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.     

Triplet Energy Transfer between the Primary Donor and Carotenoids in Rhodobacter sphaeroides R-26.1 Reaction Centers Incorporated with Spheroidene Analogs Having Different Extents of π-Electron Conjugation

Abstract— Three carotenoids, spheroidene, 3,4-dihydrospheroidene and 3,4,5,6-tetrahydrospheroidene, having 8, 9 and 10 conjugated carbon-carbon double bonds, respectively, were incorporated into Rhodobacter (Rb.) sphaeroides R-26.1 reaction centers. The extents of binding were found to be 95±5% for spheroidene, 65±5% for 3,4-dihydrospheroidene and 60±10% for 3,4,5,6-tetrahydrospheroidene. The dynamics of the triplet states of the primary donor and carotenoid were measured at room temperature by flash absorption spectroscopy. The carotenoid, spheroidene, was observed to quench the primary donor triplet state. The triplet state of spheroidene that was formed subsequently decayed to the ground state with a lifetime of 7.0±0.5 μs. The primary donor triplet lifetime in the Rb. sphaeroides R-26.1 reaction centers lacking carotenoids was 60±5 μs. Quenching of the primary donor triplet state by the carotenoid was not observed in the Rb. sphaeroides R-26.1 reaction centers containing 3,4-dihydrospheroidene nor in the R-26.1 reaction centers containing 3,4,5,6-tetrahydrospheroidene. Triplet-state electron paramagnetic resonance was also carried out on the samples. The experiments revealed carotenoid triple-state signals in the Rb. sphaeroides R-26.1 reaction centers incorporated with spheroidene, indicating that the primary donor triplet is quenched by the carotenoid. No carotenoid signals were observed from Rb. sphaeroides R-26.1 reaction centers incorporating 3,4-dihydrospheroidene nor in reaction centers incorporating 3,4,5,6-tetrahydrospheroidene. Circular dichroism, steady-state absorbance band shifts accompanying the primary photochemistry in the reaction center and singlet energy transfer from the carotenoid to the primary donor confirm that the carotenoids are bound in the reaction centers and interacting with the primary donor. These studies provide a systematic approach to exploring the effects of carotenoid structure and excited state energy on triplet transfer between the primary donor and carotenoids in reaction centers from photosynthetic bacteria.     

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.     

ETUDE PAR SPECTROSCOPIE D''ECLAIRS DU TRANSFERT D''ENERGIE CHLOROPHYLLE-CAROTENOIDE

Abstract— Energy transfer from chlorophyll A in its lowest triplet state to carotenoid pigments is demonstrated by rapid flash photolysis experiments.
Two systems are used; the first consists of chlorophyll A and p carotene in organic solvents: in diluted solutions, energy transfer is diffusion controlled. The second consists of chlorophyll A and lutein incorporated into digitonin micelles suspended in water; with this system a very rapid energy transfer is observed (< 0.4 × 10^--⁶ sec).
Energy transfer results in a carotenoid metastable state, which is supposed to be a triplet state; for lutein its half-life is 8·9 × 10^--⁶ sec, and it has an absorption peak at 518 nm. Depopulation of lutein ground state, around 450 nm, can be observed, as well as the reactivity of oxygen towards the metastable state.
Most of these results were obtained with a Q -switch ruby laser as exciting source (6943 Å). A 4350 Å flash can also be obtained by two successive non linear effects. Using this flash for exciting chlorophyll A alone, a strong signal is detected, due to its triplet state. By exciting directly B carotene or lutein, it is not possible to detect any metastable state with our technique.     

Inter- and intraspecific variation in excited-state triplet energy transfer rates in reaction centers of photosynthetic bacteria

In protein-cofactor reaction center (RC) complexes of purple photosynthetic bacteria, the major role of the bound carotenoid (C) is to quench the triplet state formed on the primary electron donor (P) before its sensitization of the excited singlet state of molecular oxygen from its ground triplet state. This triplet energy is transferred from P to C via the bacteriochlorophyll monomer B(B). Using time-resolved electron paramagnetic resonance (TREPR), we have examined the temperature dependence of the rates of this triplet energy transfer reaction in the RC of three wild-type species of purple nonsulfur bacteria. Species-specific differences in the rate of transfer were observed. Wild-type Rhodobacter capsulatus RCs were less efficient at the triplet transfer reaction than Rhodobacter sphaeroides RCs, but were more efficient than Rhodospirillum rubrum RCs. In addition, RCs from three mutant strains of R. capsulatus carrying substitutions of amino acids near P and B(B) were examined. Two of the mutant RCs showed decreased triplet transfer rates compared with wild-type RCs, whereas one of the mutant RCs demonstrated a slight increase in triplet transfer rate at low temperatures. The results show that site-specific changes within the RC of R. capsulatus can mimic interspecies differences in the rates of triplet energy transfer. This application of TREPR was instrumental in defining critical energetic and coupling factors that dictate the efficiency of this photoprotective process.     

