Photosynthesis Research 45: 203-217, 1995. © 1995 KluwerAcademicPublishers. Printedin theNetherlands.

Regular paper

Reversible photochemistry in a chlorophyll model system Gilbert R. Seely Department of Chemistry and Biochemistry and Center for the Study of Early Events in Photosynthesis, Arizona State University, Tempe, AZ85287-1604, USA Received 23 May 1995; accepted in revised form 28 July 1995

Key words: 5,5'-dithiobis(2-nitrobenzoic acid), hydrazines, imidazole surfactants, pheophytin, photosensitization, pigment association

Abstract A reversible, endothermic photochemical redox reaction, sensitized by chlorophyll a and related compounds, has been demonstrated in a heterogeneous particulate system. The oxidant, 5,5'-dithiobis(2-nitrobenzoate) (DTNB), is photoreduced to the thiolate which remains primarily in an aqueous phase. Reductants are trisubstituted hydrazines, capable of oxidation to tetrazanes in the hydrocarbon particle phase. In the course of three days in the dark, thiolate and tetrazane react to regenerate DTNB in yields approaching 100%. A novelty of the present system is that photoreaction often takes place in discrete rate regimes, which are related to the presence of spectrally identifiable associations of chlorophyll pigments, Mg-containing and free bases. Among the associations that promote photochemical activity are those of chlorophyll and pheophytin with themselves and with each other. Perhaps more active are associations of a Mg rhodochlorin allomerization product of chlorophyll with its free base. Contributing to the associations is the stabilizing presence of amphiphiles that both ligate the Mg of chlorophyll strongly and hydrogen-bond to carbonyls: 2-tridecylimidazole, 2-tridecylimidazoline, and (2-aminoethyl)myristamide. Results of this work demonstrate the possibility of generating reaction center models in an artificial heterogeneous system, and of conducting reversible photochemical reactions with them.

Abbreviations: AEMA - N-(2 aminoethyl) myristamide; Asc Palm - ascorbyl palmitate; Chl - chlorophyll a; C13-Im - 2-tridecylimidazole; C13-Imoline - 2-tridecylimidazoline; CPyCI - N-hexadecylpyridinium chloride; DEE - diethyl ester (of DTNB); DMOH - 1,1-dimethyl-2-octyl hydrazine; DPI - dodecylpyridinium iodide; DTNB -5,5~-dithiobis(2-nitrobenzoate); HB - 1,2-diphenylhydrazine; MHB - 1,2-diphenyl-l-methylhydrazine; MPBH: 2-butyl-1-methyl-1-phenylhydrazine; NMMA - N-methylmyristamide; Phe - pheophytin a; (Mg)Rhodo - an allomerized Chl derivative; SCE - KCl-saturated calomel electrode; TMA - tetramethylammonium; TRIS tris(hydroxymethyl)aminomethane

Introduction For some years a certain heterogeneous model system employing chlorophyll a (Chl) has been under investigation. It is the intent of this article to summarize the development of this model system, and conclude its investigation with presentation of evidence for a reversible, endergonic redox reaction photosensitized by the system. The goal of work with the model system was to demonstrate feasibility of photochemical ener-

gy conversion and storage in a system based on Chl and formulated with guidance from our understanding of the natural process. Inevitably, some interesting analogies to the natural process have emerged. The conversion of light into chemical free energy in vivo comprises six steps: (1) absorption of light by the photosynthetic unit; (2) transfer of the energy to a reaction center; (3) charge separation through transfer of an electron within the reaction center; (4) further separation of the charges from each other within the

204 reaction center complex; (5) separation of redox products and restoration of the reaction center complex; (6) exergonic back reaction of the redox products to generate material of use to the organism. Photosynthetic apparatuses of green plants and bacteria effect these steps in the following ways: (1) an array of up to several hundred pigment molecules absorbs sunlight; (2) excitation energy is transferred in a matter of picoseconds to a reaction center protein; (3) an electron is transferred from some kind of Chl or bacteriochlorophyll 'special pair' to another Chl or pheophytin (Phe) pigment molecule; (4) the electron is further transferred to a quinone or Fe-S protein within the reaction center complex, while the hole is filled by oxidation of a cytochrome, a tyrosine residue, or plastocyanin; (5) diffusive exchange of quinone, plastocyanin, etc. prepares the reaction center to receive another photon; (6) enzymatic reactions powered by ATP and NAD(P)H ultimately reduce CO2 by HzO, H2S et al. to carbohydrates. In previous reports on the particulate model system, evidence has been presented for processes analogous to the first five of these steps. (1) Chl is adsorbed at a concentration of one-fourth to one-half of a monolayer onto the surface of the particles (Seely and Senthilathipan 1983). (2) There is evidence for limited transfer of excitation to low-energy sites on the particle surface from the general asymmetry of absorption and fluorescence spectra (Kusumoto et al. 1983a,b; 1994), and from unpublished data on fluorescence of Chls a and b together.* (3) There is photochemical evidence of primary charge separation from the singlet excited state of a pair of pigment molecules (Seely and Senthilathipan 1985; Seely and Haggy 1987, 1988; Seely and Rehms 1991). (4) There is further transfer of the electron to an oxidant, and of the hole to a reductant (Seely and Haggy 1987, 1988; Seely and Rehms 1991, 1992). (5) There is indirect evidence for diffusive exchange of reduced oxidant for oxidant at the particle surface (Seely and Rehms 1991, 1992). All photoreactions of the particulate system described to date appear to be exergonic, or at least irreversible, and therefore unsuited to the storage of energy. It is now reported that by a change of reductant, an endergonic, reversible photoreaction can be demonstrated. The products could, in principle, be separated and recombined at suitable electrodes to generate an * This systemturned out to be more complexthan anticipated, for there wele indicationsthat energytransferfrom Chl b to Chl a was in competitionwith transferto dimericassociationsof Chl b.

