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.     

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.     

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.     

THE TRIPLET EXCITED STATES OF BILIVERDIN AND BILIVERDIN DIMETHYL ESTER

Abstract— The triplet states of biliverdin and biliverdin dimethyl ester have been generated using pulse radiolysis excitation. Biliverdin triplet was formed by energy transfer from biphenyl triplet in acetone, absorbed throughout the wavelength range studied (380–1000 nm) and had a half-life of 11.7μs under the cpnditions chosen. Biliverdin dimethyl ester triplet was formed by energy transfer from biphenyl triplet in benzene, likewise absorbed throughout the wavelength range studied (360–1000 nm) and had a half-life of 6.7μs under the conditions used. Both biliverdin and biliverdin dimethyl ester efficiently quench anthracene, naphthacene, but not μ-carotene, triplet states. On the other hand. neither μ-carotene nor oxygen were found to quench the triplet states of biliverdin or biliverdin dimethyl ester. Estimates or limits for the rate constants of all these quenching reactions were obtained. These reactivities suggest that the triplet levels of both biliverdin and biliverdin dimethyl ester lie around 90 kJ mol^-1. The triplet energy transfer rate from bilirubin to biliverdin dimethyl ester in benzene was measured to be 1.9 × 10⁹ M^-1 s^-1. The singlet-triplet intersystem crossing efficiencies of both molecules were very low, limits of 0.004 and 0.001 being found for biliverdin and biliverdin dimethyl ester, respectively, using 347 nm laser excitation.     

ACTINOMETRY IN MONOCHROMATIC FLASH PHOTOLYSIS: THE EXTINCTION COEFFICIENT OF TRIPLET BENZOPHENONE AND QUANTUM YIELD OF TRIPLET ZINC TETRAPHENYL PORPHYRIN

Abstract— The extinction coefficient ε_T, of triplet benzophenone in benzene has been directly determined by absolute measurements of absorbed energy and triplet absorbance, Δ D ⁰_T, under demonstrably linear conditions where incident excitation energy, E ₀, and ground state absorbance, A ₀, are both extrapolated to zero. The result, 7220 ± 320 M ^-1 cm^-1 at 530 nm, validates and slightly corrects many measurements relative to benzophenone of triplet extinction coefficients made by the energy transfer technique, and of triplet yields obtained by the comparative method.
As E ₀ and A ₀ both decrease, Δ D ⁰_T becomes proportional to their product. In this situation, the ratio R = (1/ A ₀)(dΔ D ⁰_T/d E ₀) = (ε_T - ε_G)φ_T. Measurements of R , referred to benzophenone, give (ε_T - ε_G)φ_T for any substance, without necessity for absolute energy calibration.
Both absolute and relative laser flash measurements on zinc tetraphenyl porphyrin (ε_T - ε_G at 470 nm = 7.3 × 10⁴ M ^-1 cm^-1) give φ_T= 0.83 ± 0.04.     

EFFECT OF TEMPERATURE AND pH ON THE QUENCHING OF TRIPLET LUMIFLAVIN IN THE PRESENCE AND ABSENCE OF FERRI- AND FERROCYANIDE

Abstract— –Flash photolysis at 450 nm over the temperature range 0.8–60°C was used to determine Arrhenius parameters for the first and second order disappearance of triplet lumiflavin (1.66 µ .M ) at a flash energy of 2 kj in deaerated phosphate buffer at varying pH:
³Lf → Lf₀
³Lf +³Lf → Lf₀+ Lf₀
Arrhenius parameters were also determined for the pseudo first-order quenching of triplet lumiflavin by 10 µ M ferri- and ferrocyanide ions,
³Lf + Fe³⁺→ Fe³⁺→ Lf₀+ Fe³⁺ (energy transfer)
³Lf + Fe²⁺→ Lf^-+ Fe³⁺ (electron transfer)
and for disappearance of the semireduced lumiflavin in the presence of ferrocyanide at pH 6.8, by the second-order reaction
Lf^-+Lf ^-→ Lf₀+ Lf⁼.     

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.     

RESONANCE RAMAN and ABSORPTION SPECTRA OF ISOMERIC RETINALS IN THEIR LOWEST EXCITED TRIPLET STATES

Abstract— The triplet-triplet absorption spectra of 9-cis-, 13-cis- and all-fraw-retinal as well as the time-resolved resonance Raman spectra of the lowest electronically excited triplet states of 9-cis-, 11-cis, 13-ciy and all-trans-retinal in aromatic solvents at room temperature have been obtained under conditions ensuring the isomeric purity of the starting materials. The triplet states were produced by triplet energy transfer from a sensitizer in pulse-radiolysis experiments. The overall results suggest that the isomeric retinals form either different relaxed triplet species or different mixtures of relaxed triplet species. The possible implications about the size of the energy barriers separating the various triplet species are discussed. The resonance Raman spectra obtained by using either anthracene (E_T= 177.7 kJ mol^-1) or naphthalene (E_T= 254.8 kJ mol^-1) as sensitizers were virtually identical for the corresponding triplet states from each of the isomers 11-cis-, 13-a's- and all-tams-retinal, suggesting that the relaxed triplet species or the mixture of relaxed triplet species formed from each isomer is independent of the energy of the sensitizer.     

