Triplet State Energy Transfer Between the Primary Donor and the Carotenoid in Rhodobacter sphaeroides R-26.1 Reaction Centers Exchanged with Modified Bacteriochlorophyll Pigments and Reconstituted with Spheroidene

Abstract— The dynamics of triplet energy transfer between the primary donor and the carotenoid were measured on several photosynthetic bacterial reaction center preparations from Rhodobacter sphaeroides : (a) wild-type strain 2.4.1, (b) strain R-26.1, (c) strain R-26.1 exchanged with 13²-hy-droxy-[Zn]-bacteriochlorophyll at the accessory bacteriochlorophyll (BChl) sites and reconstituted with spheroidene and (d) strain R-26.1 exchanged with P-vinyl]-13²-hydroxy-bacteriochlorophyll at the accessory BChl sites and reconstituted with spheroidene. The rise and decay times of the primary donor and carotenoid triplet-triplet absorption signals were monitored in the visible wavelength region between 538 and 555 run as a function of temperature from 4 to 300 K. For the samples containing carotenoids, all of the decay times correspond well to the previously observed times for spheroidene (5 ± 2 us). The rise times of the carotenoid triplets were found in all cases to be biexponen-tial and comprised of a strongly temperature-dependent component and a temperature-independent component. From a comparison of the behavior of the carotenoid-con-taining samples with that from the reaction center of the carotenoidless mutant Rb. sphaeroides R-26.1, the temperature-independent component has been assigned to the buildup of the primary donor triplet state resulting from charge recombination in the reaction center. Arrhenius plots of the buildup of the carotenoid triplet states were used to determine the activation energies for triplet energy transfer from the primary donor to the carotenoid. A model for the process of triplet energy transfer that is consistent with the data suggests that the activation barrier is strongly dependent on the triplet state energy of the accessory BChl pigment, BChl_B.     

TRIPLET FORMATION AND TRIPLET DECAY IN REACTION CENTERS FROM THE PHOTOSYNTHETIC BACTERIUM Rhodopseudomonas sphaeroides

Abstract— In the reaction center of photosynthetic bacteria, with the primary ubiquinone reduced, the triplet state P^R of the primary electron donor (a pair of bacteriochlorophylls named P) is PO ulated with a takes place in a few ns. We measured by flash absorption spectroscopy the influence of temperature on formation and decay kinetics of P^R and ³Car in the reaction center of several strains of R. sphaeroides . The rate of triplet energy transfer, measured as the decay of P^R after a flash, decreases when the temperature is lowered. Between 60 and 30 K the half-time of energy transfer becomes longer than the ³Car half-time decay (about 6 μs) and below 20 K the transfer is slower than the internal decay of P^R (about 100 μs). In several cases it is clear that P^R and ³Car decay independently and are not in thermal equilibrium. The singlet energy transfer from carotenoid to P occurs with a high efficiency at all temperatures.
The data can be accounted for on the basis of estimated energy levels of P^R and ³Car, in the context of the equilibrium ³P ←³D where ³P is the localized triplet state of P-870 and ³D is another triplet state. A reasonable kinetic scheme leads us to estimate that ³D is 0.0025 ± 0.005 eV above ³P. ³D may thus be the state observed by Shuvalov and Parson (1981). We propose that both triplet and singlet energy transfer between P and the carotenoid occur via a bacteriochlorophyll, to which the carotenoid should be tightly coupled via exchange interaction.     

KINETIC AND SPECTROSCOPIC FEATURES OF SOME CAROTENOID TRIPLET STATES: SENSITIZATION BY SINGLET OXYGEN

Abstract— Triplet-triplet absorption spectra of a series of carotenoid pigments in benzene solution have been determined by pulse radiolysis experiments. The natural lifetimes in deaerated solution have also been measured. They fall in the range 2–10 µ s as found for other carotenoids under similar conditions. Pulsed laser (337 nm) excitation of benzene solutions containing oxygen, carotenoid and a photosensitized molecule (anthracene) showed the generation of absorption spectra of the triplet states. These absorptions decayed by first order kinetics in such a way as to indicate that they were formed in reactions with singlet oxygen, itself generated by interaction with the anthracene triplet state. Bimolecular rate constants for energy transfer from O*₂ (¹△_g), to carotenoid have been evaluated.     

