VECTORIAL ELECTRON TRANSPORT ACROSS LIPID VESICLE MEMBRANES DRIVE BY TWO PHOTOREACTIONS. II. PHOTOCHEMICAL REGENERATION OF REDUCED CYTOCHROME c BY FLAVIN TRIPLET STATE AND ETHYLENEDIAMINETETRAACETIC ACID IN THE INNER COMPARTMENT AND REDUCTION OF FERREDOXIN BY CHLOROPHYLL TRIPLET STATE IN THE OUTER COMPARTMENT

We have attempted to mimic natural photosynthesis with regard to the photogeneration of a powerful reductant, using a negatively charged lipid bilayer vesicle system incorporating two photoreactions sensitized by a flavin analog (flavin mononucleotide [FMN]) and chlorophyll (chl) in their respective triplet states. Ethylenediamine-tetraacetic acid (EDTA) in the inner aqueous compartment was used as a sacrificial electron donor to the FMN triplet, and ferredoxin in the outer aqueous compartment served as the final electron acceptor (mediated via triplet electron transfer chain in this multicomponent system to be elucidated. By itself, EDTA does not function as an effective donor to membran-bound oxidized chl (chl^+.), which is formed by electron transfer from triplet chl to the viologen follwed by transbilayer electron migration. This is a consequence of electrostatic repulsive interactions with the negatively charged membrane. This limitation is avoided when FMN is used as a photomediator between EDTA and chl^+.. The overall reaction is dramatically increased in rate by enclosing cytochrom c together with EDTA and FMN in the inner compartment. The rate constant of the key step in the reaction, i.e. elctron transfer from reduced cytochrome c, generated via photoreduction by the FMN/EDTA system, to chl^+. is increased 20-fold over that obtained with cytochrome c alone as the elctron donor. One of the important constraints that limited the net electron transfer across the bilayer to 50% of the added cytochrome, i.e. inhibition by oxidized cytochrome c formed in the inner compartment, is avoided by the inclusion of the second photoreaction in this system, thus allowing photoreduction of all of the added ferredoxin to be achieved. This system provides a model for a photochemical energy storage process that utilizes two photorections operating in series resulting in electron flow across a lipid bilayer membrane.     

MECHANISM OF PHOTOREDUCTION OF THIAZINE DYES BY EDTA STUDIED BY FLASH PHOTOLYSIS—III CONSEQUENCES OF A NEWLY FOUND pKT OF THIONINE ON THE MECHANISM IN BASIC SOLUTIONS

Abstract— Evidence for an acid-base equilibrium between the neutral and monocationic forms of the triplet state of thionine, ³ T and ³ TH ⁺ (p K _T# 8–9) has been found. This equilibrium, which has no counterpart in the methylene blue system can explain the different behaviours of these two similar dyes concerning their photoreduction by ethylene diamine tetracetic acid (EDTA) in basic solutions. With reasonable assumptions about the reactivity of the various species present in solution, it is now possible to calculate over the whole pH range the quantum yield of semireduced dye formation φs, which, according to the previously proposed mechanism, must be proportional to the overall photoreduction quantum yield φR. The very good fit between the calculated curve φs = f (pH) and the experimental one φR = f (pH) for both dyes strongly supports the proposed mechanism.     

QUENCHING OF OXONINE EXCITED STATES BY EDTA VIA ELECTRON TRANSFER AND DEACTIVATION PROCESSES. A COMPARATIVE STUDY OF EXCITED SINGLET AND TRIPLET STATE REACTIVITY

The quenching of excited singlet oxonine by EDTA in aqueous solution leads mainly to deactivation of the dye to the ground state and, to a lesser extent, to electron abstraction. The rate constants for these processes have been measured and compared to those for the same reactions involving the oxonine triplet state. The rate constant of electron abstraction is about ten times greater via the singlet state than via the triplet state. However, the rate constant of deactivation to the ground state is 10³-10⁴ times greater for the excited singlet state than for the triplet state, so that the efficiency of electron transfer is much smaller for the singlet state.     