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.     

THE IN VITRO PHOTOCHEMISTRY OF BIOLOGICAL MOLECULES—II. THE TRIPLET STATES OF β-CAROTENE AND LYCOPENE EXCITED BY PULSE RADIOLYSIS

Abstract— –Pulse radiolysis has been used to excite the triplet states of β-carotene (τ# 9μ sec) and lycopene (τ= 8μsec) in hexane solution, both in the presence and absence of naphthalene as a triplet sensitiser. The absorption spectra of both triplets have been measured in the range 430–550 nm and have thus been extended into the region of the corresponding singlet absorptions. The overlap of the triplet and singlet spectra is discussed in relation to in vivo studies. Extinction coefficients of 1.3±0.1 × 10⁵ l/mole cm for β-carotene triplet 515 nm and 3.9±0.2 × 10⁵ l/mole cm for lycopene triplet at 525 nm were obtained. Isomerisation of the all- trans polyenes used was detected and preliminary measurements indicate that the yield of isomerisation was greater than the triplet yield. The rate of triplet energy transfer from naphthalene to β-carotene was estimated to be 1.5 × 10¹⁰ l/mole sec. The corresponding value for lycopene was 1.4× 10¹⁰ l/mole sec. The measured efficient quenching of triplet β-carotene by oxygen may occur by an energy transfer mechanism, leading to the formation of singlet oxygen (¹Δ_g. This would suggest that the triplet energy level of β-carotene lies between 121 and 94 kJ mole^-1.     

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.     

PHOTOCHEMICAL-LIKE DESTRUCTION OF TRYPTOPHAN IN SERUM ALBUMINS INDUCED BY ENZYME-GENERATED TRIPLET SPECIES

Abstract —Human and bovine serum albumin quench enzyme-generated acetone phosphorescence ( K _sv= ca . 10⁴ M ¹). Concomitantly, these proteins are altered as shown by diminished tryptophan absorption at 280 nm, appearance of products of the formylkynurenine type (_max= ca . 320 nm) and disappearance of tryptophan fluorescence. These alterations—which are similar to those induced photochemically—were also observed with serum albumins exposed to enzyme-generated triplet acetaldehyde. On the other hand, triplet acetone generated by the thermolysis of tetramethyldioxetane failed to induce alterations. Presumably energy transfer occurs from the enzyme-generated triplet species to tryptophan group(s) in the serum albumin associated with the acting enzyme. The detailed mechanism is, however, not yet understood.     

ELECTRONICALLY EXCITED STATES IN MICROSOMAL MEMBRANES: USE OF CHLOROPHYLL-a AS AN INDICATOR OF TRIPLET CARBONYLS

Abstract —Chlorophyll- a enhances the photoemission from the microsomeltert-butyl hydroperoxide system without affecting the rates of O² uptake and lipid peroxidation. This photoemission, due to energy transfer from triplet carbonyls to microsome-bound chlorophyll- a , shows that microsomal membranes produce a significant amount of excited triplet carbonyls during lipid peroxidation.     

TRIPLET LIFETIMES OF SOME FLAVINS

Abstract— Using flash photolysis the rate constants of the triplet decay at pH 7.0 (26°C) of 10-hydroxyethyl-isoalloxazine (2700 s⁻¹), 2'-deoxyRF (2300 s⁻¹), 5'-deoxyRF (3200 s⁻¹), 8-hydroxyRF (18 000 s⁻¹) and 8-aminoRF (28 000 s⁻¹) have been measured. The results agree with other evidence that photochemical properties of flavins are influenced by the interaction of the sidechain with the isoalloxazine nucleus. In addition, our data on RF and FMN indicate that the triplet decay rates given in the literature for these compounds have to be corrected to 3200 s⁻¹ (RF) and 4900 s⁻¹ (FMN), respectively. The rate constants for the quenching of the triplet by ground state molecules for all above compounds are given.     

REACTIVITY OF ACRIDINE DYE TRIPLET STATES IN ELECTRON TRANSFER REACTIONS

Abstract—Rate constants, k _q , for the reaction of cationic and neutral acridine orange and 10-methylacridine orange triplet states (³AOH ⁺, ³AO, ³MAO⁺) with a series of electron donors have been measured. Two different protolytic forms of the semireduced dye radical are produced by acridine orange triplet quenching at various pH^M values in methanolic solution.
It is found that k ₄ decreases with increasing oxidation potential of the reducing agent for all triplet states in a manner which is expected for electron transfer reactions. The different reactivities of the cationic and neutral triplet forms can, therefore, be attributed to the difference in reduction potentials of these species. The difference in reduction potentials is related to the p K ^M values of triplet state, p K _T^M , and semireduced dye radical, p K ^M_S , by thermodynamic consideration. p K _T^M (³AOH⁺/³AO) is determined to be 11.2. From thisp K _S^M (AOH./AO;) is estimated to be 17–18. This is in striking contrast to the protolytic equilibrium of the semireduced dye radicals found to be pK^F= 4.1. We conclude that the last value represents the second protonation equilibrium (AOH⁺₂./AOH). This conclusion is confirmed by spectroscopic data.     