PICOSECOND LASER SPECTROSCOPY AND OPTICALLY DETECTED MAGNETIC RESONANCE ON A MODEL PHOTOSYNTHETIC SYSTEM

Fluorescence-detected magnetic resonance of triplets in zero magnetic field (FDMR), fluorescence fading (FF) due to triplet-formation, both at 4.2 K, and prompt fluorescence decay kinetics (FDK) at room temperature have been measured for free pheophorbide- a (f-Pheo) and bound (b-Pheo) to a synthetic polypeptide (L-L ys -L-A la -L-A la )_n, dissolved in dimethylformamide (DMF). Fluorescence decay kinetics measurements of f-Pheo in DMF yielded 1-5 ns lifetimes, for b-Pheo in DMF a ~ 50 ps decay-component was found emitting at 730–750 nm. Zero-field splitting parameters |D| and |E| of the lowest triplet state T₁ were determined from FDMR spectra as (337 and 24) 10^-4 cm^-1 for f-Pheo and (359 and 25) 10^-4 cm^-1 for b-Pheo, both in DMF. Decay rate constants of the three spin levels of T¹ of b-Pheo ( K _x= 1200 50 s^-1, k _y= 440 25 s^-1, k _z= 80 5 s^-1) and relative steady-state populations (N_x= 28 2%, N_y= 47 2%, N_z= 26 2%) determined from FF curves predict a fluorescence decrease at the D–E and D + E FDMR transitions, whereas experimentally a fluorescence increase is observed. The FDMR sign-inversion results from singlet-singlet energy transfer from b-Pheo monomers to their aggregates, followed by fast intersystem crossing to T₁. These results indicate that aggregates are formed by two or more b-Pheo molecules at different positions on the folded polypeptide chain. This situation resembles that in chlorophyll-proteins, containing low-lying traps, resulting from interaction of chromophores with other chromophores and with the protein environment.     

TRIPLET STATES OF CAROTENOIDS FROM PHOTOSYNTHETIC BACTERIA STUDIED BY NANOSECOND ULTRAVIOLET AND ELECTRON PULSE IRRADIATION

Abstract— Absorptions of the triplet excited states of five carotenoids (15,15'-ds phytoene, all- trans phytoene, C-carotene, spheroidene and spirilloxanthin), extracted from the photosynthetic bacteria Rhodopseudomonas spheroides and Rhodospirillum rubrum, have been detected in solution using pulse radiolysis and laser flash photolysis. Triplet lifetimes, extinction coefficients, lowest energy levels and quantum efficiencies of formation have been determined. Comparison of the carotenoid triplet energy levels with that of O₂('Δ_g) suggests that spirilloxanthin, spheroidene and possibly alsoζ-carotene, would be expected to protect against photodynamic action caused by O₂ ('Δ_g), but not cis or trans phytoene. The S → T intersystem crossing efficiences of all five polyenes were found to be low, being a few per cent or less. In their protective role these triplet states can only therefore be effectively reached via energy transfer from another triplet, except in the case of O₂ ('Δ_g). The low crossover efficiencies also mean that light absorbed by such carotenoids in their possible role as accessory pigments would not be wasted in crossing over to the triplet state.     

FLUORESCENCE AND PHOTOOXIDATION OF 1—ANILINONAPHTHALENE—8—SULFONIC ACID IN REVERSED MICELLAR SOLUTIONS AND THE EFFECT OF CCl4

Abstract— The 1-anilinonaphthalene-8-sulfonic acid solubilized in dodecylammonium propionate reversed micellar cyclohexanic solutions, emitted a strong fluorescence, and was photooxidized under aerobic conditions. Carbon tetrachloride (CCl₄) highly quenched the fluorescence and remarkably enhanced the oxidation reaction. The fluorescence quenching obeyed the Stern-Volmer relation, and the photooxidation was caused by the singlet oxygen generated by the photosensitization of the dye. From the kinetic analysis, it was known that the intersystem crossing rate from the dye excited singlet to triplet was enhanced by CCl₄. Carbon tetrachloride did not quench the triplet state. The ratio of quantum yields for the oxidation in the presence and absence of CCl₄ was independent of the oxygen concentration in the reaction mixture. The fluorescence quenching constant and the intersystem crossing rate were obtained at various solubilized water contents.     