PHOTOCHEMICAL INVESTIGATIONS OF OXAZINE, THIAZINE AND SELENAZINE DYES. THE REACTIVITY OF PROTOLYTIC TRIPLET FORMS IN ELECTRON TRANSFER REACTIONS

Abstract— The photoreduction of oxonine, thionine and selenine with the reducing agent allylthiourea was investigated by flash photolysis. The oxonine triplet state was produced by triplet-triplet energy transfer with 9,10-dibromoanthracene as donor. For all three dyes the rate constant of the electron transfer is considerably higher for the acid triplet form than that of the corresponding reaction of the basic triplet form. It is shown that the higher reactivity of the acid triplet can be related to its higher reduction potential which is available from the difference of the pK values of triplet and semiquinone of the dye.     

A MECHANISM FOR THE METHYLENE BLUE SENSITIZED OXIDATION OF NUCLEOTIDES

Abstract— An investigation of the methylene blue sensitized oxidation of isolated nucleotides has been carried out, based on the behaviour of the excited dye molecule in long-term irradiation and flash photolysis experiments. It was necessary first to establish the effect of the phosphate buffer upon the triplet excited state of the dye and the consequent effect upon its photo-fading. The buffer has a salt effect which accelerates the protonation of the dye triplet, and also the photoreduction of the dye which takes place through the resulting protonated triplet. Xanthosine monophosphate (XMP), which is the nucleotide most sensitive to the photodynamic action of methylene blue, is also effective in reducing the triplet of the dye, while other nucleotides which are resistant to photooxidation do not act as reducing agents. The quantum yield of the sensitized oxidation of XMP was found to correspond to that of the anaerobic photo-reduction of methylene blue in its presence, which leads to the conclusion that the primary step of the photooxidation is electron or hydrogen abstraction from the nucleotide by the triplet excited methylene blue molecule.     

CHLOROPHYLL-SENSITIZED REDUCTION OF SAFRANINE

Abstract— The kinetics of the direct photoreduction, and the chlorophyll-sensitized reduction, of safranine by ascorbic acid in aqueous pyridine was studied. In darkness following illumination, safranine was reformed from the colorless reduced derivative by a first order reaction. In light, however, the photostationary concentration of reduced safranine was proportional to the square root of the absorbed illumination, indicating a second order back reaction. The spectrum of reduced safranine, the inhibition of chlorophyll photoreduction by safranine, and the quenching of chlorophyll fluorescence by safranine were also measured. While other mechanisms of sensitization are conceivable, the simplest explanation of the results seems to be one involving the formation of the triplet state of safranine at the expense of the triplet state of chlorophyll.     

THE REDUCTION AND QUENCHING OF PHOTOEXCITED FLAVINS BY EDTA

Riboflavin was irradiated anaerobically in aqueous EDTA solutions over the pH range 2.5–10. In other dye systems (Bonneau and Pereyre, 1975), only the trivalent anion of EDTA was found to have significant reactivity for photoreduction. For riboflavin, the reactivity begins with monoanionic EDTA, and the reactivity is markedly increased as the charge increases. This suggests that the charge on the reductant is more important to the electron transfer process for riboflavin than the formation of a nonhydrogen bonded nitrogen site on EDTA. At high concentrations of EDTA in the pH range 4–8, quenching of the photoreduction occurs, which can be explained by an energy transfer between the excited singlet state of riboflavin and trianionic EDTA, possibly as an association complex. The rate constants for the photoreduction of riboflavin by the monovalent, divalent, and trivalent anions of EDTA are 1.0 times 10⁷M^-1 s^-l, 4.8 times 10′M^-1 s^-l, and 2.0 times 10⁸M^-1s^-1, respectively. The rate constant for the singlet state quenching by trianionic EDTA is 3 times 10⁹M^-l s^-1, and the limiting quantum yield for intersystem crossing for riboflavin in aqueous solution is 0.50 ± 0.05.     

TRANSIENT SPECIES IN THE PHOTOCHEMISTRY OF EOSIN*

Abstract— Photochemical reactions of eosin in aqueous solution were studied using the flash photolysis technique. In deaerated solution the dye was converted quantitatively to the triplet state during flashing. The triplet dye decayed by first and second order reactions which partly regenerated the dye in the ground state and partly produced semioxidized and semireduced eosin. These radical species were formed in an electron dismutation reaction between two triplet molecules and also in a reaction between one triplet and one unexcited molecule. The radicals recombine rapidly to give the dye in the ground state.
An efficient reversible photooxidation reaction was observed in eosin solutions containing potassium ferricyanide. Semioxidized eosin was formed in high yield by reaction between the triplet dye and the oxidant. The dye was regenerated rapidly in a reverse reaction between the products of the oxidation reaction.
An analogous type of reaction was found to occur in eosin solutions containing p -pheny-lene diamine. This reagent reduced the triplet dye to semireduced eosin; the dye was regenerated in the ground state in a very efficient reverse reaction. The protolytic behaviour of semireduced eosin was studied by varying the pH.
Absorption spectra of the transient products were determined and rate constants for the observed reactions were measured. The results are compared with results from previous studies of fluorescein.     

