when illuminated under those conditions. This assumption leads to a simple mechanism which is consistent with the experimental data.