INTENSITY AND TIME-DEPENDENCE OF THE CAROTENOID TRIPLET QUENCHING UNDER LIGHT FLASHES OF RECTANGULAR SHAPE IN CHLORELLA

Abstract— As is known the chlorophyll fluorescence of photosynthetizing plants is strongly quenched by carotenoid triplet states if the exciting light intensity is high enough (> 10 kW/m²). This light-induced quenching process was studied by measuring the relative yield of chlorophyll fluoresccncc excited with a pulsed argon laser at 20 C in light adapted algae as function of time (within lo μ s ) and exciting light intensity (<400 kW/m²). The experimental yield against time and yield against intensity curves have been adequately explained by a statistical model of Photosystem 2 (PS 2) units based upon the existence of freely moving excitons according to which the carotenoid triplet quenchers are randomly distributed and are perfect traps for excitons. Accepting the hypothesis that carotenoid triplet quenching occurs only in PS 2 units. it could be concluded that the height of the true zero level of PS 2 fluorescence is somewhat lower than the half value of the fluorescence level of the dark adapted state.     

Aggregation of the Light-Harvesting Complex in Intact Leaves of Tobacco Plants Stressed by CO2 Deficit

Pronounced aggregation of the photosystem II light-harvesting complex (LHC II) was observed in low-lightgrown tobacco plants stressed with a strong CO₂ deficit for 2–3 days. The LHC II aggregates showed a typical band at 697–700 nm (F₆₉₉) in low-temperature emission spectra. Its excitation spectrum corresponded to that of detergent-solubilized LHC II. Formation of F₆₉₉ in stressed plants was not reversed in the dark and leaves did not contain any zeaxanthin showing that neither a light-induced transthylakoid pH gradient nor zeaxanthin was required for LHC II aggregation. The CO₂-stressed plants showed clear signs of photodamage: depression of the potential yield of photosystem II photochemistry (F,/F_M) by 50–70% and a decline in chlorophyll content by 10–15%. Therefore, we propose that the photodamage to the photosynthetic apparatus is the cause of the LHC II aggregation in plants. The F₆₉₉ exhibited a reversible decrease of its intensity upon irradiation of leaves with intensive light. There was no or only slight decrease around 700 nm in unstressed plants. The nonphotochemical quenching of chlorophyll fluorescence showed the opposite relation, being higher before than after the strong CO₂ deficit. This discrepancy was likely related to the different LHC II aggregation state in control and stressed plants.     

ESTIMATION OF ENERGY DISTRIBUTION AND REDISTRIBUTION AMONG TWO PHOTOSYSTEMS USING PARALLEL MEASUREMENTS OF FLUORESCENCE LIFETIMES AND TRANSIENTS AT 77 K

Abstract— A single-sample method for estimating energy distribution and redistribution among the two photosystems using fluorescence lifetimes and transients at 77 K is presented. In this method,α(the fraction of photons absorbed by photosystem I, PSI) is F_1(α)/(F_1(α)+ (τ_{F 1(M)}/τ_{F 2(M)}).F_2(M)) where, F_1(α) is the fluorescence intensity from PSI excited by photons initially absorbed by the latter, τ_{F 1(M)} and τ_{F 2(M)} are the maximum lifetimes of fluorescence from chlorophyll- a in PSI (1) and II (2), and, F_2(M) is the maximum fluorescence intensity from PSII (P level). Analysis of the intensities and lifetimes of wavelength resolved fluorescence of thylakoids (pH 7.0), with and without cations, leads to the following conclusions: The addition of 10 m M Na⁺ to cation-depleted thylakoids (pH 7.0) increases α by ˜ 10%, while the subsequent addition of 10 m M Mg²⁺ leads to three principal concomitant changes (in the order of importance): a 50% decrease in PSII to PSI energy transfer, a 20% increase in other radiation-less losses, and a 10% decrease in α.     

