CHLOROPHYLL PHOTOCHEMISTRY IN CONDENSED MEDIA—II. TRIPLET STATE QUENCHING AND ELECTRON TRANSFER TO QUINONE IN LIPOSOMES*

The fate of excitation energy and electron transfer to quinones within Chl-a-containing phosphatidyl choline liposomes has been investigated. The bilayer membrane of the liposome stabilizes the Chl triplet state, as evidenced by a three-fold increase in the lifetime over that observed in ethanol solution. The relative triplet yield follows the relative fluorescence yield, indicative of quenching at the singlet level. Triplet state lifetimes are markedly shortened as the Chl concentration is increased, demonstrating that quenching occurs at the triplet level as well. This process is shown to be due to a collisional de-excitation. In the presence of quinones, the Chl triplet reduces the quinone resulting in production of long-lived electron transfer products. The percent conversion of Chl triplet to cation radical when benzoquinone is employed as acceptor is approximately 60 ± 10%, which is slightly less than in ethanol solution (70 ± 10%). The lifetime of the radical, however, can be as much as 1900 times longer. With respect to potentially useful photochemical energy conversion, the magnitude of this increased lifetime is far more significant than is the decreased radical yield.     

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.     

CHLOROPHYLL PHOTOSENSITIZED ELECTRON TRANSFER REACTIONS IN LIPID VESICLES:ENHANCEMENT IN YIELD OF VECTORIAL ELECTRON TRANSFER ACROSS THE BILAYER FROM REDUCED CYTOCHROME c TO OXIDIZED FERREDOXIN BY ADDITION OF VALINOMYCIN PLUS POTASSIUM ION

Chlorophyll photosensitized electron transfer across a vesicle bilayer from reduced cytochrome c in the inner compartment to oxidized ferredoxin in the outer compartment, using propylene diquat as a mediator, has been investigated using both steady-state and laser flash photolysis methods. One of the factors limiting the quantum yield is the transmembrane potential, which is formed during sample preparation and is increased by the electron transfer process across the membrane bilayer. This limitation can be diminished by the incorporation of valinomycin into the bilayer in the presence of potassium ion. The overall quantum yield can be approximately doubled (up to a total of 22% based on the chlorophyll triplet which is quenched, and 2.8% based on the absorbed quanta) by valinomycin addition. Another quantum yield limitation arises from the accumulation of oxidized cytochrome c in the inner aqueous compartment, which is formed as a consequence of the transbilayer electron transport process and can quench triplet chlorophyll on the inner side of the vesicle. The chlorophyll cation radical generated in this way can participate in the electron exchange equilibrium between chlorophyll molecules located within the bilayer, and thus inhibit electron flow from inside to outside. This acts to limit the extent of cytochrome c oxidation to less than or equal to 50% of the original amount.     

HORSE HEART CYTOCHROME c AS AN ELECTRON ACCEPTOR FROM CHLOROPHYLL TRIPLET IN NEGATIVELY CHARGED LIPID BILAYER VESICLES*

Abstract— Cytochrome c has been shown to bind via electrostatic interactions to egg phosphatidylcholine vesicles which contain 5–30 mol percent of negatively-charged surfactant (dihexadecylphosphate) in a low ionic strength medium. Under these conditions the oxidized cytochrome can function as a direct one-electron acceptor from membrane-bound triplet state chlorophyll to produce chlorophyll cation radical and reduced cytochrome. Kinetic experiments using laser flash photolysis have demonstrated that triplet quenching and the yield of electron transfer products increase, and product lifetime decreases, with an increase in the magnitude of the negative charge on the vesicles, and with a decrease in the ionic strength of the medium. Both triplet quenching and product formation rates and yields showed saturation behavior as the cytochrome concentration was increased, and reached limiting values at 20–30 μM cytochrome when the vesicle contained 20 mol percent of the negatively-charged surfactant. This behavior is interpreted in terms of saturation of the vesicle surface binding sites. Under optimum conditions in this system, approximately 20% of the chlorophyll triplet molecules could be converted to electron transfer products which had a halftime for the reverse reaction of approximately 1.5 ms.     

