LONG-RANGE TRIPLET-SINGLET ENERGY TRANSFER FROM ENZYME GENERATED TRIPLET ACETONE TO XANTHENE DYES

Enzyme-generated and protected acetone transfers its energy to xanthene dyes by a nontrivial process. The relative populations of the S₁ state are indicative of the operation of a heavy atom effect, being 1:15:100 for fluorescein, eosine and Rose Bengal. On the other hand Stern-Volmer constants and k _ET⁰ values are similar for the three dyes and are remarkably higher than for collisional processes.
It is tentatively suggested that at least for eosine and Rose Bengal, a triplet-triplet cxciton transfer may occur to populate an upper triplet state of the dye followed by heavy-atom enhanced intersystem crossing to the S₁ state.     

DIFFERENCE SPECTRA OF THE OXIDIZED INTERMEDIATES IN THE PHOTOSYNTHETIC OXYGEN-EVOLVING SYSTEM: EVIDENCE FOR A SMALL S0→S1 ABSORPTION CHANGE

A consensus has emerged in the recent literature on the fact that the UV difference spectrum of the first oxidation step (S₀→ S₁) of the photosynthetic oxygen-evolving complex is significantly different and generally smaller than the spectra of the higher oxidation steps (S₀→ S₁and S₂→ S₃). Discrepancies still persist, however, notably in the 300 nm region where the S₀→ S₁ change was either reported to be markedly smaller than the other changes, or, at variance, to have a similar amplitude. A novel approach is proposed here for estimating the ratio of these changes, requiring no estimate of the Kok model parameters, such as the initial S₀/S₁ratio, or damping coefficients. This was achieved by comparing the absorption difference between two fully deactivated states, differing only in their S₀/S₁, distribution, with the flash-induced changes measured from these states. The results show that, at two wavelengths around 300 nm, the S₀→ S₁ change is at least 4 times, and probably 5–6 times smaller than the S₀→ S₁change.     

WEAK COMPLEX FORMATION BETWEEN PYRENE AND POLAR SOLVENTS

Abstract— Evidence of complex formation between pyrene and polar solvent molecules has been obtained by monitoring the S₁,← S₀ and S₂← S₀ absorption spectra of pyrene. The solvents employed are propionitrile, valeronitrile, dichloromethane, and dioxane. The stabilization energy is found to be small relative to general hydrogen bond energies, but somewhat high relative to the Boltzmann energy. These are in good accord with the findings of Lianos and Georghiou [ Photochem. Photobiol. 30 ,355 (1979)] that pyrene forms 1:1 molecular complexes with several dipolar solvents in the ground state.     

Photophysical properties of the lowest excited singlet and triplet states of thio- and seleno-psoralens

The decay processes of the lowest excited singlet and triplet states of five heteropsoralens (HPS) were investigated by steady-state and shift-phase fluorometry and by laser-flash photolysis in different solvents. The emission spectra of HPS are detectable only in trifluoroethanol (TFE), where fluorescence lifetimes (τ_F) and quantum yields (φ_F) were measured. The triplet lifetimes (τ_T), triplet (φ_T) and singlet-oxygen production (φ_Δ) quantum yields were determined in benzene, ethanol and TFE by laser-flash photolysis. Semiempirical (INDO/1-CI) calculations allowed the nature of the lowest excited singlet and triplet states and transition probabilities to be obtained. Theoretical and experimental results indicate that the two lowest excited singlet states S₁ and S₂ of HPS are close-lying and different in nature (π,π* and n,π*). The "proximity effect" between these two states controls the photophysical properties of HPS as it does for the other furocoumarins. However, HPS have a peculiar behavior with respect to the related compounds because they are fluorescent and have, in three cases, detectable intersystem crossing only in TFE. This behavior can be tentatively explained by a different energy gap and/or order between the S₁ and S₂ states.     

SOLVENT EFFECTS ON THE ELECTRONIC ABSORPTION AND FLUORESCENCE SPECTRA OF INDOLE-4-CARBOXYLIC ACID: PROTOTROPIC EQUILIBRIA IN AQUEOUS SOLUTIONS

Abstract— The absorption and fluorescence spectra of indole-4-carboxylic acid in various solvents have indicated that the -COOH group is more planar with respect to the indole ring in the first excited singlet state (S₁) than in the ground (S₀) state. Relatively large Stokes' shifts indicate that polarisability and dipole moment of the molecule are increased predominantly upon excitation. Prototropic reactions in the S₀ and S₁ states are the same. The -COO^- and -COOH+₂ groups are not coplanar in the S₀, but coplanar in the S₁ state. pH-dependent fluorescence spectra have revealed that both protonation and deprotonation of the -COOH group increase the basicity of the molecule upon excitation.     

