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.     

SPINACH FERREDOXIN REDUCTION BY MEMBRANE-BOUND CHLOROPHYLL TRIPLET STATE VIA A DIQUAT MEDIATOR

Abstract Laser flash photolysis experiments have shown that the diquat analog containing a propylene bridge (PDQ²⁺), when electrostatically bound to negatively-charged vesicles containing chlorophyll, is able to mediate the rapid reduction ( k = 1.1 × 10⁵ s^-1) of spinach ferredoxin via electron transfer quenching of triplet state chlorophyll. The kinetics of formation and decay of reduced ferredoxin are consistent with a mechanism involving complex formation between oxidized ferredoxin and vesicle-bound PDQ²⁺. Under optimal conditions, approximately 15% of the quenched triplets yield reduced ferredoxin. This process is a model for soluble ferredoxin reduction which occurs in green plant photosystem I, and results in an appreciable storage of electromagnetic energy in the reaction products.     

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.     

CHLOROPHYLL PHOTOSENSITIZED ELECTRON TRANSFER IN PHOSPHOLIPID BILAYER VESICLE SYSTEMS: EFFECTS OF CHOLESTEROL ON RADICAL YIELDS AND KINETIC PARAMETERS*

Abstract The quenching of the triplet state of chlorophyll a (Chl) by asymmetrically located electron acceptors was examined in vesicle systems containing egg yolk phosphatidylcholine and 0–50 mole % cholesterol. The incorporation of cholesterol had two main effects: (1) the distribution of Chl within the vesicle wall shifted from one favoring the inner monolayer to one favoring the outer monolayer, and (2) the Chi molecules (both ground and excited states) became more accessible to water and to the quencher molecules. This latter property was probably due to the creation of space between the phospholipid head groups by insertion of cholesterol. These phenomena required cholesterol concentrations in excess of 15 mol %. In general, the addition of cholesterol caused increases in the apparent bimolecular rate constant for triplet quenching, in the probability that quenching produced radicals, and in the rate of radical recombination. Some of the specific effects of cholesterol depended upon whether or not the quencher molecules were amphiphilic.     

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.     

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.     

DIRECT OBSERVATION OF ELECTRON TRANSFER ACROSS A LIPID BILAYER: PULSED LASER PHOTOLYSIS OF AN ASYMMETRIC VESICLE SYSTEM CONTAINING CHLOROPHYLL,METHYL VIOLOGEN AND EDTA*

Abstract— Suspensions of vesicles composed of chlorophyll a (Chi) and phospholipid that were asymmetric with respect to aqueous solutions of methyl viologen (MV₂₊), an electron acceptor, and EDTA, an electron donor, were investigated using both flash and steady-state photolysis techniques. It was shown that Chl-photosensitized electron transfer occurred across the walls of the vesicles from EDTA to MV₂₊. Flash photolysis indicated that MV₂₊ dissolved in the interior aqueous compartments of the vesicles oxidized only those triplet excited state Chi molecules that were dissolved in the inner monolayers of the vesicle walls. The resultant radical products, Chi₊ and MV₊, recombined with a halftime of the order of 10_-4s. EDTA, added externally to the vesicles, competed effectively with MV₊ as a reducing agent for Chl₊. This places a lower limit of 10₄ s_-1 on the rate constant for transmembrane electron transfer. Compartmentalization by the vesicle wall of the competing pathways for the reduction of Chi₊ resulted in a nonlinear dependence of the rate constant of Chl₊ decay on EDTA concentration. The magnitude of the rate constant of electron transfer through the membrane and the way that the kinetics of Chl₊ decay depended on the concentration of Chi in the membrane strongly suggest that the electron transfer occurred by electron exchange between Chi and Chl₊.     

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 BILAYERS: INSIDE VS OUTSIDE ASYMMETRY*

Abstract— We have determined triplet quenching efficiencies. radical yields and radical recombination kinetics in mixed chlorophyll (Chl)-egg phosphatidylcholine vesicle suspensions in the presence of electrically-charged electron acceptors located either in the external. continuous aqueous phase or within the internal aqueous volume of the vesicles. There was a marked asymmetry between these processes as to whether they occurred at the outer or inner bilayer-water interfaces. With methyl viologen (MV²⁺) as acceptor, 52 ± 4% of the total Chl triplet could be quenched from the inside. whereas only 16 ± 2% was quenchable from the outside. Approximately 35% of the triplet population was inacccssible to quenching by MV²⁺ from either inside or outside. Ouenching rate constants were higher from the outside than from the inside (2 × 10⁶M^?lS-^Ivs 1 × 10⁶M^?Is^?1). A similar pattern was obtained when anthraquinone disulfonate or ferricyanide were used as acceptors. Radical yields and recombination kinetics also displayed asymmetric behaviour. From the inside. only 4 ± 2% of the quenched triplets gave rise to separated radicals using MV²⁺ as acceptor, whereas from the outside the conversion yield was 32 ± 2%. The halftime for the Chl⁺ MV⁺ reaction was approximately 100 times longer at the outer surface than at the inner surface. We conclude the following: (a) Chl is distributed asymmetrically within the bilayer such that more triplet Chl is located within quenching distance of the interface at the inner surface than at the outer surface. Furthermore, an appreciable fraction of the triplet Chl is located sufficiently far from either interface so that quenching is not possible. (b) The mobility of Chl and quencher molecules is greater at the outer surface of the vesicles than at the inner surface.     

