CHLOROPHYLL ONE-ELECTRON PHOTOCHEMISTRY: LIGHT-INDUCED ABSORBANCE CHANGES AND ESR SIGNALS FOR VARIOUS PORPHYRIN-QUINONE AND HYDROQUINONE SYSTEMS IN ALCOHOL SOLVENTS*

Abstract— Light-dark optical difference spectra of degassed ethanol or pyridine solutions of chlorophyll and benzoquinone or hydroquinone at temperatures above — 50°C show only the semiquinone absorbance band. Decay of the signals is second order, with a rate constant in agreement with earlier ESR results. Light-induced optical changes due to chlorophyll can be elicited by lowering the temperature of ethanol solutions of chlorophyll and benzoquinone to a region of high viscosity. Hydroquinone is not effective in producing these optical changes. Similar results are achieved at room temperature by using as solvent a degassed mixture of the alcohols: cyclohexanol, tert-butanol, and ethanol (CBE). Difference spectra show bleaching of the chlorophyll bands and increased absorbance in the intermediate wavelength region (460–580 nm). Decay kinetics are first order, while the rise is complicated (probably biphasic). ESR signals have no hyperfine structure and also decay by first order kinetics, at a rate which is faster than that of the optical changes. The ESR signals reach a steady state more rapidly than the optical signals, without biphasic kinetics. These results demonstrate that at least two species are generated. Addition of acid increases the amount of bleaching in CBE, while small amounts of base decrease it. Larger amounts of base cause chlorophyll bleaching to completely disappear and only the semiquinone anion is observed. Activation energies for the chlorophyll a-benzoquinone photoreaction in CBE are 10–14 kcal/mole. Lower potential quinones give lower activation energies. The rate constant for quenching of the triplet state of chlorophyll a by β-carotene in CBE is 7.5±0.5×10⁸ (M set)^-1. β-carotene also quenches photoproduct formation. The bimolecular rate constant for formation of the photoproduct with benzoquinone was calculated to be 7×10⁸ (Msec)^-1. The redox potential of the quinone affects both the magnitude of the chlorophyll absorbance changes and the rate of decay. The higher the potential, the larger the changes and the slower the decay. Other porphyrin systems show similar photoreactions only if they are chelated with a group II metal, such as Mg²⁺, Cd⁺², or Zn⁺². The results are interpreted in terms of the formation, by a triplet-sensitized one-electron transfer from solvent to quinone, of a chlorophyll-semiquinone complex which is stabilized via coordination with the chelated metal.     

ESR STUDIES OF CHLOROPHYLL ONE-ELECTRON PHOTOCHEMISTRY: KINETICS AND MECHANISM OF REVERSIBLE HYDROQUINONE OXIDATION IN SOLUTION*

Abstract— ESR studies have been made of the kinetics of semiquinone radical formation and disappearance resulting from the reversible photosensitization by chlorophyll of hydroquinone oxidation in a pyridine-water solvent. The rate of radical decay was found to be second order with respect to the radical concentration, with a rate constant of 6.7 × 10⁵ l./mole sec at -30°C and an activation energy of 6900 cal/mole. The rate of radical formation was recombination-limited and, through the use of β-carotene as a quencher, the rate constant was determined to be 8.81 × 10⁵ l./mole sec at -30°C. The effect of light intensity and hydroquinone concentration on the rate of semiquinone radical formation and on the steady state radical concentration was also investigated and possible mechanisms to explain the results are discussed.     

LASER PHOTOLYSIS STUDIES OF QUINONE REDUCTION BY CHLOROPHYLL a IN ALCOHOL SOLUTION

Upon laser photolysis of chlorophyll-quinone solutions in ethanol, transients due to the chlorophyll triplet state (C_t), the chlorophyll cation radical (C⁺) and the semiquinone radical (Q^-) can be observed. The rise of Q^- parallels the decay of C_t. demonstrating the precursor role of the triplet. The decay of C⁺ is second order, consistent with reverse electron transfer, and has a rate constant which is independent of quinone potential, and an activation energy of 14kJ/mol due mainly to the temperature dependence of solvent viscosity. Triplet quenching and C⁺ yield are found to decrease with decreasing quinone potential.     