ELECTRON TRANSFER REACTIONS OCCURRING UPON LASER FLASH PHOTOLYSIS OF PHOSPHOLIPID BILAYER SYSTEMS CONTAINING CHLOROPHYLL,BENZOQUINONE AND CYTOCHROME c-I. NEGATIVELY-CHARGED VESICLES*

Abstract— In negatively-charged lipid bilayer vesicles prepared in deionized water from egg phosphatidylcholine and 25 mol % of α-eleostearic acid, and containing chlorophyll a, benzoquinone, and cytochrome c, primary electron transfer after a laser flash occurred principally from chlorophyll triplet to benzoquinone, and to a smaller extent from chlorophyll triplet to oxidized cytochrome c. Several secondary electron transfer reactions occurred subsequent to this. The most rapid of these was electron transfer from reduced cytochrome c, which was bound to the outer surface of the negatively-charged vesicle, to chlorophyll cation radical (k= 3.9 times 10³ s^-1). Subsequent to this, the cation radical was reduced by benzoquinone anion radical (k= 1.6 times 10² s^-1>) and bound oxidized cytochrome c was reduced by the remaining anion radical which was expelled into the aqueous phase by the negative charge on the vesicle surface. This latter reaction occurred at the membrane-solution interface with an observed rate constant (k= 60 s^-1) two orders of magnitude smaller than cytochrome oxidation. Net reduced cytochrome c was produced in this process. The reduced cytochrome c was slowly reoxidized by benzoquinone (k= 17 s^-1) and the system was returned to its original state. When the vesicle system was made slightly basic by adding tris(hydroxymethyl)aminomethane, the rates of both the reverse electron transfer between chlorophyll cation radical and benzoquinone anion radical (k= 5 times 10² s^-1) and the oxidation of reduced cytochrome c by chlorophyll cation radical (k= 9.4 times 10³ s^-1) were accelerated. The rate of reduction of oxidized cytochrome c by benzoquinone anion radical remained approximately the same.     

ELECTRON TRANSFER REACTIONS OCCURRING UPON LASER FLASH PHOTOLYSIS OF PHOSPHOLIPID BILAYER SYSTEMS CONTAINING CHLOROPHYLL,BENZOQUINONE AND CYTOCHROME c-II. ELECTRICALLY NEUTRAL AND POSITIVELY-CHARGED VESICLES*

Abstract— The primary and secondary electron transfer reactions which occurred upon laser flash photolysis of electrically neutral and positively-charged lipid bilayer vesicles containing chlorophyll, benzoquinone and cytochrome c were determined by time-resolved difference spectral and kinetic measurements, and compared with previous results obtained with negatively-charged vesicles (Y. Fang and G. Tollin, Photochem. Photobiol. 1988). The extent to which oxidized cytochrome c could function as an electron acceptor from triplet state chlorophyll, and reduced cytochrome c could act as an electron donor to chlorophyll cation radical, decreased from negatively-charged to electrically neutral to positively-charged vesicles, in agreement with expectations based on changes in the ability of cytochrome c to bind to the bilayer. In all three types of vesicles, cytochrome c reduction by benzoquinone anion radical occurred in the aqueous phase.     

CHLOROPHYLL PHOTOSENSITIZED ELECTRON TRANSFER IN PHOSPHOLIPID VESICLE SYSTEMS: COMPARISON OF INSIDE-OUTSIDE ASYMMETRY BETWEEN LARGE AND SMALL UNILAMELLAR VESICLES*