QUINONES AS MEDIATORS OF ELECTRON TRANSFER FROM MEMBRANE-BOUND CHLOROPHYLL TO CYTOCHROME c IN THE AQUEOUS PHASE IN LIPID BILAYER VESICLES: SYNERGISTIC ENHANCEMENT OF CYTOCHROME c REDUCTION BY LIPOPHILIC/ HYDROPHILIC QUINONE PAIRS

Laser flash photolysis has been used to determine the kinetics of cytochrome c reduction by chlorophyll triplet state in negatively-charged lipid bilayer vesicles, as mediated by quinones. Large synergistic enhancements in the yield of reduced cytochrome were obtained using a pair of quinones, one of which was lipophilic (e.g. benzoquinone, 2,6-di-f-butylbenzoquinone) and the other of which was hydrophilic (e.g. l,2-naphthoquinone-4-sulfonate). The mechanism was shown to involve initial quenching of the triplet by the membrane-associated quinone to form chlorophyll cation radical and quinone anion radical. An interquinone electron transfer process followed this reaction, which occurred at the membrane-water interface, and greatly facilitated electron transport from within the bilayer to the aqueous phase. This process formed the basis of the synergistic effect. Cytochrome c reduction occurred in the water phase by reaction with the anion radical of the hydrophilic quinone. Finally, the reduced cytochrome was reoxidized by a slow reaction with chlorophyll cation radical. Under the most favorable conditions, we estimate that the quantum yield of conversion of triplet quenching events to reduction of cytochrome approached unity. The lifetime of the reduced protein and oxidized chlorophyll could be as long as 140 ms, under the best conditions. This system has properties which are thus quite favorable for solar energy conversion in a biomimetic process.     

ZERO FIELD OPTICAL DETECTION OF MAGNETIC RESONANCE OF TRIPLET STATE OF CHLOROPHYLL a IN LIPID BILAYER VESICLES

Abstract The zero-field ODMR of triplet state of chlorophyll a incorporated in phosphatidylcholine (PC) vesicles (Chi a : PC = 1:100) has been carried out. The zero-field ODMR frequencies and intersystem crossing rate constants have been measured at various fluorescence wavelengths. The ODMR data suggest that the chlorophyll is present in mono- and biligated species. The nature of the ligand and the role of the medium (phospholipid) are also 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.     

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 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.     

TRIPLET STATE STUDIES OF FLAVINS BY ELECTRON PARAMAGNETIC RESONANCE—I

Abstract— The triplet state of flavin derivatives and d-amino acid oxidase was observed by electron paramagnetic resonance at 77°K.
Flavin triplets (Δ m =± 2) originate from the isoalloxazine ring and are resonant at 1560 guass.The half-life of the FMN triplet in 1 N HCl is 15 nisec.This life-time is prolonged indirectly by the presence of paramagnetic species, such as oxygen or free radicals.
The flavin triplet state is pH dependent.In neutral solution the nlaximum triplet yield is obtained and the longest life-time is observed.The triplet state is affected by intra-and inter-molecular complex formation, FAD is partially quenched by indirectly substituted adenine.Tryptophan quenches completely the FMN triplet.The FAD triplet of d-amino acid oxidase is enhanced but the life-time is shortened relative to a pure FAD solution.     

Triplet state quenching by ruthenocene

Ruthenocene quenches triplet states of organic molecules with energies greater than 24000 cm^?1 in benzene solution at a diffusion controlled rate , (6 ± 1) × 10⁹ dm³ mol^?1 s^?1. For triplets with energies less than this the efficiency of quenching is dependent on the energy of the triplet state being quenched but drops off less acutely than expected for endothermic energy transfer following the Arrhenius equation. This is in agreement with the lowest triplet state of ruthenocene being geometrically distorted as expected from the previously observed large Stokes shift between absorption to and emission from its lowest triplet state. Similarities to ferrocene quenching of triplet states are discussed. Quenching of the triplet state of benzil by ruthenocene does not fall on the smooth curve which exists between the quenching rate constants k_q and the energy of the triplet state being quenched. Queching of triplet benzil by ruthenocene is therefore attributed to favourable charge-transfer interactions, also in this case the behaviour is analogous to quenching of triplet methylene-blue by ferrocene where at least a proportion of electron transfer following quenching has been previously established.     

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.     

Free energy gap laws for the pulse-induced and stationary fluorescence quenching by reversible charge transfer in polar solutions

The Stern-Volmer constants for either pulse-induced or stationary fluorescence being quenched by a contact charge transfer are calculated and their free energy dependencies (the free energy gap laws) are specified. The reversibility of charge transfer is taken into account as well as spin conversion in radical ion pairs, followed by their recombination in either singlet or triplet neutral products. The natural decay of triplets as well as their impurity quenching by ionization are accounted for when estimating the fluorescence quantum yield and its free energy dependence.     

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.     

Radiolytically generated benzene triplets as sensitizers for energy and combined electron/proton transfer processes

Pulse radiolysis technique has been employed to investigate energy and electron transfer reactions involving triplets of naphthols and hydroxybiphenyls. The transient absorption spectra obtained on pulse radiolysis of N₂-saturated solution of naphthols and hydroxybiphenyls in benzene are assigned to triplet–triplet absorption. It was found that biphenyl triplets undergo energy transfer to naphthols and hydroxybiphenyls forming the acceptor triplets. On the other hand, benzophenone triplets, favor electron transfer followed by H⁺ transfer reaction forming benzophenone ketyl radical and phenoxyl radical of the acceptor. An analogous sequence mimics with 2-naphthol triplets and using benzophenone, acetophenone or chloranil as electron acceptor.