THE TRIPLET AND RADICAL SPECIES OF HAEMATOPORPHYRIN AND SOME OF ITS DERIVATIVES

Abstract— Nanosecond laser flash photolysis and pulse radiolysis have been used to generate and characterise the triplet state, and semioxidised and semireduced radicals of haematoporphyrin, and three 0 -acyl compounds derived from it (the monoacetate, the diacetate and the disuccinate).
After 347 nm irradiation in water containing 2% Triton X-100, haematoporphyrin forms the triplet state (φ_T= 0.92) and photoionises monophotonically (φ_I= 0.03). For the O -acyl derivatives, φ_T approaches unity and photoionisation is reduced. In acetone the triplet yield of all four compounds are close to unity. The difference and corrected spectra for the triplet species are presented and decay rates ( k ₁˜10⁴s^-1) and oxygen quenching constants ( k _Q˜1.5times10⁹ M ^-1s^-1) for the triplet state have been measured. The difference and corrected spectra for the semi-reduced species in methanol and semi-oxidised species in aqueous Triton X-100 are presented.
The photophysical characteristics in fluid solution of haematoporphyrin and its 0 -acyl derivatives are rather similar to those previously recorded for other photosensitising porphyrins.     

A FLASH PHOTOLYSIS STUDY OF 1-METHYLINDOLE

Abstract— The conventional flash photolysis of 1-methylindole in aqueous media was studied at Λ_excitation≥290 nm. The transients observed 20 μs after excitation consisted mainly of the radical cation (R⁺). the hydrated electron (e^-_aq) and the triplet state (T). Electron counting experiments indicate that photoionization is the only source of R⁺ with e^-_aq/R⁺= 1.07±0.09 in neutral media. Quenching of the R⁺ yield with H⁺ indicates that the fluorescent state is the precursor to 80% of the photoionization events with the remainder probably arising from a prefluorescent state. The triplet decays with a lifetime of 29 μs in deaerated neutral media. This decay is unchanged by N₂O saturation, but T reacts with acrylamide with k ≥2.8 × 10⁹ M ^-1. In 2 M Br^-, R⁺ and T yields are increased by factors of 2–3. Consideration of fluorescence quenching and T enhancement by Br-permits an estimate of φ_Isc between 0.33 and 0.49. The increased R⁺yield at high Br-concentrations cannot be accounted for by induced photoionization or triplet state reactions.     

ENERGY TRANSFER BETWEEN THE PRIMARY DONOR BACTERIOCHLOROPHYLL AND CAROTENOIDS IN Rhodopseudomonas sphaeroides

Abstract— The temperature dependencies of the primary donor triplet state spectra are presented for the phorosynthetic bacteria Rhodopseudomonas sphaeroides wild type. GIC and R26. The data suggest that energy transfer from the primary donor triplet state to the reaction center carotenoid is dependent on the type of carotenoid present, reversible in the case of strain GIC, and best understood by a model depicting the kinetic processes that can occur between two potential energy surfaces; one representing the state ³BChl₂*Car and the other representing BChl₂³Car*. Furthermore, it is shown that the onset of spin lattice relaxation in the primary donor triplet is most likely coupled to the same energy vibrational mode as that which promotes triplet state energy transfer from the primary donor to the reaction center carotenoid     

PREPARATION AND PHOTOPHYSICAL STUDIES OF PORPHYRIN-C60 DYADS

Abstract Porphyrin-C₆₀ dyads in which the two chromophores are linked by a bicyclic bridge have been synthesized using the Diels-Alder reaction. The porphyin singlet lifetimes of both the zinc (Pz_n-C₆₀) and free base (P-C₆₀) dyads, determined by time-resolved fluorescence measurements, are ≦17 ps in toluene. This substantial quenching is due to singlet-singlet energy transfer to C₆₀ The lifetime of Pzn-¹C₆₀ is -5 ps in toluene, whereas the singlet lifetime of an appropriate C₆₀ model compound is 1.2 ns. This quenching is attributed to electron transfer to yield P_zn^bull;+-C₆₀^bull;-. In toluene, P-¹C₆₀ is unquenched; the lack of electron transfer is due to unfavorable thermodynamics. In this solvent, a transient state with an absorption maximum at 700 ran and a lifetime of-10 μs was detected using transient absorption methods. This state was quenched by oxygen, and is assigned to the C₆₀ triplet. In the more polar benzonitrile, P-¹C₆₀ underoes photoinduced electron transfer to give P^•+-C₆₀^bull;-. The electron transfer rate constant is −2 × 10¹¹ s⁻¹.     