electric current. In order to clarify the present state of the model system, it will be necessary to recapitulate briefly and rationalize the development of the model up to this point. The use of plastic as support for a Chl model system was suggested by earlier interesting observations of Komissarov et al. (1963), Cellarius and Mauzerall (1966); and Kapler and Nekrasov (1966). We introduced the refinement of swelling the (polyethylene) particles with a liquid hydrocarbon in order to create a chemically inert but viscous matrix in which alkyl chains of Chl and other surfactants could be lodged (Seely 1978a). It was recognized at the outset that in order to space Chl molecules at high concentration on the particle surface and prevent excessive quenching of the singlet excited state, it would be necessary to incorporate a 'ligating amphiphile' which would coordinate to the Mg of the pigment (Seely 1978a, 1979). At first, a variety of such amphiphiles were tested, including naturally occurring lipids, with interesting results (Seely 1979; Seely and Rutkoski 1981), but attention was soon focused on variously substituted aliphatic amides. In a series of papers, the extent to which spectral properties of Chl depended on the structure of the ligating amide was investigated in detail (Seely et al. 1982; Kusumoto et al. 1983a,b, 1994). In the absence of surfactant, or in the presence only of non-ligating surfactants such as quaternary ammonium salts or soaps, Chl was found largely in the infraredabsorbing polymer 'Ch1740', which probably consists of extended sheets of pigment lying over the surface of the polyethylene particles (Worcester et al. 1986). Further experimentation with ligating amphiphiles was pursued in anticipation of the needs of photochemical investigation. At first, some amphiphiles, unique to this model system, were prepared by grafting a reducible function to an aliphatic amide. It was the intention that these 'oxidizing amphiphiles' should accept an electron from the singlet excited state of Chl, somewhat as implied by earlier concepts of the operation of green plant and bacterial photosystems (Vernon and Ke 1966). The first examples of oxidizing amphiphiles contained grafted quinones, and probably behaved as intended, but they suffered from two serious drawbacks: (1) as hindered amides, they competed poorly in ligation of Chl with other ligands that might be present, e.g. N,N-dimethylmyristamide; (2) their poor solubility in almost everything made application to the particles difficult (Seely and Haggy 1987). In view of the presence and role of histidine residues in Chl proteins, the next generation of ligat-

205 ing amphiphiles comprised variously substituted histamines (Seely and Rehms 1991). While several lines of reasoning agreed that the oxidizing amphiphiles among them behaved exactly as they should, photochemistry quantum yields were lower than when ligating but non-oxidizing amphiphiles were used. As it was becoming ever more apparent that associated species of pigments were instrumental in photochemistry, the third generation of ligating amphiphiles was prepared for the present work with varied capacity for hydrogen-bonding (to carbonyl groups of Chl) as well as ligation to Mg, viz. 2-tridecylimidazole (Cl3-Im) and two compounds on its synthetic pathway, 2-tridecylimidazoline (Cl3-Imoline) and N-(2aminoethyl)myristamide (AEMA) (Fig. 1). Derivatization only at the 2-position of the imidazol(in)e removes the ambiguity of positional isomers introduced by ring-substitution of histamine (Seely and Rehms 1991). Earlier papers on photochemistry in this series dealt with the sensitized reduction of nitroaromatic compounds, because their redox potentials cover the range of interest for Chl reactivity (Seely 1969a,b; Seely and Haggy 1988). Nitro compounds are usually reduced to the corresponding phenylhydroxylamines in a 4electron process, of which the uptake of only the first electron is reversible and related to the polarographic potential. In contrast, the nitrophenyldisulfide ion 5,5'dithiobis(2-nitrobenzoate) (DTNB) is reduced by two electrons to the thiolate, a water-soluble yellow substance that facilitates tracking the extent of reduction (Seely and Rehms 1991). DTNB continues to be used in the present work. Most Chl-sensitized reactions reported in previous work have made use of 1,2-diphenylhydrazine (hydrazobenzene (HB)) as reductant, because it is particularly well suited to application with Chl (Seely 1965). While the oxidation of HB to azobenzene is sometimes useful for following the progress of a reaction (Seely and Haggy 1988), it is also for practical purposes irreversible (although azobenzene can be reduced by Chl through a reductive cycle (Seely 1965)). To obtain reversibility, it is necessary to alter the nature of the reductant. The oxidation of mono- and disubstituted hydrazines is generally irreversible, but trisubstituted hydrazines may be oxidized reversibly to hexasubstituted tetrazanes. Quite a few of the latter are known, but all of the stable ones bear electron-attracting substituents such as aryl, acyl and trifluoromethyl. The more crowded ones are in equilibrium with hydrazyl radicals (Wilmarth and Schwartz 1955; cf. also the

stable diphenylpicrylhydrazyl radical). Comparatively few trisubstituted hydrazines have been prepared and studied. The simpler alkyl and aryl ones are oxidized chemically or electrochemically by two electrons to hydrazones (Cauquis and Genies 1971a; Cauquis et al. 1972; Kaba et al. 1975), but if this is not possible, to diazenium ions (Cauquis and Genies 1971b; Nelsen and Landis 1973). But Fuchigama et al. (1990) reported electrochemical oxidation of 1-acyl2,2-dimethylhydrazines to tetrazanes. It was hoped that by appropriate choices of substituents the redox potentials and stability of hydrazines and their tetrazanes could be varied over ranges suitable for Chl-sensitized photochemistry. Three trisubstituted hydrazines have been prepared, two of which support the reversible photochemistry reported here.

Materials and methods

Materials Amphiphiles. The most convenient route to 2substituted imidazolines is through acylation of ethylene diamine and dehydration of the products (Hill and Aspinall 1939; Kyrides et al. 1947; Doriatti et al. 1985). Reaction of methyl myristate with excess ethylene diamine at temperatures up to 170 °C yielded two products, N-(2-aminoethyl)myristamide and the diamide. AEMA was extracted from the diamide with hot methanol and recrystallized several times from tetrahydrofuran-heptane: m.p. 79-80 °C. 1H nmr in CDC13: t~ = 3.30 ppm (quartet; triplet in presence of CD3OD), CH2CH2NHCO; 6 = 2.82 ppm (triplet), CH2CI-I2NH2. N,N'-Dimyristoyl ethylene diamine was recrystallized from 1-propanol: m.p. 155.5-156 °C. Although almost insoluble in CDC13, 1H nmr: 6 = 3.14 ppm for NHCH2CH2NH. 2-Tridecylimidazoline was most conveniently prepared by fusion of dimyristoyl ethylene diamine with ethylene diamine dihydrochloride at 270-300 °C (Waldron and Chwala 1941). The imidazoline was extracted with ether from the residue rendered basic with NaOH solution and recrystallized from methanol with dilute aqueous ammonia: m.p. 87-88 °C (cf. 88-89 °C, Kyrides et al. 1947); ~H nmr: ~ = 3.574 (singlet) for (NCH2-)2, ~ = 2.220 (triplet) for C12H25CH2C3N2Hs. 2-Tridecylimidazole was prepared by BaMnO4 oxidation of the imidazoline (Hughey et al. 1980). The product was recrystallized from ligroin, but retained about 15% imidazoline starting material, by nmr esti-