ESR STUDY OF ORIENTED TRIPLET MOLECULES GENERATED BY EXCITATION WITH POLARIZED LIGHT

Abstract— The lowest-lying triplet states of a variety of aromatic molecules and complexes have been generated by the irradiation of these compounds in solvent glasses with plane-polarized light. Measurements of the allowed (Δ M=± 1) ESR transitions clearly demonstrate that the triplets so formed are oriented with respect to the external magnetic field. By this method the triplet zero-field splitting parameters, D and E , can be evaluated simply and reliably. Intramolecular energy transfer is postulated to explain the triplet spectra of Zn( o -phen)₂(NO₃)₂ and Zn( o -phen)₃(NO₃)₂. It was observed that in triplet-triplet energy transfer from benzophenone to naphthalene there is no apparent orientation requirement between the donor and acceptor molecules. Further areas of significance and application of this technique are suggested.     

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.     

CAROTENOID TRIPLET STATE AND CHLOROPHYLL FLUORESCENCE QUENCHING IN CHLOROPLASTS AND SUBCHLOROPLAST PARTICLES

Abstract— Absorption changes attributed to the triplet state of carotenoids and to primary electron donors (P-700. P-680)^: and fluorescence quenching at several wavelengths have been measured with a single apparatus. following flash excitation with a dye or a ruby laser. Spinach chloroplasts as well as subchloroplast particles enriched in Photosystem-1 (F₁), Photosystem-2 (F₁) or the light-harvesting Chl a/h (F_III) have been examined at temperatures varying between 5 and 294 K.
The triplet state of carotenoids has been identified on the basis of its difference spectrum (having a peak at 515 nm) and decay kinetics (⋍ 7 µs at low temperature; accelerated by O₂ at 294 K). It is formed in all of the materials studied. The quantum yield of carotenoid triplet formation in chloroplasts increases at low temperature, but less than the fluorescence yield.
In most cases the fluorescence quenching recovers approximately with the same kinetics as the decay of the carotenoid triplets. The fluorescence recovery is, however, significantly faster for chloroplasts at 730 nm. Fluorescence quenching occurs in all types of materials. The ratio of fluorescence quenching to the concentration of carotenoid triplets varies with the material, being maximum in chloroplasts and minimum in F_m particles.
We conclude that the formation of the carotenoid triplet state is not limited to a few sites in the chloroplast and that a carotenoid triplet is a quencher of chlorophyll fluorescence. A detailed comparison of carotenoid triplets and fluorescence quenching gives some information concerning the organization of the pigments in the photosynthetic apparatus.     

FREE RADICALS INDUCED BY PHOTOLYSIS OF FROZEN SOLUTIONS OF THYMIDINE-5'-MONOPHOSPHATE: AN ESR STUDY

Abstract— The photolysis (Δ < 220 nm) of thymidine-5'-monophosphate was studied by electron-spin resonance (ESR) in acidic and alkaline phases. In both cases, the H–addition radical at the C₆ position is detected at 77°K. At 225°K, a triplet 1:2:1 is observed, which suggests a H abstraction radical from the CH₃ group. When oxygen is present during irradiation, a peroxide–type radical is observed, which results partly from a reaction like R + O₂→ ROO and partly from an energy transfer from thymidine-5'-monophosphate to oxygen, probably in the ¹π₀ state.     

PROFLAVINE-SENSITIZED PHOTOOXIDATION OF METHIONINE

Abstract— A study was made of the kinetics of the proflavine-sensitized photooxidation of methionine to methionine sulfoxide. The reaction is inhibited by the paramagnetic ions Cu²⁺ and Mn²⁺, which suggests that the triplet state of the sensitizer is an intermediate. A mechanism is proposed in which methionine reacts with the first singlet state of oxygen, produced by energy transfer from the triplet sensitizer. The decrease in the rate of photooxidation with increasing proflavine concentration is ascribed to self-quenching of the excited singlet state of the dye.     

