Photoinduced electron transfer reactions between copper ions and

J. Phys. Chem. 1995,99, 12025-12029

12025

Photoinduced Electron Transfer Reactions between Copper Ions and Ethidium Bromide in Polynucleotides Stephen J. Atherton**+ Chemistry Department, Hutchison Hall, University of Rochester, Rochester, New York 14627

Paul C. Beaumont*?' Multidisciplinary Research and Innovation Centre, Faculty of Science, Health and Medical Studies, North East Wales Institute, Mold Road, Wrexham, Clwyd, U 1 1 2AW, U.K. Received: December 19, 1994; In Final Form: March 17, 1 9 9 9

Reaction of the first excited singlet state of ethidium with Cu2+ ions results in electron transfer to form oxidized ethidium and Cu'. The decay of these species is via reverse electron transfer, leaving the system unchanged. The reverse electron transfer reaction has been studied in solutions containing polynucleotides, leading to information conceming binding of Cu+ and electron transfer in these media. Cu+ binds strongly to calf thymus DNA, poly(deoxyadeny1ic-thymidylic acid), and poly(deoxyguany1ic-deoxycytidylic acid), in agreement with previous work. The rate of escape of Cu+ from the poly(deoxyadeny1ic-thymidylic acid) helix is measured as (1.3 f 0.3) x 105 s-l. The strongest complex is that with poly(deoxyguany1icdeoxycytidylic acid), and in this case reverse electron transfer occurs over several decades of time. Fitting these data to a summation of exponentials, ket = X v exp(-Pr), where r is the separation distance quantized in base pairs, leads to an attenuation factor, j3, of 0.73 f 0.05 A-l, for electron transfer in this medium.

Introduction It is well established that copper occurs naturally in association with DNA,' and it has been suggested to have a number of biological functions. Such functions include a major role in DNA quaternary structure by linking threads of DNA to structural proteins,2 as well as the oxidative degeneration of DNA i t ~ e l f . The ~ interaction of Cu2+ with DNA has been relatively well ~ t u d i e dhowever, ;~ considerably less information is available concerning DNA binding of Cu+, primarily because of the difficulty of producing a population of Cu+ at the DNA helix. Minchenkova and Ivanovs generated Cu+ in the presence of DNA by the addition of a reducing agent (ascorbic acid or sodium borohydride) to a solution of DNA containing Cu2+. They concluded that a complex is formed between Cu' and the N7 of guanine, resulting in proton transfer along the hydrogen bond from guanine to cytosine. The stoichiometry of the complex was shown to be one Cu+ to four bases, though this is presumably due to the DNA samples used having roughly 50:50 GC:AT ratios. More recently, Priitz et al.6 have used both steady state and pulse radiolysis methods to produce Cu+ in the presence of a variety of different nucleic acids and have shown that particularly stable complexes are formed with poly(dG-dC), poly(I), and native DNA. Binding constants for some of the polynucleotides have been determined as well as the time scales over which Cu+ can be expected to remain in the polynucleotide helix. There have been a number of studies of electron transfer reactions and electron mobilities in DNA.7 These are motivated in part by the idea that DNA may act as a conducto? and that a high electron mobility, either through the basesg or through the sugar-phosphate backbone,I0 may result. Earlier studies of Fielden et al.7aand W h i l l a n ~presented ~~ conflicting conclusions regarding an anomalously high rate of electron transfer, [email protected]

* [email protected] @

Abstract published in Advance ACS Abstracts, July 1, 1995.

which later studies have yet to resolve. Using the technique of microwave conductivity, Van Lith et al.7c.dshowed that no conducting species were formed on radiolysis of DNA at low levels of hydration; however, above a critical water concentration conducting species with mobilities similar to electrons in ice were formed. They concluded that these species were electrons confined to an ice-like sheath around the DNA and that there was no evidence for electron migration within the helix. Fromherz and Rieger7e investigated electron transfer quenching of DNA-intercalated ethidium by methylviologen and observed that the reaction occuned much faster than in homogeneous solution. However, they suggested that the reaction was actually some 100 times slower than predicted if considerations such as locally enhanced concentrations were taken into account. Conversely, Barton et al.7fand hrugganan et al.7hconcluded that DNA enhanced the electron transfer reaction between ruthenium complexes and complexes of cobalt, rhodium, and chromium. We have recently examined the interactions of Cu2+and Cu+ with DNA." In these studies our strategy has been to use DNAintercalated ethidium bromide as a probe molecule and to observe the reactions of Cu2+with the first excited singlet state of ethidium bromide (EB) whilst the reactants are bound to the DNA helix. Ethidium bromide is strongly bound to DNA,'* and for the concentrations used in the present study essentially all EB is bound. Quenching of ethidium excited singlet states by Cu2+ occurs via electron transfer to form Cu' and oneelectron-oxidized ethidium (EB+), which gives us a convenient method of producing Cu+ at the DNA helix. The details of the quenching of DNA-intercalated ethidium excited singlet states have been considered previously,!l a and in the following we concentrate on the fate of the products of this quenching reaction. The final return of the system to equilibrium involves back electron transfer from Cu+ to EB+, and we have observed this reaction in the presence of calf thymus DNA and the synthetic polynucleotides poly[dAdT]poly[dA-dT] and poly[dG-dC]poly[dG-dC]. The kinetics

