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.     

TRIPLET, RADICAL ANION AND RADICAL CATION SPECTRA OF FUROCOUMARINS

Abstract— Absorption spectra of triplets, radical anions and radical cations of four furocoumarins, psoralen (Ps), 8-methoxypsoralen (8MOP), 5-methoxypsoralen (5MOP) and 3-carbethoxypsoralen (3CPs), have been determined by laser flash photolysis and pulse radiolysis. The triplet spectra of 8MOP, 5MOP and 3CPs are strongly modified when going from an H-bonding solvent such as water to a non H-bonding solvent as benzene or acetonitrile while the triplet spectrum of Ps is solvent independent. Theoretical considerations using a CNDO/S method are able to explain the existence of these two different triplets. For 8MOP, 5MOP and 3CPs in water the triplets might be considered as triplet exciplexes ³(FC^δ-. H₂O^δ+) consistent with these triplet spectra being similar to the spectra of the corresponding radical anions.     

REACTIVITY OF EXCITED STATES OF FLAVIN AND 5-DEAZAFLAVIN IN ELECTRON TRANSFER REACTIONS

Abstract— Quenching of the excited states of lumiflavin and 3-methyl-5-deazalumiflavin by methyl-and methoxy-substituted benzenes and naphthalenes in methanol was investigated. The observed difference in the reactivity of acid and neutral lumiflavin triplets is explained thermodynamically by applying the Michaelis cycle, as being due to the higher reduction potential of the acid triplet. In this connection the p K values of lumiflavin triplet (p K ^M= 6.5) and semiquinone (p K ^M= 11.3) have also been determined in methanol. The difference in the reactivity between the singlet and triplet states of lumiflavin is found to be greater as predicted by the difference in excitation energy. The reactivities of the excited states of flavin and 5-deazaflavin differ only slightly in contrast to the marked difference in the ground state reactivities of electron transfer reactions. This is explained in terms of the model of Rehm and Weller. The pH dependence of the electron transfer quenching of 5-deazaflavin triplet was investigated in water, yielding a triplet p K of 2.5. In contrast to the flavin, this triplet p K does not significantly differ from the p K of the 5-deazaflavin ground state. From this, different sites of protonation are deduced for the photoexcited triplet states of flavin and 5-deazaflavin.     

KINETICS OF TRIPLET OXIDATION OF METALLO-PORPHYRIN COMPOUNDS TO THEIR CORRESPONDING RADICAL CATIONS

Abstract— The kinetics of photooxidation of triplets of metalloporphyrin compounds to their corresponding radical cations was investigated. Zn-tetraphenyl porphyrin (ZnTPP) and Mg-tetraphenylpor-phyrin (MgTPP) triplets were oxidized by europium salt with rate constants of 4.8 × 10⁵M^-1s^-1 and 2.1 × 10⁶M^-1s^-1, respectively. The high rate constant of oxidation of MgTPP triplet might be related to the ground state oxidation potential, being 0.54 V (SCE) for the Mg complex and 0.71 (SCE) for the Zn complex.
The rate constant of oxidation of ZnTPP excited singlet is in the order of diffusion control, i.e. ˜ 10¹⁰M ^-1 s^-1. Excitation of ferric, cupric, cobaltic, and vanadyl tetraphenylporphyrin did not result in a long-lived triplet state that would allow oxidation studies using flash photolysis.     

THE FLAVIN-SENSITIZED PHOTOOXIDATION OF NUCLEOTIDES — III. THE PARTICIPATION OF OXYGEN

Abstract— Quantum yields for the lumiflavin-sensitized oxidation of guanosine monophosphate and adenosine monophosphate in solution have been measured as functions of oxygen and nucleotide concentration. The quantum yield increases with oxygen concentration at low oxygen concentrations, but quenching of the excited flavin molecule by oxygen results in a fall in quantum yield at higher concentrations. It has also been established that the reciprocal of the quantum yield is linearly related to the reciprocal of the nucleotide concentration. A mechanism in which molecular oxygen reacts with an excited complex formed between triplet lumiflavin and the nucleotide is consistent with these observations.
A value for the second-order rate constant for the quenching of triplet lumifiavin by oxygen of 2·65 × 10⁹ M ^-1 sec^-1 has been obtained.     

