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.     

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.     

NH3R++NHR2(R=H,CH3)体系质子传递反应的从头算研究

用从头计算法在HF/6-31G^*基组水平上研究了NH₄⁺+NH₃→NH₃+NH₄⁺,NH₄⁺+NH₂CH₃→NH₃+NH₃CH₃⁺,NH₄⁺+NH(CH₃)₂→NH₃+NH₂(CH₃)₂⁺以及NH₃CH₃⁺+NH₂CH₃→NH₂CH₃+NH₃CH₃⁺等4个体系的质子传递反应的机理.结果表明:(1)上述质子传递反应均具有双阱型的势能面,质子沿N(1)、N(2)连线直接传递;(2)质子受体分子中的甲基对质子传递起促进作用,而质子给体离子中的甲基则阻碍质子的传递。     

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.     

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.     

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.     

THERMALLY STIMULATED LUMINESCENCE OF UV-AND X-IRRADIATED CAFFEINE

Abstract— Thermally stimulated luminescence studies of UV- and X-irradiated caffeine have been conducted in the interval77–300 K. The X-ray-induced glow curve exhibits peaks at 102, 128, 158, and 198 K, whereas the UV-induced curve is characterized by peaks with maxima at 120 and 208 K. Analysis of the X-ray-induced glow peaks revealed that the 102 and 128 K peaks obeyed first-order kinetics, while the 158 and 198 K ones followed orders of 1.3 and 1.8, respectively. Trapping parameters associated with the peaks were also elucidated. Concomitant emission spectra of each glow peak (both UV and X-ray induced) were identical and consisted of a band with well-defined maxima at 405 and 480 nm which are attributed to singlet (S₁→ S₀+ h v ) and triplet (T₁→ S₀+ h v ) transitions, respectively. At elevated temperatures the triplet transition is not observed due to quenching of the triplet state or to triplet-triplet interactions. An energy-level diagram depicting the thermally stimulated processes in irradiated caffeine is presented.     

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.     

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.     

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.     

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.     

LASER FLASH PHOTOLYSIS STUDIES OF DNA-COMPLEXED ETHIDIUM BROMIDE

Abstract— Laser flash photolysis studies of DNA-complexed ethidium bromide were undertaken. We have observed a singlet-singlet (S₁-S_n) absorption process for DNA-complexed ethidium bromide. The observed lowest singlet excited state lifetime was 21 ± 2 ns. The molar difference extinction coefficient was measured to be 2.4 ± 0.4 × 10³M^-1 cm^-1 at 370 nm. The assignment of this transition was confirmed by time resolved fluorescence measurements.     

BLUE LIGHT INDUCED, REVERSIBLE IN ACTIVATION OF THE TONOPLAST-TYPE H+-ATPase FROM CORN COLEOPTILES IN THE PRESENCE OF FLAVINS

Abstract— Irradiation (λ_max 447 nm; 58.5 W m^-2) of a microsomal membrane fraction of corn coleoptiles for 5 min in the presence of the in vivo concentration of riboflavin inactivates the tonoplast-type H⁺-ATPase. This inhibition is O₂-dependent, is enhanced in D₂O and suppressed by NaN₃, indicating participation of singlet molecular oxygen in the inactivating mechanism. Besides singlet oxygen, the superoxide anion (O₂^-) is generated during irradiation, which obviously has no effect on the H⁺-pumping activity. However, in the presence of superoxide dismutase (SOD), O₂^- is transformed into H₂O₂ which causes an additional strong inhibition of H⁺. ATPase activity. This inhibition can be increased by ethylenediaminetetraacetic acid (EDTA), which is known to be an electron donor of the excited flavin molecule. In contrast, catalase prevents the H₂O₂-mediated photoinactivation of the H⁺ -ATPase. The light dependent inactivation of H⁺-transport does not occur if reduced glutathion (GSH) is added prior to or after irradiation. These results indicate that the blue light mediated inhibition of the H⁺-ATPase is mediated by singlet oxygen and H₂O₂ which oxidize essential SH-groups of the enzyme into disulfides. Reduction of the formed disulfides by GSH restores the activity of the enzyme.     

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.     

Photophysical Properties of the Cationic Form of Neutral Red

Abstract— Photophysical properties of the cationic form of neutral red (NRH⁺), a phenazine-based dye of biological importance, have been investigated in several protic and aprotic solvents using optical absorption, steady-state and time-resolved fluorescence and picosecond laser flash photolysis techniques. Absorption and fluorescence characteristics of the dye in protic solvents indicate the existence of intermolecular hydrogen bonding between the NRH⁺ and solvent molecules in the ground state as well as in the excited state. Measurements of the fluorescence lifetime in normal and heavy water also support the formation of intermolecular hydrogen bonding. Time-resolved transient absorption spectra obtained in the picosecond laser flash photolysis experiments show only the absorption band due to the S_n← S₁ absorption. The picosecond transient absorption results do not indicate any spectral shifts attributable to the hydrogen bond formation dynamics between the excited NRH⁺ and the protic solvent molecules. It is inferred that the hydrogen bonding dynamics are much faster than the time resolution of our picosecond setup (∼35 ps).     