PRIMARY PRODUCTS IN THE FLASH PHOTOLYSIS OF TRYPTOPHAN*

There has been considerable interest in the photochemistry of tryptophan in connection with ultraviolet inactivation of enzymes. Earlier flash photolysis work has demonstrated that the hydrated electron (e^-_aq) is an initial product in the irradiation of indole derivatives, accompanied by a longer-lived transient absorption near 500 nm attributed to an aromatic radical species[1–5]. Similar transients were observed in a recent flash photolysis study of lysozyme[6] in which it was proposed that inactivation is a consequence of electron ejection from 1 to 2 essential tryptophan residues in the active center. However, there has been uncertainty concerning the tryptophan radical structure and its relationship to the triplet state and radical spectra reported for tryptophan photolysis in low-temperature rigid media. This note reports a flash photolysis investigation of L-tryptophan (Trp) and 1-Methyl-L-tryptophan (1-MeTrp) undertaken to clarify these points. The flash photolysis apparatus and methods employed are described in Ref. [6].     

A FLASH PHOTOLYSIS STUDY OF THE TRIPLET STATE OF ACRIDINE ORANGE IN BASIC SOLVENTS

Abstract— Photochemical reactions of acridine orange (AO) in basic aqueous and ethanolic solutions were studied using the flash photolysis technique. The absorption spectrum of AO was determined in detail (230–900 nm) and extinction coefficients were obtained. The decay of the triplet state is the result of a first order process, a triplet-triplet annihilation process and a quenching by the dye in the ground state. The main part of the triplet decays to the ground state; however the observation of semi-reduced AO shows that the decay is partly due to chemical reactions.
An efficient reversible reaction is observed on flashing aqueous solutions of AO containing tetramethyl- p -phenylenediamine: semi-reduced AO is formed in high yield by reaction between the triplet dye and the diamine. In addition, irreversible reactions of AO occur; these are shown to be due to the triplet state by the method of triplet energy transfer.     

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.     

CHLOROPHYLL ONE-ELECTRON PHOTOCHEMISTRY-II. PHOTOSENSITIZATION BY CHLOROPHYLL,PHEOPHYTIN AND BACTERIOCHLOROPHYLL OF A ONE-ELECTRON TRANSFER REACTION BETWEEN ETHANOL AND QUINONE*

Abstract— Chlorophyll, pheophytin and bacteriochlorophyll sensitize a one-electron transfer in the presence of quinones in ethanol, to produce a ternary complex of ground state porphyrin analog, alcohol cation radical and semiquinone anion radical as the primary photo-product at low temperature. A similar photo-oxidation of alcohol to produce a binary complex is caused by direct excitation of quin-one in the absence of chlorophyll. The mechanisms of these reactions and their implications for photosynthesis are discussed.     

CHLOROPHYLL-QUINONE PHOTOCHEMISTRY IN LIPOSOMES: MECHANISMS OF RADICAL FORMATION AND DECAY*

Abstract— Laser flash photolysis has been used to investigate the mechanism of formation and decay of the radical species generated by light-induced electron transfer from chlorophyll a (Chi) triplet to various quinones in egg phosphatidyl choline bilayer vesicles. Chlorophyll triplet quenching by quinone is controlled by diffusion occurring within the bilayer membrane (kq～ 10⁶M^?1 s^?1. as compared to ～ 10⁹ M^?1 s^?1 in ethanol) and reflects bilayer viscosity. Radical formation via separation of the intermediate ion pair is also inhibited by increased bilayer viscosity. Cooperativity is observed in the radical formation process due to an enhancement of radical separation by electron transfer from semiquinone anion radical to a neighboring quinone molecule. Two modes of radical decay are observed, a rapid (t_1/2= 150μ) recombination between Chi and quinone radicals occurring within the bilayer and a much slower (t_1/2= 1–100 ms) recombination occurring across the bilayer-water interface. The latter is also cooperative, which accounts for a t_1/2 which is dependent upon quinone concentration. The slow decay is only observed with quinones which are not tightly anchored into the bilayer, and is probably the result of electron transfer from semiquinone anion radical formed within the bilayer to a quinone molecule residing at the bilayer-water interface. Direct evidence for such a process has been obtained from experiments in which both ubiquinone and benzoquinone are present simultaneously. With benzo-quinone, approx. 60% of the radical decay occurs via the slow mode. Triplet to radical conversion efficiencies in the bilayer systems are comparable to those obtained in fluid solution (～ 60%). However, radical recombination, at least for the slow decay mechanism, is considerably retarded.     