STOICHIOMETRY and KINETICS OF PROTON RELEASE UPON PHOTOSYNTHETIC WATER OXIDATION

Abstract— We studied the magnitude and the rise kinetics of proton release into the interior of thylakoids by flash spectrophotometty with neutral red as pH indicator. Excitation of dark-adapted thylakoids by a series of between 4 and 11 flashes produced a complex pattern of proton release into the thylakoid lumen. Proton release upon each flash was time resolved.
A slow component of proton release oscillated weakly in magnitude with period of two as function of flash number. It exhibited a half-rise time of approximately 20 ms from the very first flash on, and it was abolished by inhibitors of plastohydroquinone oxidation. This component was attributed to the oxidation of plastohydroquinone by PS I via the Cytb₆/f complex.
Additionally, rapid and multiphasic proton release was observed with half-rise times of less than 2 ms which exhibited a pronounced and damped oscillation with period of four as function of flash number. This rapid proton release was attributed to water oxidation. A detailed kinetic analysis suggested that proton release occurred with the following stoichiometry and with the following half-rise times during the transitions S₁ S_i+1 of water oxidation: 1 H⁺(250 μs, S₀→₁): 0 H⁺(S₁→ S₂):1 H⁺(200 μs, S₂→S₃):2 H⁺(1.2 ms, S₃→ S₄→ S₀) . Proton release and proton rebinding upon oxidation and reduction of the intermediate electron carrier Z, respectively, may have influenced the kinetics of the respective proton yields but not the stoichiometric pattern.     

THERMOLUMINESCENCE EVIDENCE FOR CHLORIDE REQUIREMENT IN THE S2→S3 TRANSITION OF THE WATER-SPLITTING SYSTEM

Abstract— The role of chloride in photosynthetic oxygen evolution was investigated by means of thermoluminescence measurements. It was found that chloride depletion in isolated chloroplasts almost completely abolished the B₁ thermoluminescence band (S₃Q_B⁻ recombination) but diminished only slightly the amplitude of the B₂ band (S₂Q_B⁻ recombination). The B₂ band could be excited to full intensity by the first flash of a flash series and subsequent flashes caused no further change in the amplitude of the band. These observations suggest a block in the S₂→S₃ transition of the water-splitting system in chloride-depleted chloroplasts. Readdition of chloride provided evidence that the inhibitory effect of chloride removal is reversible.     

THE PHOTOPHYSICS OF p-AMINOBENZOIC ACID

Abstract— The lowest-lying allowed UV transition in p -aminobenzoic acid (PABA) is assigned Γ→¹L_a based on quantitative absorption and fluorescence studies, as well as semiempirical PM3 multielec-tron configuration interaction calculations. The oscillator strengths, fluorescence quantum efficiencies and lifetimes are reported for PABA in several polar, nonpolar, protic and aprotic solvents (aerated) at 296 K. Reasonable agreement is found between the observed radiative rate constant and that calculated from the absorption and fluorescence spectra. Shifts in the absorption and fluorescence spectra in aprotic solvents are analyzed in terms of the Onsager reaction field model; results are consistent with an increase in dipole moment of ca 4 D between the relaxed S₀ and S₁, states. No evidence is found for the emission from the amino-twisted form of PABA in all solvents studied although calculations show that the amino-twisted S, state is highly polar, but higher in energy by ca 35 kJ/mol ( in vacuo ). The fluorescence efficiency is excitation wavelength independent in both methylcyclohexane and water. The temperature dependence of the nonradiative rate constant (from S₁) was studied in several solvents. Nonradiative decay may be due to intersystem crossing, which would be fast enough to compete with thermally activated intramolecular NH₂ twisting. The phosphorescence spectrum and lifetime obtained in an EPA glass at 77 K are reported, and the triplet energy of PABA is estimated.     

PHOTOPHYSICAL PROCESSES IN TROPOLONE, α-METHOXY-TROPONE AND COLCHICINE

Abstract— The spectral characteristics of the emission observed from tropolone, α-methoxy-tropone and colchicine in EPA at 77 K are reported. Luminescence polarization, lifetime, quantum yield and energy transfer experiments provide evidence that the luminescence observed is S₁→ S₀ fluorescence and that the lowest excited singlet state is π, π* The two compounds α-methoxy-tropone and colchicine have been found to exhibit dual luminescence. No phosphorescence could be detected in these three molecules. The photophysical processes for each molecule are discussed and their respective energy level diagrams are also 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.     