THE FLAVIN-SENSITIZED PHOTOOXIDATION OF NUCLEOTIDES—II. THE PARTICIPATION OF THE FLAVIN TRIPLET STATE

Abstract— –A study has been made of the effects of a series of nucleotides upon the electronic excited states of lumiflavin in order to determine the mechanism of their flavin-sensitized oxidation. A hydrogen-abstraction mechanism is ruled out, because if the nucleotide acts as a reducing agent for the excited dye molecules, it should increase the rate of reduction of the dye when the irradiation is carried out in the absence of oxygen. However, each of the nucleotides studied was found to reduce the rate of anaerobic photoreduction. While oxidation by an intermediate species such as the dye 'moloxide' or singlet oxygen is not entirely ruled out, our evidence suggests that the initial reaction is between the nucleotide and the flavin triplet. This results in a loss of the triplet excitation energy and is a very efficient reaction, guanosine monophosphate shewing 36 per cent of the triplet quenching efficiency of potassium iodide. The relative rates of reaction of the nucleotides with the flavin triplet exactly parallels their quantum yields of sensitized photo-oxidation. The formation of ground-state complexes between flavin and nucleotide and the participation of the singlet excited state of the flavin are not considered to be important.     

INDICATION OF FAST TRIPLET FORMATION IN BIMOLECULAR QUENCHING OF EXCITED SINGLET STATES STUDIED BY CONVENTIONAL FLASH PHOTOLYSIS

The bimolecular quenching of the first excited singlet state of oxonine by allylthiourea leads to the formation of the triplet state of the dye. This has been proved by comparison with the triplet-triplet absorption spectrum of oxonine obtained by triplet-triplet energy transfer. The conventional flash experiments suggest that the dye triplet state is produced directly rather than by radical recombination.     

THE PHOTOREDUCTION OF METHYLENE BLUE BY ARYLAMINOMETHANE-SULFONATES

Abstract— The photoreduction of methylene blue in the presence of arylaminomethanesulfonates (RAMS = RC₆H₄NHCH₂SO₃Na) was studied by laser and conventional flash photolysis. These compounds quenched the methylene blue triplet deviating from a normal Stern-Volmer behaviour. For low quencher concentrations, a Rehm-Weller relationship was found between the k _q's and the DL G 's obtained for the electron transfer reactions. The lack of further quenching at higher [RAMS] is ascribed to the formation of a ground state ion pair between the dye and the anionic quencher which, on excitation, forms a triplet state unable to under go electron transfer for steric reasons. A second order decay rate constant was found for the semireduced species (MB') ( ca. 5 × 10⁹ M _-1 s_-1, independent of the RAMS used) and is attributed to a proton transfer from the radical zwitterion (RC₆H₄NH CH₂SO₃⁻) to MB. The overall dependence on the substituent of the bleaching observed by continuous irradiation follows the triplet behaviour.     

Photochemical reduction of phenosafranine by edta and its use in amperometric titrations

Phenosafranine in the presence of ethylenediaminetetraacetic acid is reduced to the leuco dye form on illumination with white light. The rate of photoreduction is strongly pH-dependent and exhibits a maximum at about pH 6. Variables such as wavelength and intensity of the light and the concentration of reactants have been studied. The photoreduction involves a long-lived excited state of the dye and is retarded by small amounts of p-phenylenediamine and iodide. The dye is used as an amperometric indicator in titrations of Pb(II), Zn(II) and Cu(II) with EDTA; the method is applicable in the range 0.02–0.0005 M. The current is caused by anodic oxidation of the phenosafranine reduced in the photochemical reaction with EDTA.     