ELECTRON TRANSFER FROM THE EXCITED STATE OF TYROSINE TO COMPOUNDS CONTAINING DISULFIDE LINKAGES

Abstract— The flash photolysis of aqueous solutions of tyrosine has been studied in the presence of various concentrations of the cyclic disulfide sodium lipoate (thioctic acid, Na⁺ salt). In addition to the formation of phenoxyl radicals and hydrated electrons (and possibly H atoms) from the photoionization of tyrosine, the characteristic spectrum of the radical anion RSSR^- of lipoate was also observed in neutral as well as in alkaline solutions. From the dependence of these yields upon the concentration of lipoate, it was found that a long–lived triplet excited state of tyrosine, rather than the singlet excited state, is involved in these reactions. The negative radical ions RSSR^- are formed by two distinct pathways: (a) Na⁺–lipoate reacts with the solvated electrons which are ejected from the tyrosine triplets ³Tyr → RO.+ e ^-_aq+ H⁺ followed by e ^-_aq+ RSSR → RSSR^-, and (b) by direct interaction of lipoate with triplet excited tyrosine, resulting in the transfer of a negative charge from tyrosine to the disulfide linkage. At high lipoate concentrations, the singlet excited state of lipoate is quenched, k ₄= 1.6 × 10¹⁰ M ^-1 sec^-1, but this reaction does not lead to the formation of RSSR^- radical ions.     

RADICAL CATION PRODUCTION IN THE PHOTOLYSIS OF 5-M-ETHOXY-1-M-ETHYLINDOLE

Abstract— The flash photolysis of 5-m-ethoxy-1-m-ethylindole in aqueous media was studied for the purpose of assigning the absorption spectrum of the radical cation. Transients produced in this study were analogous to those formed in the photolysis of 5-m-ethoxyindole. The major transient observed with an absorption maximum of 460 nm was O₂-s_-ensitive and had a lifetime of 20 μs in nitrogen saturated solutions. One radical species is produced with absorption maxima at 445 and 530 nm. Ionic strength effects on the reaction of this species with I⁻ confirms that it is the radical cation of 5-m-ethoxy-l-methylindole. The effect of H⁺ and Br⁻ on the fluorescence, radical cation and triplet yields is discussed in relation to the mechanism of transient formation.     

INTERACTION OF HYDROXYLAMINE WITH THE WATER-OXIDIZING ENZYME INVESTIGATED VIA PROTON RELEASE*

Abstract— Photosynthetic water oxidation is a four-step redox process which is driven by a one-quantum-one-electron reaction center. Stepwise electron Abstract—ion from the water-oxidizing enzyme is accompanied by stepwise proton release with the following stoichiometric pattern at given half-rise times: 1 H⁺ (250 μs, S₀→ S₁):0 H⁺(S₁→ S₂): 1 H⁺ (200 μs, S₂→ S₃): 2 H⁺ (1.2 ms, S₃→ S₄→ S₀). (Förster and Junge, 1985, preceding article in this issue). Hydroxylamine at low concentrations (?100 μ M) appears to compete with water at the active site of the water- oxidizing enzyme. Its interference shifts the dark state of the water-oxidizing enzyme by two steps backwards (Bouges, 1971). We found that the hydroxylamine-induced shift was also reflected in the stoichiometric pattern and in the kinetics of proton-release. In the presence of hydroxylamine, two protons per reaction center were released with a half-rise time of ? 2 ms upon the first exciting flash given to dark adapted thylakoids. This was slower than observed for each of the protons released during unperturbed water oxidation. One proton was released upon the second flash. The half-rise time of the main component observed upon the second flash in hydroxylamine-treated samples agreed with the one observed upon the fourth flash in the absence of hydroxylamine, which had been attributed to the S₀→ S₁ transition. The two protons which were observed upon the first flash in hydroxylamine-treated thylakoids may be due to hydroxylamine oxidation or to the association of water to the catalytic manganese center after detachment of oxidized hydroxylamine from its binding site.     

FLUORESCENCE CHARACTERISTICS OF INDOLES IN NON-POLAR SOLVENTS: LIFETIMES, QUANTUM YIELDS AND POLARIZATION SPECTRA

Abstract— Fluorescence lifetimes, quantum yields and polarization spectra were measured for indole, 3-methylindole and 2,3-dimethylindole in non-polar solvents. The results indicate simultaneous emission from thermally equilibrated ¹L_a and ¹L_b levels, with ¹L_a→¹ A dominating the 2,3-dimethylindole emission, and ¹L_b→¹ A dominating the indole emission. These results are consistent with previous assignments of the 0-0 transitions in absorption for these compounds. Radiative rates are: ¹L_a→¹ A , 2·0 × 10⁸ S^-1 and ¹L_b→¹ A . 0·62 → 10⁸ S^-1. In addition, the temperature dependence of the excitation and emission spectra are presented, which show that aggregation occurs with these indoles in hydrocarbons below approximately - 110°C. Possible applications to tryptophyl emission in the hydrophobic interiors of proteins are briefly discussed.     

THE USE OF NEUTRAL RED TO MONITOR THE SURFACE POTENTIAL OF THE PURPLE MEMBRANE

Abstract— The binding of neutral red to purple membrane has been studied. The intrinsic p K _a and the apparent p K _a, of bound neutral red were determined by titration and by measuring the binding ratio of neutral red to purple membrane as a function of pH. The surface potential of purple membrane was inferred from the difference between these two p K _as. The H⁺/M412 ratio at different ionic strengths was also measured and compared with the surface potential. The results show that the H⁺/M412 decreased as the surface potential increased due to decreased salt concentrations. However, this correlation holds only for KCl concentrations higher than 30 m M . At lower salt concentrations, the change in surface potential is always less than the variation in the H⁺/M412 ratio.