206 marion: m.p. 77-79 °C (cf. 81-82 °C, Kyrides et al. 1947). 1H nmr: 6 = 6.96 ppm (singlet), -CH=CH-; 6 = 2.73 ppm (triplet), CI2H25CH2C3N2H3. Cetylpyridinium chloride (CPyCI) was recrystallized from ethyl acetate-methanol. Hydrazines. 1,2- Diphenyl - 1 - methylhydrazine (MHB) was prepared by refluxing 1,2-diphenylhydrazine with dimethyl sulfate and CaO in benzene for two days (Rassow and Berger 1911). The product was extracted into benzene, washed with NaOH solution, recrystallized 3 times from hexane, chromatographed on alumina, recrystallized several times from hexane again and finally from aqueous methanol: m.p. 77.578 °C, cf. lit. 75 °C. IH nmr in CDCI3:6 = 3.16 ppm, CH3; 5.48 ppm, NH. 1,1-Dimethyl-2-octylhydrazine (DMOH) was prepared by LiAIH4 reduction of the hydrazone of 1,1dimethylhydrazine with octanal. The product was distilled at 185 °C. As DMOH is not very stable in air, it was better converted to the binoxalate and applied in that form. The nmr spectrum was consistent with the expected structure:/~ = 2.45 ppm (singlet); (CH3)2 N; = 2.76 (irregular quartet), -CH2NH. 2-Butyl- 1-methyl- 1-phenylhydrazine (MPBH) was prepared by LiA1H4 reduction of the hydrazone from 1-methyl- 1-phenylhydrazine and butanal. The product was purified through conversion to the binoxalate. 1H nmr: ~ = 3.126 ppm (singlet), CH3N; 6 = 3.04 ppm (triplet), CH2N. This hydrazine is more stable in air and could be applied to the particles from a solution in t-butyl methyl ether, as well as in the form of the binoxalate. Commercial ascorbyl palmitate (Asc Palm) was recrystallized from chloroform. DTNB was applied in the form of the tetramethylammonium salt (TMA DTNB: Seely and Rehms 1991). The diethyl ester of DTNB (DEE DTNB) was prepared by refluxing DTNB in ethanol with acetyl chloride and benzene, and removing volatiles by slow distillation. The diester was extracted from the residue with benzene, recovered with petroleum ether, and recrystallized from absolute ethanol: light yellow crystals, m.p. 99-101 °C. The nmr spectrum showed the ethyl groups: 6 = 1.355 ppm (triplet), CH3, and 4.40 ppm (quartet), CH2.

Me~o~ The apparatus and procedures used in this work are, for the most part, those described in Seely and Rehms (1991). Particles with AEMA, Cl3-Im and C13-Imoline

were prepared by a modified procedure: Chl was adsorbed as usual from a 25% aqueous methanol solution, which also contained 5 x 10-3 M CpyC1; almost all of the Chl and about 3 mmol/g of CpyC1 were adsorbed. The particles were collected by filtration and, before drying, were treated with a solution of the ligating amphiphile in methanol. Cl3-Imoline is a strong nitrogenous base; when it was applied, Chl was converted into an allomerization product identified by its mode of formation and spectrum (Holt 1958; Hynninen 1973) as Mg 31,32-didehydro-151hydroxy-151-methoxyrhodochlorin-15-acetic acid dlactone- 152-methyl ester- 173 phytyl ester, once known as Mg purpurin 7 lactone methyl ether methyl phytyl ester, or perhaps the 15t-dihydroxy derivative, which for brevity will be referred to as '(Mg) Rhodo'. Although it was not the original intention to investigate this derivative, its photochemistry turned out to be at least as interesting as that of Chl itself. Since C13-Im contained some C13-Imoline, a solution of 4(2-hydroxyethyl)- 1-piperazineethanesulfonic acid was applied along with it to neutralize the latter, stronger base. Even so, some Chl was allomerized. Fluorescence of the particle preparations was measured as usual (Seely and Rehms 1991, 1992), but the quantum yields reported are the so-called 'apparent' ones, ratios of photometric quantities which differ inconsequentially from 'true' yields when the representative fluorescence wavelength is taken to be 700 rim.

Mixtures for photochemistry were prepared by evaporating onto 0.05-0.08 g of particles in succession: Phe (if used) in t-butyl methyl ether + petroleum ether; the hydrazine base in t-butyl methyl ether, or its binoxalate in methanol together with tris(hydroxymethyl)aminomethane(TRIS); TMA DTNB, 0.01 M in methanol. The particles were then compounded with a 2% guar solution thickened with cellulose, placed in a special cell and equilibrated with a N2 stream before closure (Seely and Rehms 1991). The sample was irradiated by light from a 750 W projector lamp, collimated and passed through water and a 670 nm interference filter. Progress of the reaction was followed with a Cary 219 spectrophotometer. Quantum yields ~ for the photosensitized reduction of DTNB were calculated, as before (Seely and Rehms 1991), by the formula

[DTNB]o =

x (p.1.) x (dp/dt) 0.1 x l o x A x A p o ~

(1)

207 Table 1. Compositionand spectral properties of particle preparations cited

Prepn. no.' Amphiphile(cone., retool/g) Pigment (cone., retool/g) Amax(nm)(abs.) 102q~f(app) (Af, max) Aexc: 430 um 440 nm 450 um A1b A2 B C1 C2 D1 D2 D3

DPI (2.68) NaMyr (2.68) DPI (2.27) NaBu (,-~7) AEMA (4.4) (CPyCI)c Cl3-Imoline (9.45) C13-Imoline (6.4) C]3-Im (7.9) C13-Im (6.8) C13-Im (9.4)

Chl (0.88) Chl (0.85) Chl (1.03) Rhodo (0.99) Rhodo (0.99) Rhodo > Chl (0.55) Rhodo ,,, Chl (0.94) Rhodo < Chl (1.88)

665.0 432.2 666 430 666.2 432.6 665.4d 416 663.8d 416.5 664.2 420 670.2d 436.5 670.0 416

11.2 (671) 6.8 (669.5) 11.7 (671.5) 6.8 (668.5) 6.2 (670) 7.8 (671) 8.3 (676) 3.2 (679)

8.8 (673) 5.4 (670.5) 8.9 (674) 5.8 (670) 6.0 (672) 6.3 (674) 5.5 (676.5) 1.6 (674)