Abstract— We have determined the chlorophyll triplet quenching efficiencies, the chlorophyll cation radical yields and the conversion efficiencies of chlorophyll triplet to radical in large and small unilamellar phosphatidylcholine vesicles (LUV and SUV, respectively) in the presence of electrically-charged electron acceptors (ferricyanide and oxidized cytochrome c) located in either the inner or outer aqueous compartments of the vesicles. Both types of vesicles displayed inside-outside asymmetry, although the properties were reversed. Triplet quenching in SUV was more efficient when ferricyanide was located within the vesicle interior, whereas the reverse was true in LUV. When ferricyanide was located on the outside of the vesicles, the extent of triplet quenching in LUV was about two times that in SUV and the amount of cation radical formed in LUV was about two times that in SUV. Under these conditions, the conversion efficiencies of chlorophyll triplet to radical were 12.2% for LUV and 8.5% for SUV. With cytochrome c as an electron acceptor in negatively charged vesicles (25 mol per cent dixhexadecylphosphate incorporated) similar results were obtained. Again, the triplet quenching and radical yield inside-outside asymmetry properties were reversed between the two types of vesicles, and radical formation efficiencies when cyt c was located outside the vesicles were higher in LUV (11.7%) than in SUV (4.2%). We conclude that the inside-outside asymmetric photochemical behavior of unilamellar phosphatidylcholine vesicles is influenced by factors in addition to the difference in radius of curvature between the inside and outside surfaces. It is suggested that transmembrane electrostatic potentials may be involved. Furthermore, in the present system the properties of LUV were more favorable to photochemical electron transfer product formation than those of SUV.     

VECTORIAL ELECTRON TRANSPORT ACROSS LIPID VESICLE MEMBRANES DRIVEN BY TWO PHOTOREACTIONS. I. ETHYLENEDIAMINETETRAACETIC ACID AS AN ELECTRON DONOR via FLAVIN TRIPLET STATE IN THE INNER COMPARTMENT AND CYTOCHROME c AS AN ELECTRON ACCEPTOR FROM CHLOROPHYLL TRIPLET STATE IN THE OUTER COMPARTMENT

Negatively charged vesicle suspensions containing chlorophyll a (chl) dissolved in the lipid bilayer, flavin mononucleotide (FMN) and/or ethylenediaminetetraacetic acid (EDTA) enclosed in the inner compartment as electron sources and oxidized cytochrome c (cyt c[ox]) in the outer compartment as an electron acceptor have been studied using laser flash photolysis and steady-state irradiation methods. Cytochrome c initially quenches the chl triplet state (³chl) generating the chlorophyll cation radical (chi⁺′) in the membrane. Reverse electron transfer from cyt c(red) to chl^+. subsequently occurs in a kinetically biphasic reaction, with rate constants of 430 pT 30 and 21.9 pT 1.7 s^?1 for the fast and slow phases, respectively. In the absence of FMN, reduction of chl⁺′ by EDTA in the inner compartment can be observed during steady-state irradiation but not in a laser flash photolysis experiment. This is due to a low reaction yield, which is probably limited by the repulsive electrostatic interaction between EDTA and the negatively charged membrane. When FMN was enclosed together with EDTA in the inner Compartment, the reaction yield of vectorial electron transfer across the bilayer from EDTA to cyt c(oX) was increased by a factor of six during steadystate white light irradiation. Laser flash photolysis and steady-state irradiation experiments using red and blue light excitation have demonstrated that the enhancement mechanism involves the formation of fully reduced FMN by blue light-sensitized photooxidation of EDTA via the flavin triplet state, occumng simultaneously with red lightsensitized electron transfer to cyt c via the chlorophyll triplet state.     

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.     