Analytical applications of photochemical reduction of Azur B by EDTA-iodide determination

A kinetic study of the photochemical reaction of Azur B and EDTA (in the absence of oxygen) has been made in connection with development of a new kinetic method for iodide. The reaction is first-order with respect to the dye, EDTA and absorbed light-intensity. The rate of photoreduction is strongly pH-dependent, and maximal at about pH 6.8. The photoreduction involves a long-lived excited state of the dye and is dramatically retarded by small amounts of iodide. A tentative mechanism is proposed, and the experimental conditions have been optimized. The variable time method appear to be the most suitable. A detection limit of 1.0 mug/ml and a coefficient of variation of about 3% can be achieved. Chloride and bromide do not interfere at levels below 100-fold mole ratio to iodide. Metal ions do not interfere if enough excess of EDTA is used. Coloured species may interfere at high concentration (filter effect).     

A STUDY OF THE METHYLENE BLUE-SENSITIZED OXIDATION OF AMINO ACIDS

Abstract— A study has been made of the effects of eight amino acids and a group of compounds related to tryptophan upon the photofading of methylene blue under anaerobic conditions and upon the excited species generated by flash photolysis of the dye. These effects are discussed in relation to the ease of sensitized photooxidation of the amino acids by methylene blue, in order to test the hydrogen-abstraction mechanism proposed for this reaction.
In general, amino acids susceptible to sensitized oxidation also act as reducing agents in the photoreduction of methylene blue, while the insensitive compounds do not reduce the light-excited dye molecule. Methionine, histidine and tyrosine are exceptions, but this is probably due to our choice of pH for the photofading experiments.
The flash-photolysis experiments give no clear-cut evidence for a reaction between amino acid and dye triplet excited state leading to an enhanced yield of the semi-reduced radical, thought to be an intermediate of the photofading reaction. The lifetime of the semi-oxidized radical is reduced by many of the amino acids, and this may be the reactive intermediate in their oxidation.     

TRANSIENT PHOTOCHEMISTRY OF SAFRANIN-O

Abstract— We have characterized the spectra, acidity constants and decay kinetics of the triplet and semireduced radical species of Safranin-O. Between pH 3.0 and 10.6, there are three triplet species denoted ³DH^{2 +}₂, ³DH⁺ and ³D, the p K _as being 7.5 and 9.2. All three triplet species exhibit first order decay, the rate constant for ³DH⁺ being ca. 5-fold lower than the rate constants of ³DH⁺ and ³D. Ascorbic acid and ethylenediaminetetraacetic acid (EDTA) quench the triplet state under appropriate pH conditions and the pH dependencies of the yield of semireduced dye indicate that ³DH⁺ is more reactive than ³DH⁺ or ³D. With EDTA as the reducing agent, there is the additional requirement that at least one of the amino nitrogens be deprotonated to obtain a significant yield of semireduced dye. In these reactions, ascorbic acid is oxidized reversibly, but EDTA is oxidized irreversibly, so that with the latter reducing agent photolysis causes buildup of the leucodye, which on subsequent photolysis can reduce triplet state dye. With ascorbic acid as the reducing agent, the regeneration of the ground state dye is reversible, the decay of the semireduced radical being second order. In general, the transient photochemistry of Safranin-O resembles that of Thionine, the major difference being that the lifetimes of ³DH^{2 +}₂ and ³DH⁺ are much longer for Safranin-O than for Thionine.     

MECHANISM OF PHOTOREDUCTION OF THIAZINE DYES BY EDTA STUDIED BY FLASH PHOTOLYSIS-II. pH DEPENDENCE OF ELECTRON ABSTRACTION RATE CONSTANT OF THE DYES IN THEIR TRIPLET STATE

Abstract— The pH dependence of the apparent reactivity of thiazine dyes in their triplet states has been studied in aqueous solutions, using as electron donor HY^-3, the trianionic species of ethylene diamine tetraacetic acid (EDTA), in the pH range 4–8. The pH dependence is found to be related to a change in the degree of protonation of the triplet excited dye. The apparent reactivity and lifetime of two differently protonated forms of thionine, azur B and methylene blue were determined by classical and dye-laser flash techniques, making it possible to evaluate the rate constant for electron abstraction of these molecules in their triplet states. It is found that: (a) protonation on the ring nitrogen increases the electron-abstraction rate constant of the triplet-state species about twenty-fold, and (b) methylation on the side amino groups decreases it.     