SPINACH FERREDOXIN REDUCTION BY MEMBRANE-BOUND CHLOROPHYLL TRIPLET STATE VIA A DIQUAT MEDIATOR

Abstract Laser flash photolysis experiments have shown that the diquat analog containing a propylene bridge (PDQ²⁺), when electrostatically bound to negatively-charged vesicles containing chlorophyll, is able to mediate the rapid reduction ( k = 1.1 × 10⁵ s^-1) of spinach ferredoxin via electron transfer quenching of triplet state chlorophyll. The kinetics of formation and decay of reduced ferredoxin are consistent with a mechanism involving complex formation between oxidized ferredoxin and vesicle-bound PDQ²⁺. Under optimal conditions, approximately 15% of the quenched triplets yield reduced ferredoxin. This process is a model for soluble ferredoxin reduction which occurs in green plant photosystem I, and results in an appreciable storage of electromagnetic energy in the reaction products.     

QUENCHING OF CHEMIEXCITED TRIPLET ACETONE BY BIOLOGICALLY IMPORTANT COMPOUNDS IN AQUEOUS MEDIUM

Abstract— Thermolysis of tetramethyl-l,2-dioxetane is a convenient source of triplet acetone, which can be monitored in aerated solutions by the sensitized fluorescence of 9,10-dibromoanthracene. We have investigated the quenching of chemiexcited triplet acetone in air-equilibrated aqueous solutions containing the 9,10-dibromoanthracene-2-sulfonate ion by five classes of compounds: indoles, tyrosine derivatives, quinones, riboflavin, and xanthene dyes. Quenching rates for indoles, tyrosine and its 3,5-dihalogenoderivatives, and xanthene dyes (k_q= 10⁸-10⁹ M^-1 s^-1) are considerably smaller than the diffusion controlled rate, whereas those for quenchers with high electroaffinities, such as quinones (IP = 10–11 eV), approach the diffusion controlled rate (k_q= 10¹⁰ M^-1 s^-1). Energy transfer for riboflavin probably occurs by a triplet-singlet Förster type process.
A comparison of the present data with previous studies of quenching of enzymically generated triplet acetone (isobutanal/O₂/horseradish peroxidase) by the same classes of quenchers (except riboflavin) reveals that, independent of the nature of the quencher and the deactivation mechanism, the Stern-Volmer quenching constants ( k_q t⁰) are systematically about one order of magnitude higher in the enzymatic system. The difference is attributed to a longer lifetime of triplet acetone in the latter case, "protected" in an enzyme cavity against collisions with dissolved oxygen.     

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.     

TRIPLET EXCITATION TRANSFER INVOLVING β-IONONE. A KINETIC STUDY BY LASER FLASH PHOTOLYSIS

Abstract— Employing nanosecond laser flash photolysis, β-ionone (BI) has been examined as an acceptor and as a donor of triplet excitation. In the limit of diffusion-control as well as below it, the rate constants for the quenching of a series of sensitizer triplets by BI are2–3 times smaller than those by 2,4-hexadienal (HD), although the triplet energies (spectroscopic) of the two carbonyl-containing dienes are estimated to be the same (∼55 kcal mol^-1). We attribute the difference to a steric effect arising from ground-state geometric distortion and heavy alkyl-substitution in BI. In spite of possible exothermic energy transfer, BI triplet is nearly nonquenchable by azulene and ferrocene; this is explainable by torsional relaxation to an equilibrium geometry at which the vertical energy gap is smaller than 40 kcal mol^-1. The singlet oxygen yield from the interaction of BI triplet with oxygen in benzene is estimated to be 0.5, suggesting that spin-exchange and energy-transfer may be involved to the same extent in the oxygen quenching process.     

ELECTRON TRANSFER FROM PHOTOEXCITED SINGLET AND TRIPLET BACTERIOPHEOPHYTIN

Abstract— Nanosecond and picosecond. kinetic techniques have been used to study electron transfer from the first excited singlet state (Bph*) and the first excited triplet state (Bph ^T ) of bacteriopheophytin to p -benzoquinone. Quenching of the first excited singlet state by 40 m M p -benzoquinone results in a decrease in the lifetime of Bph* but does not lead directly to the formation of the π-cation radical (Bph†). In the presence of 8 M methyl iodide and 40 m M p -benzoquinone together, the singlet lifetime is reduced further; however, the quantum yield of Bph ^T is enhanced due to the increased rate of intersystem crossing between Bph* and Bph ^T . Electron transfer from Bph ^T to p -benzoquinone leads to the formation and detection of Bph†. The results are discussed in terms of the spin-selectivity of the reverse electron transfer process within the intermediate charge transfer complexes.