THE CHLOROPHYLL-SENSITIZED REACTION BETWEEN BENZOQUINONE AND ETHANOL*

The electron-transfer reaction between triplet excited chlorophyll and quinones has been extensively studied as a model of the primary reaction in photosystem II. There has also been reported a minor reaction in which the chlorophyll cation radical ostensibly oxidizes the alcohol solvent or even water, leading to a gradual net reduction of quinone, but the exact mechanism and even the existence of this reaction has been uncertain. We have examined the consequences of prolonged irradation of ethyl chlorophyllide and benzoquinone in acidulated ethanol, and find a chlorophyllide-sensitized reaction which is not analogous to the better-known autosensitized reduction of quinones in blue or UV light. In the chlorophyllide-sensitized reaction, benzoquinone is apparently converted to ethoxy-substituted quinones and quinols, and polymeric material. Ethyl chlorophyllide (or chlorophyll) is simultaneously oxidized to more polar products which themselves continue to photosensitize the reaction of quinones. The production of acetaldehyde could not be demonstrated in the sensitized reaction. Chlorophyllide-sensitized reaction of (l-hydroxyethyl)benzoquinone, ethoxybenzoquinone and 2.5-diethoxybenzo-quinone were examined for additional information. A reaction sequence, tentatively proposed to accommodate the known facts, starts with oxidative attack by quinone on an oxidized chlorophyllide radical formed by loss of a hydroxyl proton from alcohol bound as a ligand to Mg²⁺. It is not likely that this reaction is closely related to events at the oxidizing side of photosystem II.     

CHLOROPHYLL PHOTOCHEMISTRY IN CONDENSED MEDIA—I. TRIPLET STATE QUENCHING AND ELECTRON TRANSFER TO QUINONE IN CELLULOSE ACETATE FILMS*

Chlorophyll-a was incorporated into cellulose acetate films and the triplet state decay kinetics and electron transfer from triplet to p-benzoquinone in aqueous solution was studied using laser flash photolysis and EPR. The triplet was found to decay by first order kinetics with a rate constant which was independent of Chl concentration. The triplet yield, however, was concentration dependent. These properties are due to quenching which occurs only at the singlet state level. In the presence of quinone, the triplet is quenched and, when the quinone is in an aqueous solution in contact with the film, Chl cation radical (C^±) as well as the semiquinone anion radical (Q^±) can be observed. The C decays by second order kinetics with a rate constant of 1.5 × 10⁶M^-1 s^-1. Although triplet conversion to radicals is slightly lower in the films as compared to fluid solutions (? 3 times), the lifetimes of the radicals are greatly increased (? 10³ times).     

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.     

PHOTOINDUCED ONE-ELECTRON TRANSFER BETWEEN BACTERIOCHLOROPHYLL AND QUINONE IN ACETONE*

Abstract— Illumination with red light of a degassed solution of bacteriochlorophyll and benzoquinone or ubiquinone in dry acetone at low temperatures (< - 105°C) leads to the formation of the bacteriochlorophyll cation radical and the quinone anion radical, as detected by ESR spectroscopy. At temperatures around - 110°C, the quinone radical signal corresponds to an emission of microwave radiation. These results are interpreted in terms of a one-electron transfer from a spin-polarized bacteriochlorophyll triplet state to the quinone, producing an anion radical which is predominantly in the upper spin state.     

PHOTOCHEMICAL CHARACTERIZATION OF COVALENTLY-LINKED PORPHYRIN-QUINONE COMPLEXES*

Abstract— The fluorescence properties of a covalently-linked porphyrin-quinone complex and its zinc derivative were studied in a variety of organic solvents. The kinetics of fluorescence decay for both the quinone and hydroquinone oxidation states were measured in acetonitrile, dichloromethane, dimethyl-formamide, and pentane. The fluorescence yield and kinetics of decay at room temperature were little affected in the porphyrin or zinc porphyrin complexes when the attached quinone was reduced. However, for these complexes the fluorescence yield and lifetimes were both substantially decreased in acetonitrile and dichloromethane when the quinone was in its oxidized state. These latter decay kinetics were not explainable by a process having a single exponential decay. On the other hand, little fluorescence quenching or lifetime shortening was observed in dimethylformamide or pentane, indicating unique solvent dependencies for the quenching process. Evidence was obtained for photoproduced charge separation from EPR measurements on the covalently-linked zinc porphyrin-quinone complex. The EPR data showed equivalent concentrations of a Zn porphyrin cation radical and a benzoquinone anion radical in acetonitrile or dichloromethane at both room temperature and 77 K. The charge separated state rapidly decayed at room temperature (in sub-millisecond times) but was quite stable at 77 K. It is concluded that light-induced charge separation in acetonitrile and dichloromethane at room temperature may occur from the excited singlet state with a high quantum efficiency. A photoproduced charge separated state also occurred when the covalently-linked complexes were incorporated into egg yolk phosphatidylcholine liposomes. The quantum yield for radical formation in this latter system was 0.1 and the lifetimes of the radical species formed were many minutes.     