TRIPLET STATES OF CAROTENOIDS BOUND TO REACTION CENTERS OF PHOTOSYNTHETIC BACTERIA: TIME-RESOLVED RESONANCE RAMAN SPECTROSCOPY

Time-resolved, low-temperature resonance Raman spectra of triplet states of the carotenoids specifically present in bacterial reaction centers in a strained cis conformation have been obtained, thus demonstrating the possibility of studying intermediate transient states of these structures using resonance Raman spectroscopy. Resonance Raman spectra of triplet cis spheroidene and cis methoxyneurosporene present in reaction centers of Rhodopseudomonas spheroides, (strains 2.4.1. and Ga, respectively) exhibit marked differences with those of triplet, all- trans carotenoids previously studied in vitro. These differences, together with the frequency shifts measured for the v ₁ modes, indicate that triplet carotenoids bound to reaction centers retain a cis conformation, and that probably no isomerization occurs to all- trans carotenoids upon T ← S₀ excitation. P_i electron distributions along the polyene backbone are probably less regular in the triplet state than in the singlet ground state, although probably not to the extent suggested by previous theoretical calculations. The apparently anomalous behaviour of the v ₂ bands of all- trans carotenoids upon T ← S₀ excitation is shown to result largely from the actual complexity of this region of the Raman spectra, together with a weak participation of the v _c—–c internal coordinate in the corresponding modes. Finally, the Raman scattering efficiency of triplet spheroidene bound to reaction centers is lower than that of the singlet, ground state form, under equivalent excitation conditions.     

KINETIC PROPERTIES OF THE TRIPLET STATES OF METHYLENE BLUE AND OTHER PHOTOSENSITIZING DYES

Abstract— The long-lived (> 1 μsec) transients formed in the flash excitation of the representative photosensitizers methylene blue, eosin Y and pyrene have been investigated and various criteria have been used to distinguish between triplet state intermediates and chemical intermediates. Previous assignments of the triplet transients of methylene blue appeared less secure in view of the photochemical reactivity of this dye and its lack of phosphorescence. Earlier assignments of monomeric and dimeric triplet transients of methylene blue are substantiated, however, by the observations that the rate constant for quenching by oxygen is approximately 1/9^th diffusion controlled and the formation rates are commensurate with singlet decay rates and by the observation of triplet-triplet annihilation. Additional evidence in support of monomer triplet assignments for methylene blue and eosin Y is provided by the effect of heavy atom quenchers Cs⁺, Hg²⁺ and T1⁺ on decay rates. Due to chemical reactivity, quenching by I⁻appears less suitable as a diagnostic test for triplet state intermediates. The effect of N₃⁻, which is known to quench singlet oxygen molecules and to alter the course of photosensitized oxidations, on the triplet decay of methylene blue, eosin Y and pyrene is also investigated.     

QUENCHING OF CHLOROPHYLL FLUORESCENCE BY NITROBENZENE

Abstract—Nitrobenzene quenching of chlorophyll fluorescence in ethanol has been investigated. Steady state relative quantum yields have been measured and fluorescence decay rates were determined using both nanosecond photon counting and picosecond pulses from a mode-locked Nd³⁺ glass laser.
The fluorescence decay is described by
1( t )= I ₀ exp (- t/τ−At^1/2 )
the form predicted for decay governed by the kinetics of the continuum model of diffusion controlled reactions. From the parameters of the fluorescence decay, the encounter distance is 5–7 A° the mutual diffusion coefficient is 0.62 × 10^--⁵ cm2s^-1± 12%.
Some of the fluorescence quenching is also attributed to static quenching by a nitrobenzene-chlorophyll, ground-state complex. The equilibrium constant for formation of this ground-state complex was determined to be 4.1 M ^-1. The combined dynamic and static quenching model allows calculation of quantum yields of fluorescence in good agreement with the experimentally determined quantum yields.     