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.     

WAVELENGTH EFFECTS ON THE PHOTOPROCESSES OF INDOLE AND DERIVATIVES IN SOLUTION

Abstract— The two main primary photoprocesses (electron ejection and H-atom release) for indole, 5-methoxyindole and N-methylindole in various polar and nonpolar solvents were studied as a function of the excitation energy and were correlated with the corresponding fluorescence quantum yields. In hydrocarbon solvents, N–H bond cleavage is the main primary photoprocess from the ¹B_b band of the substrates with the exception of N-methylindole. In alcohols, both processes are of negligible importance. Hydrated electrons (e⁻_aq) are ejected from the relaxed singlet states of all three compounds in aqueous solutions with a similar yield for excitation at 280 and 254 nm (¹L_a and ¹L_b states). The yield increases when the excitation is into the ¹B_b band. The quantum yields of the two primary processes from the higher excited states are generally lower than the fraction of molecules not converting to the fluorescent state. This is explained by an efficient back reaction in competition with a thermally activated radical release from an intermediate state or radical pair formed from the S₂ (¹B_b) state. The non-occurrence of a photoionization energy threshold is discussed.     

A Photophysical Study of Purines and Theophylline by Using Laser-Induced Optoacoustic Spectroscopy

Abstract— The photophysics of purinic compounds (purine, 6-meth-ylpurine, 6-aminopurine [adenine], 6-chloropurine, 6-methoxypurine) and theophylline in acetonitrile solution were studied by pulsed laser-induced optoacoustic spectroscopy (LIOAS) exciting at 266 nm. The effect of O₂, Xe and MnCl₂ on the photophysical behavior of these compounds was studied; as well, the formation quantum yield of purine and 6-methylpurine triplet states were determined, with φ_T= 0.88 ± 0.03 for both compounds. Multiphotonic and depletion processes were observed at high laser fluences. In order to explain this behavior, theoretical UV-visible absorption electronic spectra from both the S₀ and S₁ state have been calculated for purines and theophylline by using the semiempirical PM3 and ZINDO/S methods.     

PHOTOLYSIS OF SUBSTITUTED NAPHTHALENES ON SiO2 AND Al2O3

Abstract— Photolysis of naphthalene on the surface of SiO₂ under an atmosphere of air produces phthalic acid as the only major photoproduct, accounting for 49%o of the consumed naphthalene. Photolysis on Al₂O₃ also produces phthalic acid, in 31% yield. Photolysis of 1 -methylnaphthalene on SiO₂ proceeds under similar conditions to produce 2-acetylbenzoic acid (35%) as the major photoproduct with the production of a small amount of I-naphthaldchyde (6%). 1-Cyanonaphthalene does not photooxidize under similar conditions. The presence of oxygen is necessary for the photodecomposition of naphthalene and 1-methylnaphthalene to proceed. Superoxide formed from the photolysis of naphthalene at the SiO₂/air interface is readily observed by electron paramagnetic resonance spectroscopy. In the absence of naphthalene no superoxide is observed. A mechanism involving electron transfer from the S₁ state of the naphthalene to O₂ is proposed on the basis of these observations and related literature precedent.     

LIGHT-DEPENDENT AND LIGHT-INDEPENDENT PRODUCTION OF HYDROGEN GAS BY PHOTOSYNTHESIZING RHODOSPIRILLUM RUBRUM MUTANT C

Abstract. Rhodospirillum rubrum mutant C grew photosynthetically in the light and produced copious amounts of H₂. During light-growth mutant C produced 7.9mmol of H₂ in medium with 9mmol of Na-pyruvate per mg protein. When parent strain R. rubrum S ₁ was grown similarly, these cells only produced a trace amount of H₂. Light-grown mutant C evolved H₂ by H₂-nitrogenase and formic hydrogenlyase. Although both hydrogenases were previously detected in R. rubrum S₁, the activities of the reactions in light-grown mutant C were higher and they operated under different conditions. In the parent strain S₁, the production of nitrogenase was strongly repressed during growth in medium enriched with organic nitrogen and the cells only reduced 0.06 pmol of acetylene per mg protein after 30 min in the light. Under similar conditions, nitrogenase activity measured by the acetylene reduction test in mutant C was 10-fold greater. In addition to nitrogenase, mutant C also produced large amounts of H₂ with formate as an intermediate when the cells were grown with Na-pyruvate. Formic hydrogenlyase in mutant C operated equally well in anaerobic light or dark conditions. The analogous formate oxidation reaction in parent strain S₁ only functioned in the dark. These data, compared with results with R. rubrum S₁ suggested that C was a regulatory mutant. Additional observations suggested that formic hydrogenlyase occurred constitutively in R. rubrum . Pyruvate formate-lyase, however, which produced formate for formic hydrogenlyase, was only detected in the cells after growth in media with Na-pyruvate. The reaction was not formed when R. rubrum was grown in the light in media with dl-malate as the sole carbon substrate.     

