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.     

FLUORESCENCE QUENCHING OF INDOLIC COMPOUNDS BY ALIPHATIC AMINO ACIDS. EVIDENCE FOR EXCITED STATE COMPLEXES

Abstract The fluorescence quenching of indole, tryptophan, tryptamine and indole-3-acetic by aliphatic amino acids was studied. The bimolecular rate constant ( k _q) for the deactivation of the excited state was determined. The k _q values were in the range 0.6 × 10⁸–1.6 × 10⁹ M ^–1 S^–1 and they increased in the order tryptophan < tryptamine < indole ≈ indole-3-acetic acid. When the rate constant was corrected for diffusion al effects a good linear correlation was found between the log ( k '_q) and the ionization equilibrium constant of the carboxylic group of the amino acid (p ^k _a1). This was interpreted as arising from a charge transfer mechanism in which the indole moiety acts as an electron donor and the carbonyl group of the amino acid as the acceptor.
The activation parameter for the quenching processes were also determined. The ΔH^≠ values were in the range —4.0 to +4.0 kcal/mol and the ΔH^≠ in the range –7 to –37 e.u. For the systems with lower values of k _q negative values for ΔH^≠ were observed. A good enthalpy-entropy compensation was found with an isokinetic temperature of 229 K. These results suggest that a common mechanism is operating for all the systems and that it involves the formation of an excited state complex between the indolic compound and the amino acid.     

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.     

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.     

QUENCHING OF THE FLUORESCENCE OF 8-METHOXYPSORALEN BY PROTONS

Abstract— The quenching of 8-methoxypsoralen (8-MOP) fluorescence by protons was observed to occur at the diffusion controlled rates in aqueous solutions at room temperature. Enhanced basicity of 8-MOP in the excited state compared to the ground state is expected on theoretical grounds. The fluorescence yield. which we determined as 6.3 × 10^--⁴ at pH 1 is surprisingly low and indicative of extremely fast radiationless decay pathways. The fluorescence lifetime of 8-MOP in neutral aqueous solution is on the order of 1–2 ns.     

DELAYED FLUORESCENCE AND THE REVERSAL OF PRIMARY PHOTOCHEMISTRY IN RHODOPSEUDOMONAS VIRIDIS

Abstract— Delayed fluorescence from chromatophores of the photosynthetic bacterium Rhodopseudomonas viridis was measured at temperatures below 0°C. A component with a decay half-time of about 7 ms was found. Its intensity was directly proportional to the number of reaction centers in the P985⁺·A^- state. During prolonged illumination it faded as electrons moved forward along the electron transport chain from the primary acceptor, A, (P985⁺·A^-→P985⁺·A), and its decay in the dark paralleled the disappearence of the P985⁺ electron paramagnetic resonance absorption. The data suggest that this component of delayed fluorescence results from a direct reversal of the primary light reaction. While the rate of the P985⁺middot;A^-→P985·A reaction was almost independent of temperature, delayed fluorescence intensity displayed an apparent activation energy of 0°2 eV. It is concluded that the P985⁺·A^-→P985·A reaction proceeds by parallel radiative and nonradiative routes. The direct proportionality between delayed fluorescence and the concentration of P985⁺·A^- pairs seems to preclude an involvement of triplet-triplet annihilation or dependence of delayed fluorescence upon the variable prompt fluorescence yield.     

LASER FLASH PHOTOLYSIS OF INDOLE IN THE PRESENCE OF AMINO ACIDS

The laser flash photolysis of indole at 265 nm in the presence of glycine, proline and hydroxy proline was studied. The relative yields of c _aq, triplet state, and indole cation radical were determined in the absence and in the presence of the amino acids. The yields were determined as a function of laser intensity and the values at very low intensity were compared with the fluorescence quenching results. It was concluded that in these conditions the photoionization of indole occurs via the fluorescent state. From the curves of triplet yield vs laser intensity, the triplet quantum yield extrapolated at low laser intensity was obtained, φr = 0.55 φ 0.05, relative to the literature value of 0.15 for φ_eag. This gives φ_F+φ_eaq= 1.0 ± 0.1 at room temperature. When proline and hydroxy proline were used as singlet quenchers, the yield of In was greater than the yield of c_aq. This was considered as evidence that a fraction of the quenching processes leads to complete electron transfer from indole to the amino acids.     

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.     

RELATIVE REACTIVITIES OF THE EXCITED STATES OF FUROCOUMARINS FOR [2+2] PHOTOCYCLOADDITION REACTION WITH TETRAMETHYLETHYLENE

Abstract— The Stern-Volmer constants for fluorescence quenching by tetramethylethylene decrease in the order DMC ≫ DHP > F-2 > 8-MOP. The same order was observed for the quantum yields of [2+2] cycloaddition reaction with tetramethylethylene on direct irradiation. In [2+2] photocycloaddition of F-2 with tetramethylethylene in ethanol, the ratio of quantum yields deduced from singlet and triplet states of F-2; φ₃⁰/φ₁⁰, is about 5. The excited triplet state is the reactive state for the [2+2] photocycloaddition of F-2 with tetramethylethylene in solution but the excited singlet state of F-2 becomes very important in biological conditions.     