5.0 (677.5) 2.3 (676.5) 5.2 (680) 2.2 (676.5) 3.5 (676) 3.2 (677) 3.6 (678) 1.1 (680)

aSuecessive columns list the amphiphile and pigment content of the particles, positions of the stronger absorption bands, and yields and peak wavelength of fluorescenceexcited at the indicated wavelengths. 'Apparent' quantumyields (~) are expressed as percent of absorbed exciting light. bPreparations of the 'A' series were described in Scely and Rehms (1992). Prepn. A1 - Prepn. IV of that paper and A2 - III. DPI: dodecylpyridiniumiodide; NaMyr, NaBu - sodium myristate, butyrate. eThis and followingprepns contain CPyC1at about 3 mmol g-1. din some preparations for fluorescence,bands of the 'teuamer' aggregatewere seen: In C1 and C2, absorptionat 674.5 and 686.6 urn, fluorescenceat 688 nm; in 1)2, absorption at 684 rim, fluorescenceat 686 urn.

where [DTNB]0 is the initial concentration of DTNB, (p.1.) the thickness (cm) o f the layer in the reaction cell, I0 (mol m -2 s -1) the incident light intensity and A the fraction absorbed. The rate of absorbance change due to thiolate production (dp/d0 and the total absorbance increase Apoo were previously evaluated from the sum of absorbance increases at 405,420 and 450 nm (Seely and Rehms 1991). Here, because Phe is present in many of the reactions and is consumed, absorbance of the thiolate was measured only at 470 nm, clear of the region of strong Phe absorption. Plots of A p at 470 nm vs. A p at other wavelengths were generally close to linear over a wide range. Quantum yields determined under these conditions are naturally not as accurate as those measured in homogeneous solution. Since reasonable efforts have been made to standardize conditions of reaction, differences in excess o f 20% should be regarded as probably significant. When a reaction was slow and not pursued to completion, Apo~ had to be inferred from reactions of similar constitution, and greater allowance for error must be made.

N.H2

NMMA

A E M A CBh~oLINE: C~IM

Fig. 1. The three ligating, hydrogen-bonding amphiphilcs used in

the present work are comparedwith N-methylmyristamide.

Results The pigment and surfactant concentrations in the particles used for the present work are listed in Table 1, along with absorption and fluorescence spectral data. Fluorescence was excited at three wavelengths in the Soret region to span the range from mainly isolated to mainly aggregated pigment absorption. In that

208 2..5

3.0 THIOLATE

2.0

P 2£ 1.5

1.0

1.0

0.5

x (nm) Fig. 2.

Reversibility of the sensitized reduction of DTNB by DMOH. Traces from the spectral record of reaction (C. l.d) of Table 2 recorded before (1) and after 1000s (2) and 5000s (3) of 670 nm irradiation, and then after 66 h in the dark (4, dashed line). The sensitizer is Mg Rhodo.

sequence, the peak fluorescence wavelength increases and the quantum yield diminishes, as before (Seely and Rehms 1991, 1992). This demonstrates the presence of fluorescent associated, probably dimeric, species of Chl in these preparations also. In absorption spectra of Chl preparations, the red band maximum falls in the range 665--670 nm. When Rhodo predominates (samples C1, C2 and D1), it is nearer 664 nm. The Soret bands of Chl and Rhodo appear at about 432 and 416 nm, respectively. There appears to be little systematic difference in fluorescence spectra or quantum yields between particles with Chl and those with Rhodo. One of the reasons for selecting amphiphiles related to 2-alkylimidazole is their resemblance to Nmethylmyristamide (NMMA) with regard to the directionality of their coordination and hydrogen-bonding functions (Fig. 1). Some years ago, it was found that NMMA had the ability to induce Chl on particles to form a remarkable sort of aggregate characterized by sharp bands at 687 and 678 nm (Kusumo-

X (rim) Fig. 3.

The fastest recorded sensitized reduction of DTNB by

DMOH, reaction (C.l.a.) of Table 2. Spectra plotted were recorded before irradiation (0), and after 20 (1), 40 (2), 60 (3), 100 (4), 200 (5), and 510s (6) exposure to 670 nm light. The dashed trace (7) was recorded after 5d in the dark. Some of the thiolate produced is in the form of a narrow-band 450 nm species which may be a sort of J-aggregate held together by cations on the surface of the particles. The 0-100 s difference spectrum (short-dashed trace) represents loss of H2 Rhodo at 678 nm.

to et al. 1983b). Extreme circular dichroic phenomena led to its assignment as a cyclic tetramer of Chl's linked by NMMA. Since then, the same aggregate has been observed in surfactant micelle solutions, apparently in purer form, and characterized by Raman spectroscopy (Kusumoto et al. 1987; Kurawaki and Kusumoto 1989). Observation of this unusual sort of aggregate with AEMA, C13-Im or C13-Imoline would support the hypothesized linking mechanism and indirectly the tetrameric model. In fact, narrow peaks or shoulders in the 683-687 nm range are detected on occasion in preparations with these three amphiphiles. Rhodo appears more prone than Chl to form this kind of aggregate (686.5 and 674.5 nm in C2) and the fluorescence of C1 and C2 contains a narrow band at 688 nm which corresponds to the former absorption band.

209 4

O

o~ 2.5

p

0.6 I

0.4 2.0

0.2

o

.50)0

600

x, nm

~

700

Reaction(B.a), with Chl only. Spectra plotted before (0) and after 500 (1), 1030 (2), 2505 (3) and 4020s (4) of irradiation at 670 nm. The differencespectrumshows pigment loss during the first 500s; there is no appreciableloss afterthat.

Fig. 5.

l

1.0 I

0.5

,,..,/

/

I I

I/

I i I t

Ii

i I

./

300

400

500

~OU

CUU

x (nm)

Fig. 4. Reaction (C.2.a) sensitized by particles that contain some 'tetrameric' Mg Rhodo. Spectral traces recorded before irradiation (0) and after 500 (1), 1000 (2) and 2000s (3) irradiation at 670 nm. For comparison, the spectrum of an extract of C.2 particles into l-propanol is shown (dashed trace). The difference spectrum corresponding to pigment loss during the first 500s (short-dashed trace) peaks at 680 nm; there is little loss of pigment after that. In the dark, bands of 'tetramer' intensify (not shown).