0022-365419512099-12025$09.00/0 0 1995 American Chemical Society

Atherton and Beaumont

12026 J. Phys. Chem., Vol. 99, No. 31, 1995

of back electron transfer are found to be dramatically different depending on which polynucleotide is the host medium, whereas the forward electron transfer reaction rate is essentially unchanged. We interpret the differences in the back electron transfer reactions and their kinetics in terms of the modes of binding of Cu+ to the polynucleotides and in terms of electron transfer in the DNA helix.

r

0.015t

Experimental Section The synthetic polynucleotides poly(deoxyadeny1ic-thymidylic acid) (poly[dA-dT]poly[dA-dT]) and poly(deoxyguany1icdeoxycytidylic acid) (poly[dG-dC]poly[dG-dC]) and ethidium bromide were obtained from Sigma Chemical Co. and used as received. Water was purified via a Millipore filtration system. All other materials were of the best available grade. All polynucleotide samples were made up in 5 x M Na2S.04, in order to prevent denaturation, and concentrations were determined by measuring the absorbance at 260 nm using an extinction coefficient of 660 m2 mol-' for both calf thymus DNA and poly[dA-dT]poly[dA-dT] and 840 m2 mol-' for poly[dG-dC]p~ly[dG-dC].'~Concentrations of polynucleotides are expressed throughout in moles of phosphate. Steady state absorption and emission spectra were measured with a Hewlett-Packard 8540A UV-visible spectrophotometer and a Perkin-Elmer LS5 fluorescence spectrophotometer, respectively. Time-resolved emission measurements were made by time-correlated single-photon counting. The 532 nm output of a Coherent Antares Nd:YAG laser was used to pump a Spectra Physics 375B dye laser and home-built cavity dumper combination containing pyridine 2. The dye laser was tuned to 720 nm, and a Camac Systems Inc. Bragg cell driver was adjusted to produce a train of pulses ca. 6 ps wide at a repetition rate of 1.9 MHz from the cavity dumper. The dye laser pulses were then frequency doubled to 360 nm to provide the excitation source. Fluorescence photons were detected perpendicular to the excitation by a Hamamatsu R2809U-07 proximity focus microchannel plate. Wavelength selection for the fluorescence was achieved by placing a monochromator between the sample and the microchannel plate. Start and stop pulses were amplified by a Phillips Scientific Model 774 amplifier before being passed to a Tennelec TC 454 constant fraction discriminator. The discriminator outputs were led to the start and stop channels of an Ortec 457 time to amplitude converter whose output was passed to a Tracor Northem TN 7200 multichannel analyzer (MCA). Data from the MCA was collected by an IBM compatible personal computer, which was also used for data analysis, storage, and hardcopy. For time-resolved absorption measurements, excitation was provided by the second harmonic (532 nm, 11 ns pulse) from either a Quantel YG481 or Quantel YG580 Q-switched Nd: YAG laser. The beam energy was attenuated as necessary using either calibrated metal screens or crossed calcite Glan-Laser polarizers. Energies up to a maximum of 30 mJ cm-* were used. Transient absorptions were measured perpendicular to the excitation beam by using a conventional lamp, monochromator, photomultiplier tube arrangement. Signals were captured by a Biomation 8100 transient digitizer and were sent to an IBM compatible personal computer for analysis, storage, and hardcopy. The apparatus for pulse radiolysis has been described previously. l 4

Results The decay of ethidium fluorescence in the presence of DNA or synthetic polynucleotides was well fit to a single exponential. In these experiments the polynucleotide concentration was

380

480

580

Wavelength/nm

Figure 1. Absorption spectra obtained 200 p s ( 0 )and 4.4 ms (0) after 532 nm laser flash photolysis of an air-saturated solution of 7.3 x M ethidium, 7.5 x M Cuz+, 1.8 x M poly[dGM Na2S04. dC]poly[dG-dC], and 5 x