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.     

PRIMARY PROCESSES IN THE PHOTOCHEMISTRY OF AQUEOUS SULPHACETAMIDE: A LASER FLASH PHOTOLYSIS and PULSE RADIOLYSIS STUDY

Abstract— The spectra have been measured of the transient species formed in the nanosecond flash photolysis of aqueous solutions of sulphacetamide under a variety of conditions. In addition to the excited triplet state, the cation radical and the solvated electron were observed. The ionisation of aqueous sulphacetamide was found to occur by a biphotonic process. The extinction coefficient of the cation radical of sulphacetamide was determined by both laser flash photolysis and pulse radiolysis techniques, a value of 1.9 times 10₃ dm₃mol_-1cm_-1 being obtained. The rate of electron reaction with sulphacetamide and the anion radical spectrum were also determined by the two techniques, good agreement being obtained. The spectrum of the product of the reaction of the superoxide anion radical and the corresponding rate constant have also been determined. A possible mechanism of photosensitized skin reaction due to sulphacetamide is discussed.     

CHLOROPHYLL ONE-ELECTRON PHOTOCHEMISTRY-I. PHOTOPRODUCTION OF THE CATION RADICALS OF CHLOROPHYLL AND BACTERIOCHLOROPHYLL IN SOLUTIONS AT LOW TEMPERATURES*

Abstract— Electron spin resonance studies have shown that chlorophyll and bacteriochlorophyll can be photo-oxidized in a variety of solvents via their lowest excited singlet states to produce cation radicals. Pheophytin does not undergo this reaction. The mechanism of this photoprocess and its implications for photosynthesis are discussed.     

FLASH PHOTOLYSIS STUDIES OF PROTEINS AND INDOLE DERIVATIVES IN SOLUTION

Abstract— Transients obtained upon flash photolysis of a number of proteins in aqueous solution appear to derive from electron ejection from tryptophyl residue side chains. These decay by a second order process. Oxygen is an effective quencher for the protein transients but is less so for the flash-induced signals obtained from simple indole derivatives. Experiments using other quenchers indicate that the signals are not due to an indole triplet state, but that the triplet state may be a precursor of the flash-induced metastabie species. Compounds which bind to the active site of chymotrypsin were found to exert only non-specific effects on the flash-induced signals.     

MECHANISM OF THE PHOTOHYDRATION OF PYRIMIDINES: A FLASH PHOTOLYSIS STUDY*

Abstract— Based on comparisons between the microsecond flash photolysis of uracil and substituted uracils and the radiation chemistry of the corresponding 5,6-dihydro-2,4-dioxopyrimidines, it is proposed that the mechanism of photohydration of pyrimidine bases involves the formation of a pyrimidine carbocation. The effects of substitution in the pyrimidine ring and of pH are consistent with a proton transfer from water to the excited singlet state at theC–5 position of the pyrimidine ring. The resultant carbocation is thought to undergo solvolysis to form the photohydrate or eliminate a proton at N-l producing an intermediate isomeric form of the pyrimidine (isopyrimidine) which re-arranges to the parent pyrimidine by a first-order process.     