An example of the reversible reduction of DTNB by DMOH is presented in Fig. 2. The photoreaction, which was rather slow, is marked by the fall of DTNB absorption at 317 nm and rise of thiolate at 413 nm. After three days in the dark, the spectrum had regressed to its original state, apart from a minor loss of pigment. In these reactions, reversal begins immediately after the light is shut off, but is only about 30-50% complete overnight; not all reactions reverted completely but those that did required three days. (Lack of complete reversibility in this time might be due to loss of tetrazane through some side reaction.) It was not practicable to follow all reversions to completion. Before undertaking a detailed investigation of the reaction with DMOH and MPBH, a few other available hydrazines were tested, using particle sample A1, which lacks ligating surfactants. Phenylhydrazine

reduced DTNB very slowly, and 2-hydrazinopyridine even more so, but both could be driven to completion in unfiltered lamplight. Reversibility was not checked. 1-Methyl-l-phenylhydrazine sustained a slow and incomplete reaction, with limited reversibility. Probably the tetrazane formed by its oxidation is unstable. MHB sustained no reduction of DTNB whatsoever. Rates with DMOH were similar to those with HB. In some cases, Phe appeared during dark reversion, an indication that some Chl had been reduced (Seely and Folkmanis 1964). After a number of reactions had been carried out with various particle samples and with DMOH and MPBH as reductants, two general observations emerged. (1) Although initial rates varied by almost 100-fold, reduction of DTNB was fastest when, for some reason, Phe (or free-base Rhodo (H2 Rhodo)) was present along with Chl or Mg Rhodo. (2) Whether or not Phe was present, the red band of the pigment usually became narrower during the photoreaction, owing to selective loss of a long-wave component. Phe (if present) was reduced during the photoreaction, and was about half regenerated in the dark. The rate of dark regeneration was much faster than that of DTNB and apparently independent of it; disproportionation of reduced Phe (PheH2) is the likely route. The photoreaction is further illustrated in Figs. 3 and 4, using samples that contain Mg Rhodo and CI3Imoline. Particulars of these and other photoreactions are collected in Table 2. In the course of learning the conditions for incorporating DMOH from the binox-

210 o

I.C

P

O6

P

0.~

O.E

/

(22 ¢

/ 0

'

560

a

x,, nm

6

0.4 700 0.~

Fig. 6. Reaction (B.e), with Phe prepared in situ only. Spectra plotted before (0) and after 100 (1), 200 (2), 500 (3) and 1060s (4) of irradiation at 670 nm. Difference spectra are plotted for 0-100s (solid trace) and 50001060s (dashed trace) intervals.

alate into the reaction mixture, sometimes M g was lost leaving a mixture with some free base Rhodo on the particles, as in Fig. 3. Reaction (C. 1.a) shown therein was the fastest on record. Difference spectra showed loss o f a H2 Rhodo band at 676-8 nm. In contrast, the reaction mixture of Fig. 4 contains no H2 Rhodo, but sharp bands of the 'tetrameric' aggregate are evident. DTNB is reduced rather slowly, and the pigment difference spectrum corresponds neither to the 'tetramer' nor to the monomer of Mg Rhodo. In order to understand the role of Phe better, some photoreductions were carried out with Phe deliberately added to particles of sample B, which contained Chl and A E M A . With the particles as constituted with Chl only, the photoreaction was quite slow (Fig. 5). The difference spectrum during the first 500 s is best interpreted as loss of one component of a Chl dimer near 684 nm and blue-shift of the other near 663 nm. After that there was little further loss of Chl. W h e n the Chl o f sample B was converted to Phe (by treatment with trifluoroacetic acid), the photoreaction was much faster (Fig. 6). The difference spectrum displays a band at 679 nm initially, which is replaced by one at 670 nm as the reaction concludes. There is little further reduction of Phe after the DTNB is gone, even though M P B H remains in excess. When Phe was added to particles already coated with Chl, reduction of DTNB was faster than with Chl alone, though not as fast as with Phe alone (Fig. 7). The pigment difference spectrum is rather complex at first; in (B.b) and (B.d) it seems to consist of loss of

OO

~j n m

Fig. 7. Reaction (B.d), with Phe added to particles with Chl. Spectra plotted before (0) and after 200 (1), 400 (2) and 2000s (3) irradiation at 670 nm. Differencespectra are plotted for the 0-200s (solid trace) and 400-2000s (dashed trace) intervals. To obtain the (000-0) trace, enough of the difference spectrum of Fig. 5 was subtracted from the 00200 s trace to eliminate the blue shift responsible for the dip at 654 nm.

~_e/? t~' ~ - \ ~ °

a

s6o

iobo T/me~ s

Semilog~witbmic plot of the rate of tl~olate production, Ap/At, measured at 470 nm, against time of irradiation at 670 nm, for reactions of Prepn. B, Table 2. Legend: B.a (O), B.b (A), B.c (O), B.d (~7) and B.e (O). Rates for all reactions are represented as the supeq~ositionof two exponential decays. There is usually a brief induction period during which residual 02 reduced. Fig. 8.

211 Table 2. Spectral and quantum yield datafor photosensitized reduction of DTNB Reactiona no.

Reductantb (cone., mM)

[DTNB]0 c (raM)

fphed

~max(t - 0)¢ (rim)

103~f (t - 0)

-BandsS (nm)

Pigment identity

Reversion (%) (time(h))

A.l.a

DMOH (18) DMOHox

0.78

0.07

665.0

0.07

666.2

674 673 674

Phe Chl Pbe

91 (66)

0.41

6.1 2.5 2.0

1.7

h Phe Phe Chl, Phe Chl Chl Chl Chl

39 (17)

A.l.b

(3.6) A.l.c A. 1.d A.2.a B.a B.b B.c B.d B.e B.t5

DMOHox (3.6) DMOH (3.6) DMOH (7) MPBH (2.9) MPBH (2.5) MPBH (3.4) MPBH (2.6) MPBH (2.7) MPBH

0.42 0.56

1.0i (tfa) 0.07

671.4 665.9

0.61

0

666.2

0.96

0

667.8

0.85

0.18

669.0

1.18

0.39

670.0

0.91

0.43

670.0

0.94

1.0~

670.9

0

0.35

0.82

30 (18)

669.0

10.3 6.3 0.54 0.22 1.0 0.22 1.76 1.15 5.9 3.0 8.2 6.1 7.5 5.3 13.0 9.8 0.28

677 672 662, 672 667 684 664 684 h 679 674 672, 684 675 681 673 679 670 675

Phe, Chl Pbe /'he, Chl Pbe Pbe, Chl Pbe Phe Phe Pbe

0.12

665.3

46

678; 675

H2 Rhodo

0.53

0.26

670.0

12.6

680; 676

H~ Rhodo

36 (24) 70 (114) 52 (20)