PHOTOINDUCED REDUCTION OF CYTOCHROME c IN THE PRESENCE OF HYDROPEROXIDE DERIVATIVE OF BIPHENYL

Abstract The photosensitive hydroperoxide derivative of biphenyl (BPP) was synthesized by the ozonolysis of phenanthrene in methanol. When cytochrome c (cyt c ) was illuminated by UVB light in the presence of BPP (BPPUV), it was reduced both under aerobic and anaerobic conditions. The action spectrum of the reduction was consistent with that of photolytic decomposition of BPP. Both gave maximum reactions at wavelengths around 300–310 nm. Electron spin resonance studies, using 5,5-dimethyl-1-pyrroline N -oxide as a spin-trapping reagent, revealed the generation of hydroxyl radicals in the BPPUV system. Product analysis of adamantane oxidation by BPPW also suggested the generation of hydroxyl radicals rather than singlet oxygen. However, the effects of scavengers were complicated. Singlet oxygen scavengers significantly inhibit the reaction while none of the hydroxyl radical scavengers tested was effective in inhibiting the BPPUV-mediated cyt c reduction. Deuterium oxide, which extends the lifetime of singlet oxygen, inhibited rather than enhanced the reaction. Reduction of cyt c was inhibited by salts, and their activities were correlated to the electron-donating nature of the anions. These results suggest that reduction of cyt c is mediated by electron transfer from a light-induced product of BPP, rather than by free hydroxyl radicals or singlet oxygen.     

CHLOROPHYLL PHOTOSENSITIZED ELECTRON TRANSFER REACTIONS IN LIPID BILAYER VESICLES: VECTORIAL ELECTRON TRANSFER ACROSS THE BILAYER FROM REDUCED CYTOCHROME c IN THE INNER COMPARTMENT TO OXIDIZED FERREDOXIN IN THE OUTER COMPARTMENT

A negatively charged large unilamellar vesicle system containing a membrane-bound photo-sensitizer (chlorophyll, Chi), a reduced redox protein [cytochrome c, cyt c(red)] in the inner aqueous compartment, an oxidized redox protein [ferredoxin, Fd(ox)] in the outer aqueous compartment, and propylene diquat (PDQ²⁺) as a mediator, was investigated using both flash and steady-state photolysis techniques. The results demonstrate that the light-generated triplet state of Chi (³Chl) was initially quenched by PDQ²⁺ at the outer membrane surface to form Chi cation radical (Chl⁺) and the reduced diquat (PDQ⁺). This was succeeded by a biphasic recombination between Chi⁺ and PDQ⁺. The slow phase of the recombination process, which represents reverse electron transfer between Chl⁺ and those PDQ⁺ molecules which escaped from the membrane surface, could be suppressed effectively both by the reduction of Chl^ in the inner monolayer of the vesicles by cyt c(red), and by the reoxidation of PDQ⁺ by Fd(ox) in the outer aqueous compartment. These reactions lead to the permanent accumulation of oxidized and reduced product proteins, i.e. cyt c(ox) in the inner compartment and Fd(red) in the outer compartment. The yields of such accumulation were 11%, based on the ³Chl quenched, and 1.4%, based on absorbed quanta, under the conditions used in the present study. This system mimics one of the key events in natural photosynthesis and results in an appreciable storage of electromagnetic energy in the reaction products.     