PHOTOCHEMISTRY OF THE REACTION CENTERS OF SYSTEM II UNDER REPETITIVE FLASH GROUP EXCITATION IN ISOLATED CHLOROPLASTS

Abstract— Absorption changes induced in isolated chloroplasts by excitation with repetitive flash groups have been measured at 690 nm, indicating the photochemical turnover of chlorophyll-a_II (Chl-αn), and at 480 nm and 513 nm respectively, reflecting via electrochromic effect the formation of a transmembrane electric field. The data are compared with measurements of oxygen evolution. In chloroplasts with practically fully intact oxygen evolving capacity it was found: 1. The initial amplitude of the 690 nm absorption change induced by the second flash as a function of the time t_v between the first and second flash of a group increases with a half life of about 35 µs. On the other hand, the average oxygen yield due to the second flash as a function of the time t_v rises with a half life of about 600 µs (and a kinetics in the ms-range of minor extent), confirming the data of Vater et al. (1968). 2. Under far red background illumination, where contributions due to PS I in the µs-range can be excluded, the difference spectrum in the red of the absorption changes induced by the first flash corresponds with that of the absorption changes induced by the second flash fired 200 µ after the first flash. 3. The pattern of the absorption changes at 690 nm induced by repetitive double flash groups at t_v= 200 µs does not markedly change in normal chloroplasts by the presence of DBMIB?. Similar 690 nm absorption changes occur in trypsin treated chloroplasts, independent of the presence of DCMU. 4. The fast regeneration in the µs-range of Chl-a_n is also observed in the third flash of a triple flash group at a time t_v= 200 µs between the flashes of the group. 5. The initial amplitudes of the absorption changes with a decay kinetics slower than 100 µs induced by the second flash at 480 nm and 513 nm, respectively, as a function of the time t, between the first and second flash of a group, are characterized by a recovery half-time of about 600 µs, confirming earlier measurements at 520 nm (Witt and Zickler, 1974). On the basis of these results it is inferred that there does exist a photoreaction of Chl-α_n., with an electron acceptor, referred to as X_a, other than the ‘primary’ plastoquinone acceptor X320, if X320 persists in its reduced state. Under conditions of X320 being in the reduced state, this photochemical reaction was shown to be highly dissipative with respect to charging up the watersplitting enzyme system Y. Furthermore, this Chl-a_n-photoreaction with X_a does not lead to a vectorial transmembrane charge separation, which is stable for more than a few microseconds. Different models for the functional and structural organization of PS II are discussed.     

QUANTUM YIELDS OF THE LUMINOL CHEMILUMINESCENCE REACTION IN AQUEOUS AND APROTIC SOLVENTS*

Abstract— Quantum yields for luminol (3-aminophthalic hydrazide) chemiluminescence reactions fall into two classes depending on oxidizing conditions. In aprotic solvents the quantum yield is high and the excitation yield which allows for the fluorescence quantum yield of the product, is 0·09 and is unaffected by changes in solution temperature or polarity, or the presence of quenchers. In aqueous solution under optimum pH conditions (11–13), hydrogen peroxide oxidation results in a high chemiluminescence quantum yield with an excitation yield of 0·04 again unaffected by temperature, viscosity or quenchers. Other oxidizing conditions produce lower quantum yields probably by the introduction of competing chemical pathways. The luminol chemiluminescence light standard has been used to calibrate a spectrofluoro-meter with results in good agreement with the quantum yields of the ferrioxalate actinometer and the fluorescence of quinine sulfate and diphenylanthracene.     

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.     