0.52

0

674.6k

4.5

680

Mg Rhodo

75 (66)

0.63

0

666.9

1.0

670, 680

Mg Rhodo

96 (66)

0.42

0

674.&

2.3

680

Mg Rhodo

28 (18)

6.8 2.6 1.2 5.0 4.1 1.4 2.7 1.4 3.1

673 673 670 675 671 670 675; 673 671 680; 671

Phe Phe Chl? Phe Pbe Chl? Phe Chl? Phe

29 (17)

93 (66) 85 (23) 42 (17) 30 (17) 98 (67) 59 (20) 52 (41) 50 (90)

(3.1) C.l.a C. 1.b C.I.c C.l.d C.2.a

DMOHox (29) DMOHox (38) DMOHox (37) DMOH ( < < 40) DMOHox

(30) D.l.a

MPBH (2.8)

0.99

0.35

668.2

D.l.b

MPBH (2.8)

0.97

0.21

667.4

D.2.a

MPBHox (5) MPBH (3.2)

0.71

0.35

669.8

I.I0

0.39

671.5

D.3.a

81 (66)

72 (65) 51 (24)

aRe,action numbers are built up on sample numbers of Table 1. bWhen the hydrazine is added in the form of the binoxalate (ox), the base TRIS is also added in excess. CTypical pigment cone. (raM) in the reaction mixtures, exclusive of added Phe, were for series A: ,-,0.07, B: 0.05, C: ,-,0.07, DI: 0.035, I)2: 0.05, D3: 0.09. Incident light intensity (I0) at 670 nm was 2.9-6.5 x 10- 4 tool m -2 s - 1. The estimated fraction absorbed (A) ranged from 0.45 to 0.70. dThe fractionof Pbe (or H2 Rhodo) is estimatedby comparison of the strengthof the 535 n m band with thatnear 620 nm. eThe initiallocationof the pigment red band peak is given for comparison with the locationof lostpigment bands during the reaction. fQuantum yields for DTNB reduction are estimated by extrapolation to t - 0 of line segments in plots such as in Fig. 8 of iog(AP470/At) vs. t. speaks in the pigment difference spectra during successive reaction stages are listed, together with the probable identity of the pigment responsible for them. If there is only one entry, there is only one such reaction stage. Commas separate bands that appear to be lost together; semicolons, bands that am lost successively without obvious change in the slope of log (AP47o/At) vs. t. The last column gives the percent reversion of thiolate to DTNB, estimated at 470 nm, during the dark interval specified. hpigment loss insufficient to determine a difference spectrum. IChl was converted to Phe in situ by treatment with triiluoroacetic acid, and evaporation of the excess. JData in this row pertain to the reduction of Phe in the absence of DTNB. kTbe peaks of Rhodo 'tetmmer' were prominent.

212 Chl 663,684 dimer, superimposed on loss of Phe in a broad band around 680 nm. Later, Phe is lost from a band near 674 nm. Both the 680 and the 674 nm bands probably represent Phe in association with Chl. Again, reduction of Phe stops when DTNB is exhausted. When Phe and MPBH were added but not DTNB (reaction (B.f)), Phe was reduced but with a rather low quantum yield. Still, the reduction of Phe is about four times as fast as it is when DTNB is present. The difference spectrum band for Phe reduction in the presence of Chl is at 675 nm. When pheophytin was added but neither DTNB nor MPBH, there was gradual loss of a pigment that appeared to be a species of Chl (678, 445 nm), which would be consistent with the results of Seely and Senthilathipan (1985). Further insight into the significance of the difference spectra can be had by plotting rates of reaction (Ap47o/At) logarithmically against time (Fig. 8). Exponential decay of the rate with time implies kinetic dependence on the concentration of some component of the reaction. All plots of this series appear to consist of two line segments, representing an initially faster rate regime, and a later, slower one until the substrate DTNB approaches exhaustion. It turns out that the rate regimes are correlated in time with difference spectra such as are shown in Figs. 5-7. Thus, the early period of faster rates for (B.a) corresponds to the period over which the 684 nm Chl band disappears, that for (B.e) to the bleaching of Phe near 679 nm, and those for (B.b-d) to the early, complex difference spectra as in Fig. 7. The primary implication is that initially faster rates require the presence of certain associated species of pigment, which are gradually consumed leaving less actively sensitizing species to complete the reduction. Reactions were run with various amounts of Phe added to samples of(D. 1) and (D.3), and with free-base pigment present in (D.2). The results were complex as might be imagined from the complexity of the particle preparations themselves, and only a synopsis is given; a sampling of the data is included in Table 2. As in Fig. 8, plots of log(Ap47o/At) vs. time consisted of at least two straight line segments, which upon extrapolation to t = 0 gave the quantum yields tabulated. The presence of Phe (or H2 Rhodo) roughly triples the initial quantum yield over that in its absence. Faster initial rates are accompanied by loss of a band at >673 nm belonging to an associated species of Phe. In the absence of Phe, pigment loss is slow and the position of the band in the difference spectrum hard to ascertain. In the D. 1 series, the band of lost Phe appears

4

0

O.E P

O.~

I O~

I

500

J

I

600

""

. . . . . .

x., n m

7(:

Fig. 9. Excerptsfromthe spectralrecordof reaction(D. l.b). Spectra shown were recorded before (0) and after 100 (1), 500 (2), 1515 (3) and 6000s (4) irradiation at 670 nm. Differencespectra are for 0-100s (--), 100-500s (--) and 1515-6000x (. . . . ) intervals. The last probablyrepresentsa loss of Chl, ratherthan Mg Rhodo.

superimposed on a broader 687 nm band, which is tentatively assigned to a dimer of Rhodo, because it is not evident in the D.2 and D.3 series. Especially in the D. 1 and D.2 series, after Phe is reduced, there is further loss of pigment in a band near 670 nm which is tentatively identified as Chl associated with Rhodo by the progressive blue shift of the pigment absorption band peak to 662-3 nm. These phenomena are illustrated for (D. 1.b) in Fig. 9. When HB was used as reductant, the Chl difference spectrum was irregular but there was a net red shift of the band to ca. 670 nm.