THE PHOTOPHYSICS OF 5-METHOXYINDOLE. A NON-EXCIPLEX FORMING INDOLE: ADDITIONAL EVIDENCE FOR TWO CLASSES OF EXCIPLEXES*

Abstract— 5-Methoxyindole is a non-exciplex forming indole, and its excited state behavior is qualitatively different from that of indole and its methyl substituted derivatives. This fact supports the idea that there are two limiting classes of exciplexes, charge-transfer and dipole-dipole stabilized. The fluorescence quantum yield in water is 0.29 with a lifetime of 4.0 ns at 25d?C. The activation energy for fluorescence quenching in water is 15.9 pM 0.5 kJ/mol, which is smaller than for indole and the methyl substituted indoles which have been measured. In cyclohexane at 25d?C, the fluorescence quantum yield is 0.63 with a lifetime of 6.7 ns. The fluorescence is efficiently quenched by electron scavengers, as is the case for other indoles. Some electron ejection to solvent probably occurs in both solvents.     

Energetics of Photoinduced Electron Transfer in the Indole-O2 Cluster in Gas Phase: Possible Consequences for Photoexcited Tryptophan in Solution

Abstract— A direct process for an activationless electron transfer from photoexcited tryptophan to molecular oxygen is proposed. By photodetachment of mass-selected indole-O_2- clusters in gas phase a neutral indole₊ O_2- charge-separated exciplex state is found at 2.25 0.2 eV above the neutral ground state. By theory also, the existence of an excited charge separated state at 3.05 0.2 eV is postulated. In gas phase both charge-separated cluster states are energetically below the first singlet states ₁L_b and ₁L_a and the lower even below the first triplet state T₁ of indole. In gas-phase clusters these energetics imply a very efficient quenching of photoexcited indole by fast electron transfer to oxygen. We discuss a similar mechanism for tryptophan-O₂ in aqueous environment and find it without activation barrier and presumably extremely fast. In the collisional tryptophan_*-O₂ complex the efficiency and the time scale of the charge transfer process should be mostly solvent independent. In polar solvent a complete charge separation and free superoxide formation are expected. We correlate this model with previous fluorescence and phosphorescence quenching data of excited tryptophan by O₂ and propose electron transfer to be the relevant process.     

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.     

REDUCTION OF THE EXCITED SINGLET STATE OF CHLOROPHYLL AND PHEOPHYTIN AT THE ORGANIC CRYSTAL/AQUEOUS SOLUTION INTERFACE

Abstract— Reduction of the excited singlet state of chlorophyll a , chlorophyll b , and pheophytin a at the surface of perylene, anthracene, and chrysene single crystals has been measured as hole injection current. The dependence of the quantum yield on the standard free-energy difference of the reaction was in accordance with theoretical expectations without correcting for an interaction energy. The maximum quantum yield of only about 10⁻² holes per absorbed photon is ascribed to a very small effective lifetime of the excited singlet state due to concentration quenching of the excited singlet state in the adsorbed dye layer.     

ELECTRON TRANSFER FROM CHLOROPHYLL a TO QUINONE IN MONO- AND MULTILAYER ARRAYS

Abstract— Mono- and multilayers of chlorophyll a (Chl a )– lecithin have been prepared on quartz slides, by means of the Blodgett-Langmuir technique, for fluorescence studies. Self-quenching of the Chl a fluorescence has been observed in Chl a -lecithin single layer excited with a laser light at 632.8 nm. The fluorescence yield is reduced by 50% at a concentration of 7 ± 10¹² Chl a molecules cm⁻². Chl a fluorescence quenching, by adding N,N -distearoyl-1,4-diaminoanthraquinone (SAQ), has been studied. in a single layer, in pure Chl a and also at various dilutions of Chl a in lecithin. The results are explained in terms of a dynamic quenching rather than in terms of a permanent complex formation, at the ground state, between Chl a and SAQ. The fluorescence quenching has been interpreted as the result of an electron transfer from excited Chl a to SAQ, and rate constants of 8.3 ± 10⁻⁵ cm² molecule⁻¹ S⁻¹ and 2.4 ± 10⁻⁴ cm² molecule⁻¹ s⁻¹ have been found for pure diluted Chl a , respectively. Ten per cent of the diluted Chl a fluorescence always remains unquenchable and independent of the quinone concentration. In multilayers, where SAQ and Chl a are in different layers, there is no fluorescence quenching for pure or diluted Chl a even when the chromophores are in two adjacent layers. This happens only if SAQ is not able to diffuse from one layer to another. A minimum value of 22.4 nm has been found for the singlet exciton diffusion length in pure Chl a multilayers.     