AN ULTRAVIOLET PULSE GENERATOR IN THE SUBMICROSECOND RANGE FOR FLASH PHOTOLYSIS STUDIES

Los métodos experimentales que se emplean en la actualidad par identificar el electrón hidratado y otras especies transientes no pueden extenderse a escala de tiempos más cortos (10^-7–10^-8 seg), que se requiere para la observación de moléculas excitadas de corta vida, que son precursoras de estas especies transientes, sin sustancial desarrollo. Para alcanzar estos tiempos, los pulsos de excitation sean de electrones, rayos X o luz, así como la luz pulsada para el análisis espectro-fotométrico debe acortarse en varios órdenes de magnitud. Hemos diseñado un generador de luz ultravioleta y visible que emite pulsos menores que 10^-7 seg y potencia de miles de megavatios. El principio básico consiste en aumentar la tensión de chispa hasta próximo a 100 kV y decrecer la capacidad del circuito de descarga como √V, para mantener la energía de la descarga. Suponemos que estos no son los límites finales de intensidad y tiempo. Para obtener pulsos de luz netos, con tiempo de decrecimiento corto y sin cola se han mejorado considerablemente el circuito elétrico y el montaje del generador. Los primeros resultados en el estudio de transientes de corta vida muestran que esta fuente de luz mejorada permite extender el estudio de transientes en la región de 10^-7 seg.     

FLASH PHOTOLYSIS STUDIES OF HEMATO-AND COPRO-PORPHYRINS IN HOMOGENEOUS AND MICROHETEROGENEOUS AQUEOUS DISPERSIONS

This paper describes an experimental study of the photo properties of the triplet (T,) states of hematoporphyrin (HP) and coproporphyrin (CP), particularly in relation to their medium dependence and reactivity towards oxygen. Triplet-triplet absorption spectra of HP and CP have been determined in aqueous buffer at pH = 7.4 and in water-methanol and water-formamide mixtures. The spectra corrected for ground state contributions show major absorption peaks near 400 nm and lesser peaks near 500 nm. Extinction coefficient measurements have been made and their dependences on solvent composition investigated. Natural lifetimes of the T₁ states of HP and CP and the bimolecular quenching constants with oxygen have been determined. The quantum yields of T₁ formation are ca. 0.6 in buffer rising to 0.8 and higher in predominantly organic media. Incorporation of the porphyrins into micellar phases similarly causes φ_T, to increase. Quantum efficiencies of O^?₂ and O₂(^lΔ_g) formation have been determined for HP in buffer, some binary mixtures and micellar dispersions. Superoxide yields are low and may result from photo-ionization processes. O₂(^lΔ_g) yields are large but appear to have an unexpected dependence on the medium.     

LASER PHOTOLYSIS STUDIES OF QUINONE REDUCTION BY PHEOPHYTIN a IN ALCOHOL SOLUTION

Abstract- Upon laser photolysis of pheophytin-benzoquinone solutions in ethanol, transients due to the pheophytin triplet state (P_t), an exciplex (Pδ⁺ Qδ^-), the pheophytin cation radical (P⁺) and the semiquinone radical (Q^-) can be observed. Kinetic analysis indicates that the evolution of these transients at times longer than one microsecond is due to the decay of the exciplex with the concomitant formation of P⁺ and Q^-, reverse electron transfer to form P and Q, solvent oxidation by P⁺, and Q^- disproportionation. In support of the suggested solvent oxidation reaction, a large deuterium isotope effect is observed upon changing the solvent from methanol to its fully-deuterated counterpart. Comparisons are made between these results and those obtained with chlorophyll as described in the preceding paper.     

PHOTOCHEMISTRY OF FLAVINS—I. CONVENTIONAL AND LASER FLASH PHOTOLYSIS STUDY OF ALLOXAZINE

Abstract— The photobleaching of alloxazine in buffered aqueous solution has been studied by means of flash photolysis using conventional and laser excitation sources. Several transient species have been characterized. The alloxazine triplet state (Λ_max 420 nm and 550 nm, times; = 9 μs) was identified with the aid of low-temperature comparison experiments in ethanol. Transient absorption with Λ_max 440 nm, which appears after decay of the triplet state, and whose second-order decay is pH-dependent, is postulated to be due to the semiquinone radical (AH₂*) and a radical derived from alloxazine by addition of water and loss of a hydrogen atom (HAOH*), which are in equilibrium with their conjugate cation radicals. The results of experiments in the presence of oxygen indicate that these species are not primarily formed from the triplet state. The enhanced second-order decay of the flavin radicals in oxygen-containing solutions is interpreted in terms of their reaction with the peroxy radicals. The proposed mechanisms account for the production of hydroxylated alloxazines.