Effect of surface charge For the present work, a cationic surfactant (DPI in the A series, otherwise CPyC1) had been adsorbed to the polyethylene particles along with Chl in order to make their surface positively charged. The intent was not only to assist dispersal of the particles in aqueous media, but also to attract the oxidant DTNB into the double layer for easier access to excited Chl. Reasons were advanced to believe that this might have been successful (Seely and Rehms 1991). If so, then the opposite effect might be expected if the particle surface were charged negative. To investigate this, samples of (D.2) were treated with an overwhelming excess of Asc Palm (175 mmol g - l ) + TRIS, and used to reduce the TMA salt of DTNB and its diethyl ester. The photoreduction of the ester is almost 4 times as fast as that of the anion (initial quantum yield 3.7 × 10 -3 vs.

213

._toe \ NO2/|

chl, Phe~ (Mq, Hz)Rhod o

/~-----0--~" -xC

_ -~NOz

+ C~

+ /t4

CI-L oR H -B~ ....

-do'

"

"0'3

....

~"

' ;o:s'

~

/ ,..3d

"N-N /

\R

' 'E

Fig, 11. Proposed scheme for the reversible reduction of DTNB by tfisubstituted hydrazines.

Fig. 10.

Comparison of voltamraograms of MPBH and DTNB. Upper trace: the nth of a series of cycles of MPBH in acetonitrile at a Pt electrode, scanned between +0.7 and - 1.4 V(SCE) at 400mV/s, with tetrabntylammonium hexafluorephosphate as elec~olyte. Lower trace: voltammogram of TMA DTNB (1.2 raM) in acemnitdle at a glassy C electrode, scanned at 200 mV/s, in the presence of methyl p-nitrobenzoate (0.4 mM) as catalyst. Cathodic reduction of DTNB and anodic oxidation of the thiolate are shown.

0.96 x 10 - s for the anion), which tends to confirm the expected effect of surface charge. There is no initial period of more rapid reduction associated with loss of particular pigment species, nor is there conspicuous blue-shift or narrowing of absorption bands. Probably Chl is ligated by Asc Palm anions in these reactions. There is a gradual conversion of Chl to Phe, but this is not unexpected since this reaction can occur with ascorbate through a photoreductive mechanism (Seely and Folkmanis 1964). Although the reaction is reversed in the dark, it may not be very endergonic since there is also a very slow forward reaction (yellowing) between starting materials in the dark.

Cyclic voltammetry The voltammetry o f T M A DTNB and DEE DTNB was reexamined after it was discovered that their reduction could be catalyzed by methyl p-nitrobenzoate (unpublished results), which led to a revised estimate of - 0 . 8 2 V (SCE) for TMA DTNB (cf. Seely and Rehms 1991), and a similar value, - 0 . 8 3 V, for the ester in acetonitrile. The trisubstituted hydrazines were investigated by cyclic voltammetry at a Pt electrode, in acetonitrile with 0.1 M tetrabutylammonium hexafluorophosphate as electrolyte.

MHB showed an anodic wave at +0.70 V vs. SCE and a cathodic wave at ca. +0.12 V which was more sensitive to scanning speed. They are assigned to formation and reduction of 1,4-dimethyl-l,2,3,4tetraphenyl tetrazane. Since the anodic wave is at a higher potential than that of HB (+0.53 V) and the E1/2 of Chl, which is +0.562 V under the same conditions of solvent and electrolyte (cf. also values listed by Seely 1978b), it is clear why MHB does not serve as a reductant in the photoreaction. DMOH shows two irreversible anodic waves, the first at ca. +0.25 V but dependent on scanning speed, the second at +0.882 V and less dependent on scanning speed. The second wave, but not the first, is succeeded on reverse sweep by a broad, weak wave near - 0 . 9 V. The anodic waves are ascribed to formation of octanal dimethylhydrazone and its oxidation. Electrochemical precedents for this sequence have been reported by Cauquis et al. (1972) for 1,1,2-trimethylhydrazine and by Cauquis and Genies (1971a) for 1,l-dimethyl-2(2-butenyl) hydrazine. An aged (non-reducing) sample of DMOH showed only the second of the anodic waves. MPBH also shows two anodic waves, at +0.41 V and at +0.915 V. (There is also a prewave at ca. +0.25 V which disappeared on repeated cycling, or at a glassy C electrode.) When scanning was reversed before the second wave, there were two broad cathodic waves near - 0 . 3 6 and -0.61 V, which with the first anodic wave form a reversible system on cycling (Fig. 10). They are therefore assigned to formation and reduction of 2,3-dibutyl-l,4-dimethyl-l,4diphenyl tetrazane. Tetrazanes have similarly been formed from N-acyl hydrazines by Fuchigama et al. (1990). The second anodic wave is therefore assigned to oxidation of the tetrazane. Voltammograms of MPBH and DTNB are compared in Fig. 10. Although both redox cycles are

214 reversible, they are far from Nernstian, and it is not possible to estimate their thermodynamic standard potentials with much accuracy. However, the general disposition of the traces suggests that the net photochemical reaction is endergonic by at least 0.3 V.

Discussion With appropriate choice of reactants, it has been possible to demonstrate an almost completely reversible and therefore endergonic redox reaction, sensitized by Chl and related pigments in a particulate model system. The presumed reaction is outlined in Fig. 11. From cyclic voltammetry, the energy stored in the primary radical products is about 1.2 eV for MPBH and DTNB; when the products are stabilized by dimerization and dissociation, respectively, the energy stored is reduced to perhaps 0.3 eV. Distribution of the products into different phases probably helps to complete the photoreaction, and would certainly be useful in isolation of products if that were intended. Probably the most interesting finding of this work is the correlation of certain associated pigment species with elevated rates of photosensitized reduction. Several species have been identified with some measure of confidence. (1) A species containing Phe with Chl (or Rhodo), 6 7 3 - 5 nm, in reaction series (A1) (B), (D1), and (D3). The Phe band is assigned to an associated species, because it lies to the red of the normal position for Phe alone on particles, 670-671 nm. The association could be with another Phe, but it is more likely with Chl, because it is found even when the fraction of Phe is small. (2) A long-wave Chl species, 684-687 nm, in (A.2.a) and (B.a), whose bleaching is correlated with blue-shift of a band near 665 nm. This species, which is associated with a brief spurt of photoactivity, resembles spectrally the anhydrous dimer (Cotton et al. 1974). Bleaching of the 684 component, through oxidation or reduction, allows the other component to shift to the blue. (3) A long-wave Phe species, 677679 nm, in (A. 1.c) and (B.e). This species is probably different from the broad-banded 680 nm species (Fig. 7) of (B.b,d) in which Phe is more likely associated with Chl. Since it appears when Chl is treated with CF3 COOH in situ, it might be produced from a Chl dimer such as the 684 nm species. (4) An association, perhaps of Chl with Mg Rhodo in which the Chl (670 nm) is preferentially degraded (D. 1.a,b). Usually there is little loss of Chl or Rhodo in later stages of a reaction, or when free base pigments are gone. That there is