FLASH PHOTOLYSIS STUDIES OF OROTIC ACID

Abstract— The triplet state of orotic acid has been studied by flash photolysis. The rate for dimerization has been observed to vary from 2 × 10⁹ M ^-1 sec^-1 at pH 1 where both the triplet and ground state molecules are neutral, to under 10⁸ M^-1 sec^-1 above pH 9 where both the triplet and ground state molecules are doubly ionized. The p K of the triplet state has been measured as 4.6. The rate of oxygen quenching for the triplet is 2–3 × 10⁹ M^-1 sec^-1 while the rate of radiationless decay in solution is 0.73 × 10⁴ sec^-1. The triplet absorption spectra have been measured for the two ionic forms of the triplet.     

ULTRAVIOLET-INDUCED REACTIONS OF THYMINE AND URACIL IN THE PRESENCE OF CYSTEINE

Abstract —Ultraviolet-radiation photolysis of thymine in the presence of cysteine gives rise to four isomeric dimers, dihydrothymine, and at least five cysteine addition products. Similar reactions occur for uracil but the products have not all been characterized in detail. The addition reactions arise from the triplet state of the pyrimidine. The initial step is production of a hydropyrimidine radical, which then reacts with cysteine to give the addition products. The triplet is quenched by cysteine with a rate constant of about 2 times 10⁸ M^-1 s^-1 for thymine and 2–9 times 10⁸ for uracil. The total yield of products gives a lower-limit estimate of the triplet yield and hence of the intersystem-crossing efficiency. These studies, combined with earlier determinations of dimer yields, show that 93% of the thymine triplets which interact with another thymine molecule are quenched without forming stable dimers. For uracil, the corresponding figure is 75%.     

CAROTENOID TRIPLET STATE AND CHLOROPHYLL FLUORESCENCE QUENCHING IN CHLOROPLASTS AND SUBCHLOROPLAST PARTICLES

Abstract— Absorption changes attributed to the triplet state of carotenoids and to primary electron donors (P-700. P-680)^: and fluorescence quenching at several wavelengths have been measured with a single apparatus. following flash excitation with a dye or a ruby laser. Spinach chloroplasts as well as subchloroplast particles enriched in Photosystem-1 (F₁), Photosystem-2 (F₁) or the light-harvesting Chl a/h (F_III) have been examined at temperatures varying between 5 and 294 K.
The triplet state of carotenoids has been identified on the basis of its difference spectrum (having a peak at 515 nm) and decay kinetics (⋍ 7 µs at low temperature; accelerated by O₂ at 294 K). It is formed in all of the materials studied. The quantum yield of carotenoid triplet formation in chloroplasts increases at low temperature, but less than the fluorescence yield.
In most cases the fluorescence quenching recovers approximately with the same kinetics as the decay of the carotenoid triplets. The fluorescence recovery is, however, significantly faster for chloroplasts at 730 nm. Fluorescence quenching occurs in all types of materials. The ratio of fluorescence quenching to the concentration of carotenoid triplets varies with the material, being maximum in chloroplasts and minimum in F_m particles.
We conclude that the formation of the carotenoid triplet state is not limited to a few sites in the chloroplast and that a carotenoid triplet is a quencher of chlorophyll fluorescence. A detailed comparison of carotenoid triplets and fluorescence quenching gives some information concerning the organization of the pigments in the photosynthetic apparatus.     

PHOTOCHEMICAL HYDROXYLATION OF 7,8-DIMETHYLALLOXAZINE (LUMICHROME)-A FLASH PHOTOLYSIS STUDY

Abstract— Flash photolysis experiments on the hydroxylation of lumichrome (L) in aqueous 0.5 M H₂SO₄ solution in the presence of O₂ or Ni²⁺ as triplet quenchers and quantum yield measurements confirm the assignment of the photoreactive species to the protonated form of the excited singlet state. A mechamism concerning the photochemical step is proposed, accounting for the formation of protonated 9-hydroxy-5,10-dihydrolumichrome (LOH₃⁺). This primary stable photoproduct was characterized by spectral and kinetic data. The dark reactions originating from LOH₃⁺ were investigated, and data regarding the successive steps are presented. The reaction LOH₃⁺ L→ LO + LH₃⁺ is demonstrated to be a two-electron reduction. The rate constant for the reaction of LH₂⁺ with O₂ is much larger than that for the oxidation of LH₃⁺ by oxygen.     