PHOTOCHEMISTRY OF FLAVINS. II. PHOTOPHYSICAL PROPERTIES OF ALLOXAZINES AND ISOALLOXAZINES

Abstract. Pulsed laser photolysis at 347nm has been used to study the transient spectroscopy of alloxazine, lumichrome, lumiflavin, and riboflavin in acidic (pH 2.2) aqueous solution and in ethanol. Intersystem crossing quantum yields (φ_ISC) were determined by a modification of the comparative laser excitation method which utilizes the variation of the triplet yield with intensity in conjunction with a kinetic model for the various photophysical and photochemical processes occurring during the pulse. Fluorescence quantum yields and lifetimes are also reported. Correction for quenching of the excited singlet state by H⁺ ions shows that, in neutral aqueous solution, intersystem crossing for flavins is an efficient process (φ_ISC˜ 0.7) which, in conjunction with fluorescence, accounts for the fate of all absorbed photons. For alloxazine (φ_ISC˜ 0.45) and lumichrome (φ_ISC˜ 0.7) the results are more difficult to interpret owing to interconversion between alloxazine and isoalloxazine structures in the singlet excited state. For all four compounds, the quantum yield of products derived from the singlet excited state is estimated as ˜0.04. There is evidence of biphotonic product formation at high laser energies. In ethanol, where φ_ISC for lumichrome is about twice that of lumiflavin, internal conversion between the excited singlet and ground states appears to be a significant process. Complete triplet-triplet absorption spectra in the region 260–750nm are reported. For lumichrome at pH 2.2 there is spectral evidence for isomeric triplet states which appear to be in equilibrium.     

EFFECT OF METAL IONS ON THE LUMINESCENCE OF ACRIDINE DYES BOUND TO DNA

Abstract— The luminescence of acridine dyes intercalated in DNA was studied as a function of the concurrent binding of metal ions to DNA, in an effort to deduce specific site interactions of the dyes. Two dyes, proflavine (PF) and acridine orange (AO), and two metal ions, silver and mercuric, were used. Both ions quench the fluorescence of the dyes in aqueous solution at room temperature. The metal ions have a different effect on the fluorescence of these dyes when they are intercalated between the base pairs of DNA. The fluorescence of AO is decreased when silver is bound, while the fluorescence of PF is enhanced. Since Ag⁺ initially binds to GC sites in DNA, which quench the PF fluorescence, it ostensibly 'turns off' the quenching by DNA at these sites, and this effect is greater than the quenching effect of the silver ion itself. Hg²⁺ ion initially binds to AT sites in DNA. Since both dyes fluoresce from AT sites, Hg²⁺ is expected to quench their fluorescence. This behavior is observed at low r (metal ion/base). At higher r values, however, where Hg²⁺ is expected to begin binding to GC sites, the fluorescence of PF is enhanced. These quenching turn-off effects are tentatively interpreted in terms of a change in the structure of the dye/DNA complex which occurs when a metal ion binds at the intercalation site. At 77 K. no fluorescence enhancement is observed when metal ions bind; Ag⁺ quenches the fluorescence and enhances the phosphorescence of both dyes. Qualitatively similar results are obtained with Hg²⁺.     

INTENSITY AND TIME-DEPENDENCE OF THE CAROTENOID TRIPLET QUENCHING UNDER LIGHT FLASHES OF RECTANGULAR SHAPE IN CHLORELLA

Abstract— As is known the chlorophyll fluorescence of photosynthetizing plants is strongly quenched by carotenoid triplet states if the exciting light intensity is high enough (> 10 kW/m²). This light-induced quenching process was studied by measuring the relative yield of chlorophyll fluoresccncc excited with a pulsed argon laser at 20 C in light adapted algae as function of time (within lo μ s ) and exciting light intensity (<400 kW/m²). The experimental yield against time and yield against intensity curves have been adequately explained by a statistical model of Photosystem 2 (PS 2) units based upon the existence of freely moving excitons according to which the carotenoid triplet quenchers are randomly distributed and are perfect traps for excitons. Accepting the hypothesis that carotenoid triplet quenching occurs only in PS 2 units. it could be concluded that the height of the true zero level of PS 2 fluorescence is somewhat lower than the half value of the fluorescence level of the dark adapted state.     

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.     

PREPARATION AND PHOTOPHYSICAL STUDIES OF PORPHYRIN-C60 DYADS

Abstract Porphyrin-C₆₀ dyads in which the two chromophores are linked by a bicyclic bridge have been synthesized using the Diels-Alder reaction. The porphyin singlet lifetimes of both the zinc (Pz_n-C₆₀) and free base (P-C₆₀) dyads, determined by time-resolved fluorescence measurements, are ≦17 ps in toluene. This substantial quenching is due to singlet-singlet energy transfer to C₆₀ The lifetime of Pzn-¹C₆₀ is -5 ps in toluene, whereas the singlet lifetime of an appropriate C₆₀ model compound is 1.2 ns. This quenching is attributed to electron transfer to yield P_zn^bull;+-C₆₀^bull;-. In toluene, P-¹C₆₀ is unquenched; the lack of electron transfer is due to unfavorable thermodynamics. In this solvent, a transient state with an absorption maximum at 700 ran and a lifetime of-10 μs was detected using transient absorption methods. This state was quenched by oxygen, and is assigned to the C₆₀ triplet. In the more polar benzonitrile, P-¹C₆₀ underoes photoinduced electron transfer to give P^•+-C₆₀^bull;-. The electron transfer rate constant is −2 × 10¹¹ s⁻¹.     

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.