loss when Chl and Rhodo are present together suggests electron transfer within an excited state complex. These species are detected by their disappearance during the reaction. There may be other active species that are not degraded and so are not identified. In the above list of active associated species, the detection of associated Phe absorbing at 679 nm (Fig. 6) raises the interesting question as to how the species promotes photoreduction. It has generally been assumed that Phe sensitizes redox reactions through a reductive cycle which is accessible to the triplet state. As evidence in support of this, the photoreduction of Phe is generally retarded or even blocked altogether by the presence of oxidants, including DTNB. While a 35% increase in triplet yield in the species Phe 679 (operating as a perfect energy trap) is sufficient to account for the results of (B.e), association of porphyrin pigments rarely increases anything but the internal conversion rate. One alternative is electron transfer from one molecule to another within the complex, similar to that proposed for Chl-Chl and Chl-Phe associations in this and earlier papers. However, the energy of the singlet excited state (1.84 eV) is barely sufficient to drive this reaction, according to published redox potentials for Phe (Seely 1978b). Cyclic voltammetry of our sample of Phe in acetonitrile confirmed this conclusion in that the formation ofPhe + + Phe- from (Phe)2 would require 1.90 eV. However, if electron transfer were accompanied by protonation of Phe.-, or deprotonation of N+H of Phe +, or both, there might be enough stabilization to render the reaction exergonic. What is clear is that the role of Phe associations in photochemistry would likely repay further investigation. The fact that it requires three days for reversion of products to starting materials is advantageous from the point of view of energy storage, since in principle it allows time for isolation of products from their respective phases before they are allowed to react, e.g. at electrodes to generate current. Of course, only reversion of thiolate to DTNB is observed directly; the assumption that tetrazanes are formed reversibly from hydrazines is reasonable, and is based on electrochemical behavior. This may seem inconsistent with the observation that DMOH is oxidized electrochemically to the hydrazone rather than to the tetrazane, but one must take into account an essential difference between electrochemical and photochemical oxidation. At an electrode, oxidation of DMOH by one electron may be followed by immediate loss of a proton, to leave a radical which may be oxidized again at the existing poten-

215 tial to the hydrazone in what is technically an ECEC reaction (cf. Nelson and Landis 1973). In the photochemical sequence only one electron can be removed from DMOH by Chl .+. The reaction is concluded only when the resulting hydrazyl radical encounters another hydrazyl. Then it could react by disproportionation to give hydrazone and hydrazine, but the reaction should be rapid only if one of them is still protonated, which is unlikely at the ambient pH. There is no obvious barrier to dimerization of the two neutral hydrazyls to a tetrazane, so this is therefore the expected product. Back reaction of tetrazane with thiolate probably proceeds through nucleophilic displacements of hydrazine by thiolate. The initial quantum yield of DTNB reduction in Table 2 varies greatly, but is highest in (C. 1.a). This yield, which appears to be genuine, is related to the presence of a complex between Mg and H2 Rhodo. Since the reduction of DTNB is a two-electron process, and in an endergonic reaction both electrons must be provided by light, the initial quantum yield per electron approximates 9%, which is getting into a useful range. However, there is another issue to address. Since the pigment associations responsible for fastest initial rates typically constitute no more than ,,~10% of the total pigment, then either they are efficient collectors of excitation energy from surrounding less active pigment molecules, or they are intrinsically more active (by ,,~10 times) than calculated yields suggest. If the former, then small, efficient antenna systems exist on the particles; if the latter, then 'reaction centers' of high intrinsic reactivity have indeed somehow been created. Probably both situations contribute to some extent. Improvements of these systems would include better definition of reactive pigment associations, and of efficient antenna systems surrounding them. The existence of photochemically active though perishable pigment molecule associations in the particulate system appears connected with the use of amphiphiles that both ligate and hydrogen-bond strongly to Chl. There is little precedent for this sort of behavior in work previously reported from this laboratory. Probably much could be learned from the use of other amphiphiles in which the distance and angles between these functions are varied. The associations also appear connected with the use of DMOH and MPBH as reductants, instead of HB. Electron transfer within these associations is energetically possible only from the singlet excited state. The use of trisubstituted hydrazines introduces a class of reversible reducing agents of possibly wide utility in photochemistry. The

electrochemical potential for one- electron oxidation, and the stability of the central bond of the resulting tetrazane, could be varied by the choice of substitutents in foreseeable ways. The dimerization of hydrazyls to tetrazane, and the scission of disulfides to thiolates, represent two ways in which the immediate products of a photochemical reaction can be stabilized for energy storage. Nitrophenyl disulfides such as DTNB really contain two reducible functions, the nitro groups and the disulfide. The product of one-electron reduction of DTNB is apparently stable: the electron is probably confined to one of the nitro groups. When the second nitro is reduced, both electrons can be transferred to the disulfide linkage. DTNB therefore acts as a primitive electron accumulator and an example of intramolecular electron transfer. The potential, and the disulfide bond stability of nitrophenyldisulfides could be affected by substituents as with hydrazines, but a more interesting approach might be to investigate the properties of cyclic disulfides, which would open on reduction but not completely dissociate. The primary limitation on quantum yield in these reactions is probably the rate of return of the electron transferred in the initiating photoreaction. A further role expected of ligating amphiphiles is retardation of this reaction by maintaining a separation between charged pigment molecules. The wide range of quantum yields experienced in these reactions might be traceable to the degree of success in achieving this objective. In natural photosynthetic units, all primary, photo-initiated electron transfers take place within pigment associations. That there is evidence for such transfers within artificially generated pigment associations should be encouraging for the future of photochemical energy conversion.

Acknowledgements The earliest phases of this work were supported in part by the Division of Chemical Sciences, Office of Basic Energy Sciences, U.S. Department of Energy, under Grant No. DE-FG02-86ER13620. This is publication No. 245 from the Arizona State University for the Center for the Study of Early Events in Photosynthesis. The Center was established by the U.S. Department of Energy as part of the USDA/DOE/NSF Plant Science Center Program under Grant No. DEFG02-88ER13969.

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