ENERGY TRANSFER FROM ENZYME-GENERATED TRIPLET CARBONYLS TO THYLAKOID MEMBRANE FRACTIONS ENRICHED IN PHOTOSYSTEMS I and II

Abstract— Enzyme-generated triplet species transfer energy very efficiently to thylakoid membrane fractions enriched in either photosystem-I or photosystem-II. Independent of the nature of the triplet donor, the emission is always more intense with photosystem-I. Since the fluorescence quantum yield of chlorophyll in PS-I is lower and the rate of energy transfer usually smaller than to chlorophyll in PS-II, the yield of ¹S chlorophylls in PS-I is substantially higher. This is tentatively attributed to more favorable reverse intersystem crossing from an upper triplet state in PS-I.     

ENERGY CONVERSION IN THE DECAY OF TRIPLET LUMIFLAVIN IN THE PRESENCE OF FERRI- AND FERROCYANIDE

Abstract— Flash photolysis at 450 nm has been used to study the quenching of the excited triplet state of lumiflavin and the transient species formed in subsequent reactions in deaerated phosphate buffer (pH 6.9).
The effect of the presence of ferricyanide on the life time of triplet lumiflavin has been studied. The results suggest an energy transfer reaction without concurrent electron transfer reactions. The rate constant for the process was 2.8 times 10⁹ M ^-1 s^-1. The analogous reaction with ferrocyanide could not be observed because of the efficient electron transfer reaction (δG = -20.6 kcal mol^-1) leading to the formation of the semireduced lumiflavin and ferricyanide. The rate constant for this reaction was 3.3 times 10⁹ M ^-1 s^-1. The semireduced lumiflavin radical was found to disappear in a second order reaction with a rate constant of 1.7 times 10⁹ M ^-1 s^-1. It was found to react with ferricyanide with a rate constant of 0.7 times 10⁹ M ^-1 s^-1.
A model for the various photochemical and photophysical processes involved in the decay and quenching of the lumiflavin triplet state is suggested and discussed.     

QUENCHING OF ENZYME GENERATED TRIPLET ACETONE BY 2-METHYL-1,4-NAPHTOQUINONE

Abstract 2-Methyl-1,4-naphtoquinone (vitamin K₃) quenches the phosphorescence from enzyme-generated triplet acetone. Concomitantly the vitamin undergoes a photochemical-like alteration as the result of transfer of the electronic energy ( k _ET– 1 ± 10⁵ M^-1). This transfer appears to be of the long-range type.     

PHOTOOXIDATION AND ELECTRON-TRANSFER PROCESSES IN FREE-BASE TETRAPHENYLPORPHIN PROBED BY RESONANCE RAMAN SPECTROSCOPY

Abstract
We report here the resonance Raman studies of photooxidation of free base tetraphenylporphin (H₂TPP) in the presence of external electron acceptors such as CCl₄ and chloranil under selective laser irradiation. From the dependence of photooxidation on the concentration of electron acceptors, polarity of solvents, excitation lines and temperatures, we have inferred that a weak triplet exciplex formed between the excited H₂TPP and electron acceptor in non-polar solvents serves as transient species and the light-induced intermolecular charge transfer from H₂TPP to the electron acceptor is the primary process involved in photooxidation. Observation of partial photooxidation in the rigid matrix at low temperatures has been interpreted to be due to long-range quantum mechanical electron tunneling process. Almost complete photooxidation is observed in a soft matrix as the donor and acceptor molecules can attain favorable relative orientation and separation for electron transfer during the excited state lifetime of the exciplex.     

RED EMISSION FROM CHLOROPLASTS ELICITED BY ENZYME-GENERATED TRIPLET ACETONE AND TRIPLET INDOLE-3-ALDEHYDE

—Enzyme-generated triplet acetone and triplet indole-3-aldehyde transfer energy very efficiently to chloroplasts, as indicated by the intensity of the sensitized red emission that is observed. The intermediacy of excited species of oxygen (¹O₂, O₂⁻, HO) has been excluded. Our results open the way for investigating energy transfer in architecturally organized systems in the absence of light.     