265 NM LASER FLASH PHOTOLYSIS OF SOME ORTHO-SUBSTITUTED ANILIDES AND RELATED N-FORMYLKYNURENINE DERIVATIVES

Abstract— The physical and chemical properties of the triplet state of eight ortho-substituted anilides including N -formylkynurenine (FK), the major trp UV-photooxidation product and a remarkable photodynamic agent, have been investigated using both pulse radiolysis and 265 nm laser flash photolysis techniques. The molar extinction coefficient, the intersystem-crossing quantum yield and the oscillator strength of the T ₁→ T _n absorption band (Λ_max˜ 450 nm) have been determined. It is shown that anilides having n,π* triplets readily react with most solvents whereas those having π ,π* triplets slowly react with alcohols. In both cases, the semi-reduced species are formed. In water, the formation of the semi-reduced. species most probably involves the first excited singlet state. The triplet state properties of the FK derivatives (i.e. ortho-substituted anilides having a side chain bearing charged groups such as carboxylic or amino groups) are strongly modified by the ionization state of the charged side chain. In the case of the FK derivatives possessing an uncharged amino group, quenching of the triplet state occurs via a fast reversible electron transfer reaction from the NH₂ to the triplet anilide.     

pH DEPENDENCE OF SINGLET OXYGEN PRODUCTION IN AQUEOUS SOLUTIONS USING THIAZINE DYES AS PHOTOSENSITIZERS

Abstract— The production of singlet oxygen by thiazine dye photosensitization, as measured by the rate of photooxidation of tryptophan, was found to be very sensitive to changes of pH in the range 5–9. For methylene blue in aerated solutions, the production of ¹O₂* is approximately five times more efficient in basic than in acidic medium. This was shown to be related to the p K 's of the triplet dyes, by evaluating the yields of ¹O₂* from the lifetimes and the quenching rate constants for the two ionic species of sensitizer triplets measured by laser flash photolysis. Changes in the quenching rate constants of the thiazine triplet states can be correlated with the triplet energies.     

LIGHT-INDUCED QUENCHING OF PHOTOSYSTEM II FLUORESCENCE AT 77 K

Abstract— Light-induced quenching of the low temperature fluorescence emission from photosystem II (PS II) at 695 nm ( F ₆₉₅) has been observed in chloroplasts and whole leaves of spinach. Photosystem I (PS I) fluorescence emission at 735 nm ( F ₇₃₅) is quenched to a lesser degree but this quenching is thought to originate from PS II and is manifest in a reduced amount of excitation energy available for spillover to PS I. Differential quenching of these two fluorescence emissions leads to an increase in the F ₇₃₅/ F ₆₈₅ ratio on exposure to light at 77 K. Rewarming the sample from -196°C discharges the thermoluminescence Z-band and much of the original unquenched fluorescence is recovered. The relationship between the thermoluminescence Z-band and the quenching of the low temperature fluorescence emission ( F ₆₉₅) is discussed with respect to the formation of reduced pheophytin in the PS II reaction center at 77 K.     

QUENCHING OF THE TRIPLET STATE OF CHLOROPHYLL a BY ASCORBIC ACID AND ASCORBATE IN ETHANOLIC SOLUTION

Abstract— Ascorbic acid and ascorbate in chlorophyll ethanol solution were found to be fairly efficient quenchers of the chlorophyll triplet state; comparable to the efficiency of ascorbic acid as a quencher in aqueous pyridine solution.
It has been well established that ascorbic acid quenches the triplet state of chlorophyll in aqueous pyridine solution.^(1,2) The bimolecular quenching constant, kQ , is very much less than that for O₂ or quinine.^(3,4)
Information regarding the quenching of the triplet state of chlorophyll by ascorbic acid in ethanolic solution is lacking. There has been some question as to whether ascorbic acid reduces photoexcited chloro-phyll-ethanolic solution because of its high oxidation potential, or because like the ascorbate ion, it acts only as a quencher; both ascorbic acid and ascorbate in high concentrations gave low quantum yields.⁽⁵⁾ The quenching of the triplet state by ascorbic acid and ascorbate was determined by the flash-photolytic method.