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.     

TRANSIENT PHOTOCHEMISTRY OF NEUTRAL RED

Abstract— Flash photolysis of neutral red between pH 1.3 and pH 11 yields the triplet species ³DH₂⁺²³DH⁺ and ³D. Both ³DH₂⁺² and ³D exhibit first order decay with rate constants of 1.6 ± 0.3 × 10⁴ s^-1 but ³DH⁺ decays within the lifetime of the flash. Over the entire pH range, ascorbic acid quenches the triplet, forming the semireduced radicals DH₃⁺² DH₂⁺ and DH, all of which exhibit second order decay with k = 1.8 ± 0.4 ± 10⁸ M ^-1s^-1 most probably by recombination with semioxidized ascorbic acid. The dependence of the rate of decay of radical neutral red on the identity of reversible reductants supports the back-electron transfer mechanism, as does digital simulation of complex radical disproportionation schemes. In contrast to the efficient reduction of triplet neutral red by ascorbic acid, its reduction by EDTA is quite inefficient.     

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.     

A PULSE RADIOLYSIS and PULSED LASER STUDY OF THE VITAMIN D3 TRIPLET STATE: LIFETIME, RELAXATION and 'NONVERTICAL'EXCITATION

Abstract— The triplet state of vitamin D3 in benzene has been characterised in terms of its absorption spectrum, Λ_max 315 nm, its lifetime, 300 ns, its rate constant for reaction with oxygen, 4.2 times 10⁹mol⁻¹ s⁻¹ and the efficiency with which it sensitizes the formation of singlet oxygen, 25%. There is a large difference in the electronic excitation energies of the spectroscopic and relaxed triplets, ˜ 237 kJ mol⁻¹ and147–168 kJ mol⁻¹, respectively. It has been shown that, in the endothermic situation, the vitamin D3 molecule is a 'nonvertical'acceptor of triplet energy. This is in accord with the non-planar character of its acyclic conjugated Il-system.     

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.     

LUMIFLAVIN-SENSITIZED PHOTOOXYGENATION OF INDOLE

Abstract— The lumiflavin-sensitized photooxygenation of indole in aqueous solutions has been investigated by means of steady light photolysis and flash photolysis. The semiquinone of lumiflavin and the half-oxidized radical of indole were formed by the reaction between triplet lumiflavin and indole (3.7 times 10⁹ M ^-1 s^-1). The semiquinone anion radical of lumiflavin reacted with oxygen to form superoxide radical. The triplet state of lumiflavin also reacted with oxygen forming singlet oxygen, ¹O₂. But the reaction between ¹O₂ and indole (7 times 10⁷ M^_l s^_1; estimated from steady light photolysis using Rose Bengal as a sensitizer) was far less efficient than the reaction between indole and triplet lumiflavin. The quantum yield of the lumiflavin-sensitized photooxygenation of dilute indole via radical processes was much higher than that via ¹O₂ processes, though appreciable ¹O₂ was formed.     

A THEORETICAL TREATMENT OF THE ELECTRONIC STATES OF THIONINE AND RELATED MOLECULES

Abstract— –Problems associated with the protolytic equilibria of thionine and related molecules in their lowest excited electronic states were investigated. The theoretical arguments are based on semi-empirical SCF MO (CI) calculations for the π-electronic system of these molecules; all singly excited configurations were included in the CI. The results indicate that the basic form of thionine in its ground, first excited singlet and lowest triplet state is protonated at the heterocyclic N atom. The difference of the p K values of these three states can be explained in terms of the calculated charge densities. The photochemical reactivity of the lowest triplet of the acidic form of thionine (³TH₂²⁺) differs greatly from that of the lowest triplet of the basic form (³TH⁺). Some arguments for the assignment of nπ* character to ³TH₂²⁺ and ππ* character to ³TH⁺ are advanced.     