ON THE MECHANISM OF QUENCHING OF SINGLET OXYGEN IN SOLUTION

Abstract— Bimolecular rate constants for the quenching of singlet oxygen O*₂(¹Δ_g), have been obtained for several transition-metal complexes and for β-carotene. Laser photolysis experiments of aerated solutions, in which triplet anthracene is produced and quenched by oxygen, yielding singlet oxygen which then sensitizes absorption due to triplet carotene, firmly establishes diffusion-controlled energy transfer from singlet oxygen as the quenching mechanism in the case of β-carotene. The efficient quenching of singlet oxygen by two trans-planar Schiff-base Ni(II) complexes, which have low-lying triplet ligand-field states, most probably also occurs as a result of electronic energy transfer, since an analogous Pd(II) complex and ferrocene, which both have lowest-lying triplet states at higher energies than the O*₂(¹Δ_g), state, quench much less effectively.     

PHOTOOXIDATION AND ELECTRON-TRANSFER PROCESSES IN FREE-BASE TETRAPHENYLPORPHIN PROBED BY RESONANCE RAMAN SPECTROSCOPY

Abstract —We report here the resonance Raman studies of photooxidation of free base tetraphenylporphin (H₂TPP) in the presence of external electron acceptors such as CCl₄ and chloranil under selective laser irradiation. From the dependence of photooxidation on the concentration of electron acceptors, polarity of solvents, excitation lines and temperatures, we have inferred that a weak triplet exciplex formed between the excited H₂TPP and electron acceptor in non-polar solvents serves as transient species and the light-induced intermolecular charge transfer from H₂TPP to the electron acceptor is the primary process involved in photooxidation. Observation of partial photooxidation in the rigid matrix at low temperatures has been interpreted to be due to long-range quantum mechanical electron tunneling process. Almost complete photooxidation is observed in a soft matrix as the donor and acceptor molecules can attain favorable relative orientation and separation for electron transfer during the excited state lifetime of the exciplex.     

SENSIBILISIERTE PHOTOOXYDATION DURCH METHYLENBLAU THIOPYRONIN UND PYRONIN-III. MITTEILUNG: ÜBER DEN MECHANISMUS DER PHOTOSENSIBILISIERTEN OXYDATION DES GUANOSINS DURCH THIOPYRONIN

Abstract— The thiopyronin-sensitized photooxidation of guanosine was investigated, using flash photolysis techniques. The reaction kinetics of three short-lived intermediates (the triplet state, the reduced radical, and the oxidized radical) were followed by spectroscopy. The influence of guanosine concentration on the reaction rate shows that only the oxidized radical (reaction 7) of thiopyronin is affected by guanosine. This suggests that the primary step in the photodynamic destruction of DNA is the oxidation of guanosine by the oxidized radical of thiopyronin (reaction 9). This mechanism is discussed in terms of the redox potentials of the donor and the acceptor for three different cases: (1) where the oxidation potential of the donor is more negative than that of the triplet state, the acceptor being the triplet state ( F^T ); (2) where the oxidation potential of the donor is between the potentials of the triplet state and the oxidized radical, the acceptor being the oxidized radical (F_ox); and (3) where the oxidation potential of the donor is more positive than the reduction potential of both the triplet state and the oxidized radical; in this case no electron exchange takes place. The thiopyronin-guanosine system is an example of the second case.     

THE PHOTOREDUCTION OF METHYLENE BLUE BY ARYLAMINOMETHANE-SULFONATES

Abstract— The photoreduction of methylene blue in the presence of arylaminomethanesulfonates (RAMS = RC₆H₄NHCH₂SO₃Na) was studied by laser and conventional flash photolysis. These compounds quenched the methylene blue triplet deviating from a normal Stern-Volmer behaviour. For low quencher concentrations, a Rehm-Weller relationship was found between the k _q's and the DL G 's obtained for the electron transfer reactions. The lack of further quenching at higher [RAMS] is ascribed to the formation of a ground state ion pair between the dye and the anionic quencher which, on excitation, forms a triplet state unable to under go electron transfer for steric reasons. A second order decay rate constant was found for the semireduced species (MB') ( ca. 5 × 10⁹ M _-1 s_-1, independent of the RAMS used) and is attributed to a proton transfer from the radical zwitterion (RC₆H₄NH CH₂SO₃⁻) to MB. The overall dependence on the substituent of the bleaching observed by continuous irradiation follows the triplet behaviour.