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.     

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.     

ABSORPTION AND EMISSION STUDIES OF ELECTRONIC STATES OF N-ARYLBENZAMIDES

Abstract— Absorption and emission spectra of several N-arylbenzamides have been measured. The quantum yields for their fluorescence were found to be dependent on matrix viscosity and temperature. Singlet-triplet splittings for these compounds were determined from their emission spectra and found to be abnormally small for π. π* states (˜ 1500 cm^-1). Indeed, the phosphorescence maxima of N-arylbenzamides occur slightly to the blue relative to their fluorescence maxima. Intersystem crossing efficiencies were determined for several of these compounds and are consistent with S₁→ S ₀ radiationless decay.     

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.     

ENERGY TRANSFER FROM CHEMIEXCITED ACETONE TO SUBSTANCES THAT DISPLAY ANOMALOUS FLUORESCENCE

Abstract— Excited acetone generated in the thermolysis of tetramethyldioxetane elicits the anomalous S₂→ S₀ fluorescence from azulene and from xanthione. In the case of azulene it could be demonstrated that (i) only the acetone singlets transfer energy to the S ₂ state and (ii) the acetone triplets are quenched. These energy transfer processes are diffusion-controlled.     

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.     

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.     

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.     

INTERACTIONS AMONG PHOTOSYNTHETIC ANTENNA EXCITED STATES

Abstract. The data of Kung and DeVault (1978) showing high-order fluorescence from chromatophores of photosynthetic bacteria are analyzed in relation to other data on first-order fluorescence of photosynthetic systems, particularly that of Monger and Parson (1977). The wavelengths of emission observed (down to 445 nm) require energy equivalent to two lowest singlet-excited states. The dependence on excitation intensity is best explained by any of the following third-order processes: (a) 3 S ₁→3 S ₀; (b) 2 S ₁+ T , → 2 S ₀+ T ₁; (c) S ₁+ 2 T ₁→ 3 S ₀. However, (c) is ruled out because it predicts heavy T ₁-destruction which is not observed. Contribution from the second order process: 2 S ₁→ S ₀ is probable, but even the data of Monger and Parson show that it is insufficient by itself. Two-photon absorption: S ₀+ hv ₁→ S ₁; S ₁+ hv ₁→ S _n; S _n S ₀+ hv ₂ could also account for the high-order fluorescence and its dependence on excitation intensity. [ S ₀, S ₁ S _n are ground, first excited and a higher excited singlet states, respectively, of antenna bacteriochlorophyll, T _t is the lowest triplet state, c/v , is the exciting wavelength (694 or 868 nm) and c/v ₂ the wavelength of the high-order fluorescence (445, 535. or 600 nm), where c = velocity of light.] Maximum values are estimated for some of the rate constants.     

POTENTIAL PHOTOSENSITIZERS FOR PHOTODYNAMIC THERAPY—II. PHOTOPHYSICAL PROPERTIES OF [26] PORPHYRIN

Abstract— –Photophysical properties of [26] porphyrin (26 P) were investigated in chloroform. The quantum yields of fluorescence, of S₁→ T₁ intersystem crossing and singlet oxygen formation were measured. The purity, stability, the strong absorption in the red (δ_max= 783 nm; ε_max= 28 000 M ¹ cm^-1) and the ability of singlet oxygen formation recommend 26 P as potential photosensitizer for tumor therapy.     

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.     

Photostability and Spectral Properties of Fluorinated Fluoresceins and their Biarsenical Derivatives: A Combined Experimental and Theoretical Study

New fluorinated biarsenical derivatives with improved optical properties based on highly photostable analogs of fluorescein were recently introduced. The photophysical parameters of the triplet excited states as well as photosensitized oxidation reactions of these dyes were determined in order to investigate the influence of molecular structure on the exceptional photostability of these fluorophores. The lack of correspondence between triplet quantum yields and lifetimes with the photobleaching rates of some of the fluorophores of the series suggests that differential reactivities of the excited states with ground state oxygen accounts for the different photodegradation resistances. The UV–visible absorption and emission spectra of the fluorinated fluoresceins and their biarsenical derivatives were evaluated using a TD-DFT/BP86/6-31G** approach, taking bulk solvent effects into account by means of the polarizable continuum model. The calculated properties are in good agreement with experimental data. The S₀→S₁ vertical excitation energies in the gas phase and in water were obtained with the optimized geometries of the excited states. This type of calculation could be used in the rational design of new dyes.     

EXCITED TRIPLET-SINGLET INTERSYSTEM CROSSING IN ISOALLOXAZINES

Abstract— Higher excited triplet states originating from the lowest triplet state of isoalloxazines by absorption of light with Λ - 600 nm undergo "inverse" intersystem crossing to the singlet manifold [φ( T_n-S_m ) = 8 × 10^-3] followed by rapid internal conversion and "normal" fluorescence S₁-S₀ with Λ_m= 540 nm.     

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.     

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.     

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.     

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.     

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.     

Depopulation of Highly Excited Singlet States of DNA Model Compounds: Quantum Yields of 193 and 245 nm Photoproducts of Pyrimidine Monomers and Dinucleoside Monophosphates

Abstract— Formation of uracil and orotic acid photodimers, uridine and 5'-UMP photohydrates, TpT photodimers and (6-4)photoproducts, dCpT photohydrates and (6-4)photo-products and UpU, CpC and CpU photohydrates were studied in neutral deoxygenated aqueous solution at room temperature upon irradiation at either 193 or 254 nm. The photoproducts were identified and quantified and the contribution from photoionization to substrate decomposition, using λ_irr= 193 nm, was separated. The ratio of the quantum yields of respective stable products,η=φ¹⁹³/φ²⁵⁴ is indicative of the yield of internal conversion from the second to the first excited singlet state, S₂→ S₁. For the observed photodimers η decreases from 0.94 for uracil to 0.7 for TpT and further to 0.55 for orotic acid. For the (6-4)photoproducts of TpT and dCpT T| = 0.5-0.8 and for the photohydrates in the cases of UpU, CpC, CpU and dCpT TJ ranges from 0.55 to 1.     

REEVALUATION OF THE SPECTRAL AND KINETIC PROPERTIES OF HO2 AND O2- FREE RADICALS

Abstract— The spectra and molar absorbances of the HO₂ and O₂^- free radicals have been redetermined in aqueous formate solutions by pulse and stopped-flow radiolysis as well as by ⁶⁰Co gamma-ray studies. The extinction coefficients at the corresponding maxima and 23°C are ²²⁵= 1400 ± 80 M ^-1 cm^-1 and ²²⁵= 2350 ± 120 M ^-1 cm^-1 respectively. Reevaluation of earlier published rate data in terms of the new extinction coefficients yielded the following rate constants for the spontaneous decay of HO₂ and O₂^-: K _Ho2+HO2= (8.60 ± 0.62) × 10⁵ M ^-1 s^-1; K _Ho2+O2-= (1.02 ± 0.49) × 10⁸ M ^-1 s^-1; K _Ho2+O2- < 0.35 M ^-1 s^-1. For the equilibrium HO₂→ O₂^-+ H⁺ the dissociation constant is K _Ho2= (2.05 ± 0.39) × 10^-5 M or p K _HO2= 4.69 ± 0.08. G (O₂^-) has been evaluated as a function of formate concentration.