THE INFLUENCE OF THE NITRO GROUP ON THE DISSOCIATIONS OF SOME AROMATIC ACIDS AND BASES IN THEIR LOWEST EXCITED TRIPLET STATES*

Abstract— –Estimation of lowest excited triplet and singlet state dissociation constants of some nitro-aromatic acids and bases, from shifts in their phosphorescence and absorption spectra, respectively, indicate that intramolecular charge transfer to the nitro group is much more important in the lowest excited singlet state than in the ground or lowest excited triplet states. As a result, the effect of a nitro group on the acidity of the lowest excited singlet state of an acid or base is more exaggerated than that on the ground or lowest excited triplet state of the same compound. Furthermore, the basicity of the nitro group is greatly enhanced in the lowest excited singlet state. On this basis the increased rate of photoreduction of nitrobenzene in acidic solutions is found to be thermodynamically unfeasible in the lowest excited triplet state. Although the reaction is thermodynamically feasible in the lowest excited singlet state, the short lifetime of that state may make the reaction kinetically unfeasible. Rate-Hammett acidity profiles are therefore inadequate to alone establish the mechanism of photoreduction of nitrobenzene.     

THE PHOTOREDUCTION OF METHYLENE BLUE BY AMINES—II. AN INVESTIGATION OF THE DECAY OF SEMIREDUCED METHYLENE BLUE

Abstract— The decay of semireduced methylene blue (MB') formed in the photoreduction of the dye by aryl- and alkylamines was examined by flash photolysis. The second order decay of MB in methanol produced using arylamines and 1,4-diazabicyclooctane as photoreducing agents led to complete regeneration of the dye and amine. The rate constants for a series of N.N-dimethylanilines showed a small substituent effect ( p = 0.69 ± 0.16) compatible with recombination of MB' with arylamine derived radical cations. A study of the solvent effect on the recombination process revealed the importance of the stability of the radical cation toward the solvent and also indicated that the reaction approaches the rate of diffusion. The photoreduction of the dye by most alkylamines examined resulted in permanent bleaching of methylene blue. The second order decay of MB' produced using tertiary aliphatic amines led exclusively to formation of the leuco-dye with no apparent regeneration of methylene blue; this process was tentatively assigned to a reaction of MB' with α-amino radicals formed from the amine radical cation. It was concluded that the methylene blue-sensitized photooxidation of amines involves a Type I process and possible mechanisms are discussed.     

ANION AND SOLVENT EFFECTS ON THE RATE OF REDUCTION OF TRIPLET EXCITED THIAZINE DYES BY FERROUS IONS

Abstract— The lifetimes of the triplet excited states of thionine and methylene blue were measured in aqueous and 50 v/v% aqueous acetonitrile solutions acidified with 0.01 N sulfuric or trifluoromethyl-sulfonic acid. The rate constants for reaction of the triplet excited dyes with ferrous ions were measured in the same solutions. The triplet lifetimes in the absence of added quenchers were insensitive to a change in acid from trifluoromethylsulfonic to sulfuric or to a change in solvent from water to 50v/v% aqueous acetonitrile (τ for triplet thionine ˜7.5 μs, τ for triplet methylene blue ˜4.5 μs). In contrast, the rate constant for reaction of the triplet dyes with ferrous ions increased by nearly a factor of 10 with a change in acid from trifluoromethylsulfonic to sulfuric. In solutions containing sulfate ions this reaction rate constant increased with increasing sulfate concentration and with a change in solvent from water to 50 v/v% aqueous acetonitrile. The results are discussed in terms of the possibility of association of the positively charged reactive ions with sulfate anions. Quenching of the triplet excited dyes by ferric ions or by ground state dye molecules was shown to be negligible at the concentration used for the ferrous ion quenching study.     

A FLASH PHOTOLYSIS STUDY OF INTERMEDIATES IN PHOTOCHEMICAL REACTIONS OF CHLOROPHYLL

Abstract— The photochemical reactions of chlorophyll intermediates in vitro have been studied by the flash photolysis method. The flash excitation of pigment solutions has been shown to involve the population of a chlorophyll triplet state where the oxidation-reduction processes occur. The mechanism and kinetics of pigment triplet decay have been investigated from 20°to — 50°C and the ability of chlorophyll molecules to carry out triplet-triplet energy transfer has been established. The latter phenomenon has been used to show up the role of chlorophyll triplets in the reversible photooxidation reaction with P -quinone. There have been studied initial products of pigment photoreduction with ascorbic acid and phenylhydrazine. Experimental data of the mechanism of the initial oxidation and reduction in chlorophyll photosensitized reactions have been analysed. There have been also obtained the differential spectra of chlorophyll triplets and radicals. A calculation has been made of rate constants for a few elementary reactions.