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.     

LUMINESCENCE STUDIES OF QUINIZARIN AND DAUNORUBICIN

Abstract— The absorption and emission spectra of quinizarin (1,4-dihydroxy-anthraquinone) have been investigated in hydrocarbon and alcoholic solvents. Fluorescence spectra in 3 different Shpolskii matrices were recorded at 14 K. Vibrational analyses of these spectra revealed the presence of 3, 8, and 9 sites in octane, heptane, and hexane matrices, respectively. The fluorescence lifetime was found to be 6.5 ns in hexane and EPA. Fluorescence photoselection measurements in EPA (77 K) showed that the first 4 electronic transitions of quinizarin are polarized parallel, parallel, perpendicular, and parallel to the long molecular axis and can be assigned, in order of increasing energy, to ¹B₂, ¹B₂, ¹A₁ and ¹B₂ (ππ*)→¹A₁(C_2v,) transitions, respectively. The fluorescent transition is assigned as ¹B₁ (ππ*)→¹A¹. The absence of phosphorescence is attributed to the intramolecular hydrogen bonding present which displaces the parent anthraquinone n →* states above the ππ* states, thereby rendering the intersystem crossing (S₁-T₁) radiationless pathway inefficient.
Photoselection measurements on daunorubicin, a substituted quinizarin and known anticancer drug, revealed an absorption band polarization pattern identical to that of quinizarin. These results are in part at variance with assumptions used in previous work on the intercalation specificity of daunorubicin with DNA.     

ACID DEPENDENT FLUORESCENCE QUANTUM YIELD AND TETRAMETHYL- 1,2-DIOXETANE CHEMI-ENERGIZED FLUORESCENCE OF ERGOSTATETRAENONE*

Abstract. –In acidified acetonitrile, ergostatetraenone (E) efficiently quenches the tetramethyl-1,2-dioxetane (TMD) chemiluminescence at 410 nm, with the appearance of a new chemiluminescence at ca. 530 nm due to ergostatetraenone fluorescence. Control experiments exclude energization by triplet acetone and the trivial process (emission and reabsorption), establishing singlet-singlet energy transfer between excited singlet acetone (A) and ground state ergostatetraenone as major pathway. From the kinetics the rate constant for singlet-singlet energy transfer ( K ^ss_A,1,) was estimated to be ca. 10^{1 2}S^-1. The chemiluminescence yield (φ^CL) for the TMD/ergostatetraenone system was determined to be ca. 12 × 10^-8. The fluorescence efficiency of ergostatetraenone (φ) in acidified acetonitrile is a function of acid concentration, ranging between 0.06-0.82. The efficiency of singlet acetone production (α) was found to be 500-fold lower than in neutral medium due to a competing acid-catalysed, dark decomposition of TMD. A 60-fold light enhancement has been established for this binary chemiluminescent system.     

STOICHIOMETRY OF THE REDOX CHANGES OF MANGANESE DURING THE PHOTOSYNTHETIC WATER OXIDATION CYCLE

Abstract— The UV absorbance change associated with the oxygen-evolution cycle is investigated as to its correlation with the successive redox (S-) states of the system. This change is probably due to a manganese oxidation within a multinuclear Mn-cluster. The stoichiometry of its formation and destruction during the S-cycle has been a matter of controversy: (0,1,0,-1) according to velthuys, B. R. (1981) in Photosynthesis II , (Edited by G. Akoyunoglou), pp. 75–85. Balaban International Science Services, Philadelphia, or (1, 1, 1, -3) according to Dekker, J. P., Van Gorkom, H. J., Wensink, J. and Ouwehand, L. (1984) Biochim. Biophys. Acta 767, 1–9. We show that the data obtained in flash-series experiments on dark-adapted material may be fitted as satisfactorily by both models. More discriminating tests were devised, varying parameters such as the double-hits probability or the initial distribution of the S-states. These experiments clearly indicate that no (or little) absorbance change occurs both upon S₀→S₁ and S₂→S₃ transitions. This suggests that the correct stoichiometry is (0,l,0,-1). The possibility of a non-integer stoichiometry may also be considered.     

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.