INTERMEDIATES IN THE ROOM TEMPERATURE FLASH PHOTOLYSIS AND LOW TEMPERATURE PHOTOLYSIS OF PURINE SOLUTIONS

The flash photolysis of purine in acetonitrile and in water at different pH was studied. The transients produced on flash excitation of degassed aqueous solutions have been identified as the triplet excited state, the hydrated electron, a purine radical cation and radical anion on the basis of quenching experiments and comparison to transients observed in low temperature photolysis.     

CHLOROPHYLL ONE-ELECTRON PHOTOCHEMISTRY: FLASH PHOTOLYSIS STUDIES OF THE CHLOROPHYLL-QUINONE REACTION IN ALCOHOL SOLVENTS*

Abstract— Flash photolysis of chlorophyll a alone in CBE (cyclohexanol-t-butanol-ethanol) yields a difference spectrum similar to those obtained upon steady illumination of chlorophyll a-quinone mixtures in this solvent. Decay kinetics in CBE and dimethylsulfoxide are faster at the Soret band than at 460–580 nm and red band regions. This difference is not obtained in other solvents (CHCI₃, CCI₄, t-butanol, ethanol), implying that two or more species are obtained in CBE and DMSO. β-Carotene in CBE increases the rate of decay of the flash-induced chlorophyll transients at 430 and 660 nm but only decreases the magnitude of the signal at 470 nm. This implies that the 470 nm absorbance is due to a product formed from the triplet state. This effect is not observed in ethanol. Adding quinone to chlorophyll solutions results in slowly decaying species being generated by flash excitation in CBE. Three components can be distinguished: the first (t_1/2? 0.2 msec) corresponds to the triplet state; the second (t_1/2= 5–10 msec) is quinone concentration and species independent; the third (t_1/2= several seconds) is dependent upon quinone concentration and species (rate is faster for higher concentrations and lower potential quinones). The ESR signal decay rate is approximately equal to the third component flash decay rate when the chlorophyll and quinone concentrations are equal. With excess quinone, the flash decay rate becomes faster, and the ESR decay rate decreases slightly. These slowly-decaying species are not produced when quinone is added to chlorophyll a in ethanol or t-butanol, or to pheophytin in CBE. One observes merely a decrease in signal height with no accompanying increase in decay rate. Mechanisms to account for all of these phenomena are presented which involve an initial chlorophyll triplet-solvent reaction with the subsequent formation of several species of chloro-phyll-quinone radical complexes.     

REDOX PROTEINS AS ELECTRON ACCEPTORS FROM CHLOROPHYLL TRIPLET STATE IN LIPID BILAYER VESICLES: KINETICS OF REDUCTION OF MEMBRANE RECONSTITUTED CYTOCHROME c OXIDASE MEDIATED BY 2,5-DI-t-BUTYL BENZOQUINONE AND CYTOCHROME c

Laser flash photolysis was used to determine the kinetics of electron transfer between membrane-bound triplet chlorophyll (3C), cytochrome c (cyt c) located in the external water phase, and vesicle-reconstituted cytochrome c oxidase (CCO). 2,5-Di-t-butyl benzoquinone (2,5 TBQ) was used as an electron transfer mediator between 3C and cyt c. A light-induced cyclic electron transfer sequence between the redox components was observed (3C----2.5 TBQ----cyt c----CCO----C+.). Under optimum conditions of membrane surface charge and ionic strength, the overall efficiency of CCO reduction (based on 3C generated by the laser flash) was 14%. Under the anaerobic conditions used, CCO reoxidation (occurring via electron transfer to C+.) was quite slow (halftime approx. 1 s at 75 mM ionic strength). The multicomponent system displayed a high level of stability, as indicated by its ability to undergo many cycles of reduction and reoxidation without any apparent degradation of the components. These results demonstrate the feasibility of constructing complex electron transfer chains, including both soluble and membrane-bound redox proteins, in artificial lipid bilayers, whose properties can be readily controlled by manipulating parameters such as ionic strength and membrane composition.     

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.