The photoinduced triplet of flavins and its protonation states

The photogenerated triplet states of riboflavin and flavin mononucleotide (FMN) have been examined by time-resolved electron paramagnetic resonance (EPR) spectroscopy at low temperature (T = 80 K). Because of the high time resolution of the utilized EPR instrumentation, the triplets are for the first time observed in the nonequilibrated electron-spin polarized state and not in their equilibrated forms with the population of the triplet sublevels governed by Boltzmann distribution. The electron-spin polarization pattern directly reflects the anisotropy of the intersystem crossing from the excited singlet-state precursor. Spectral analysis of the resulting enhanced absorptive and emissive EPR signals yields the zero-field splitting parameters, |D| and |E|, and the zero-field populations of the triplet at high accuracy. These parameters are sensitive probes for the protonation state of the flavin's isoalloxazine ring, as becomes evident by a comparison of the spectra recorded at different pH values of the solvent. The three protonation states of the flavins can furthermore be distinguished by the kinetics of the transient EPR signals, which are dominated by spin-lattice relaxation. The fastest decays are observed for the protonated FMN and riboflavin triplets, followed by the deprotonated flavin triplets. Slow decays are measured for the triplet states of neutral FMN and riboflavin. Because proton transfer is found to be slow on the time scale of spin-polarized triplet detection by transient EPR, the pH-dependent spin-relaxation and zero-field splitting parameters offer a novel approach to probe the protonation state of flavins in their singlet ground state through the characterization of their triplet-state properties.     

Dynamics of Flavin in Flavocytochrome b2: A Fluorescence Study

Abstract— The dynamics of the flavin bound to the flavocytochrome b₂ from Hansenula anomala were studied by fluorescence intensity quenching and quenching emission anisotropy with iodide. The fluorescence intensity of bound flavin is decreased 13-fold as compared to the free molecule. The remaining fluorescence decays with two lifetimes equal to 0.963 ± 0.040 and 4.635 ± 0.008 ns and fractional intensities of 0.036 ± 0.002 and 0.964 ± 0.002, respectively. The bimolecular diffusion constant was found to be 3.33 × 10⁹ M ^-1 s^-1 when the flavin is bound to the enzyme and 8.3 × 10⁹ Mv s^-1 when the flavin is free in solution. Thus, the flavin in flavocytochrome b₂ is accessible to the solvent, but the amino acid residues of the binding site inhibit the diffusion of iodide. The rotational correlation time of bound flavin was found to be 2.015 ± 0.365 ns, a value higher than that (155 ps) of free flavin in solution. Our results are discussed on the basis of local dynamics of the flavin.     

PICOSECOND LASER SPECTROSCOPY AND OPTICALLY DETECTED MAGNETIC RESONANCE ON A MODEL PHOTOSYNTHETIC SYSTEM

Fluorescence-detected magnetic resonance of triplets in zero magnetic field (FDMR), fluorescence fading (FF) due to triplet-formation, both at 4.2 K, and prompt fluorescence decay kinetics (FDK) at room temperature have been measured for free pheophorbide- a (f-Pheo) and bound (b-Pheo) to a synthetic polypeptide (L-L ys -L-A la -L-A la )_n, dissolved in dimethylformamide (DMF). Fluorescence decay kinetics measurements of f-Pheo in DMF yielded 1-5 ns lifetimes, for b-Pheo in DMF a ~ 50 ps decay-component was found emitting at 730–750 nm. Zero-field splitting parameters |D| and |E| of the lowest triplet state T₁ were determined from FDMR spectra as (337 and 24) 10^-4 cm^-1 for f-Pheo and (359 and 25) 10^-4 cm^-1 for b-Pheo, both in DMF. Decay rate constants of the three spin levels of T¹ of b-Pheo ( K _x= 1200 50 s^-1, k _y= 440 25 s^-1, k _z= 80 5 s^-1) and relative steady-state populations (N_x= 28 2%, N_y= 47 2%, N_z= 26 2%) determined from FF curves predict a fluorescence decrease at the D–E and D + E FDMR transitions, whereas experimentally a fluorescence increase is observed. The FDMR sign-inversion results from singlet-singlet energy transfer from b-Pheo monomers to their aggregates, followed by fast intersystem crossing to T₁. These results indicate that aggregates are formed by two or more b-Pheo molecules at different positions on the folded polypeptide chain. This situation resembles that in chlorophyll-proteins, containing low-lying traps, resulting from interaction of chromophores with other chromophores and with the protein environment.     

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.