typically around low3M, with a ratio of phosphate to ethidium of 20. For both DNA and poly[dG-dC]poly[dG-dC] the measured lifetime was 22 f 1 ns; however, when intercalated into poly[dA-dT]poly[dA-dT], the ethidium fluorescence lifetime was consistently somewhat longer, at 25 f 1 ns. In the presence of Cu2+,at a ratio of phosphate to Cu2+of ca. 2, the fluorescence decay in all cases was fairly well fit to a double exponential with both components having shorter lifetimes than in the absence of Cu2+. This situation is the same as that previously observed in DNA.' l a Integration of the fluorescence curves showed that the fraction of singlet states quenched by Cu2+ in these conditions was ca. 0.8 independent of the polynucleotide. Figure 1 shows the absorption spectra obtained 200 ps and 4.4 ms after 532 nm laser flash photolysis of an air-saturated solution of 7.3 x M ethidium, 7.5 x M CuZf,1.8 x M poly[dG-dC]poly[dG-dC], and 5 x M Na2S04. The decay of the absorption is oxygen insensitive, and the spectrum is the same as that previously observed after flash photolysis of ethidium and Cu2+ in DNA solution1Iband that observed after one-electron oxidation of DNA-intercalated ethidium via pulse radi01ysis.I~ The spectra are therefore attributed to one-electron-oxidized ethidium, formed via quenching of the first excited singlet state of ethidium by CU*+. An essentially identical spectrum was observed after 532 nm laser flash photolysis of a corresponding sample in which poly[dGdC]poly[dG-dC] was replaced with poly[dA-dT]poly[dA-dT]. For each of the polynucleotides and for native DNA the absorption of the first excited singlet states of ethidium was measured in the absence of Cu2+. Then, under identical conditions of geometry and beam energy and in the presence of Cu2+,the absorption of one-electron-oxidized ethidium was measured. From a knowledge of the extinction coefficients of excited singlet' I b and one-electron-oxidized ethidium,' we calculated the concentration of each species. The concentration of oxidized ethidium was considerably less than the 80% of excited singlet states that are quenched by Cu2+, and we calculate that ca. 3% of quenching events result in oxidized ethidium which is observable subsequent to the excited singlet decay. This is independent of polynucleotide. The majority presumably decays via geminate recombination on shorter time scales. The eventual retum of the system to equilibrium occurs with kinetics which are highly dependent on the nature of the polynucleotide, the extreme cases being the synthetic polynucleotides, with calf thymus DNA as an intermediate case. Figure 2 shows the decay of oxidized ethidium in poly[dAdT]poly[dA-dT] and poly[dG-dC]poly[dG-dC] over time scales

Reactions between Copper Ions and Ethidium Bromide

4

1

40

J. Phys. Chem., Vol. 99, No. 31, I995 12027

I

aw

((00

nme/ns

Figure 2. Decay of oxidized ethidium observed at 420 nm after 532 nm laser flash photolysis of solutions containing 7.3 x M ethidium, 7.5 x M Cu2+,5 x lo-' M NaZS04 and 1.8 x M of either poly[dG-dC]-poly[dG-dC] or poly[dA-dT]poly[dA-dT]. The decay is shown on four time scales. In all cases the upper traces correspond to poly[dA-dT]poly[dA-dT] and the lower to poly[dG-dC]poly[dG-dC].

.-+x

0.02-

x .--+ v,

u) 0)

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.-cx.

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0.01 ..

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0.05

0

.--w0

Q

0

0.00l4 Time/ms

9

0.00

-7

-6

-5

-4

-3

-2

-1

Log t Figure 4. Plot of optical density vs log(time) for oxidized ethidium in poly[dG-dC]poly[dG-dC] and poly[dA-dT]poly[dA-dT].

200

400

Time/ms

Figure 3. Long time scale decay of oxidized ethidium in solutions containing (a) poly[dA-dT]poly[dA-dT], (b) calf thymus DNA, and (c) poly[dG-dC]poly[dG-dC]. Concentrations of reactants are the same as those for Figure 2. Trace (d) shows the decay of oxidized ethidium produced by pulse radiolysis as described in the text.

to 400 ps, and Figure 3 shows the decay on longer time scales for the synthetic polynucleotides and DNA. Essentially identical decays, but with reduced absorption, were observed when the samples were diluted by a factor of 2 with 5 x M Naz-

so4.

DNA-intercalated oxidized ethidium may be produced in the absence of copper via pulse radiolysis. Using this technique,

with experimental conditions similar to those used previously, DNA-bound oxidized ethidium was formed by reaction of ethidium with the azide radical.I4 Figures 3d shows the decay of oxidized ethidium formed in this manner. The decay rate is considerably slower than that in the presence of Cu+ and demonstrates that in the latter case decay of oxidized ethidium via processes other than reverse electron transfer from Cu+ is negligible. A clearer demonstration of the differences in kinetics between poly [dA-dT]poly [dA-dT] and poly [dG-dC]poly [dG-dC] can be seen if the absorption is plotted against the logarithm of time, as shown in Figure 4. Although the decay is initially faster in poly[dG-dC]poly[dG-dC], the curves cross at ca. 1 ms, and the absorption in poly[dA-dT]poly[dA-dT] decays to zero before that in poly[dG-dC]poly[dG-dC]. The decay of oxidized ethidium in the presence of calf thymus DNA falls somewhere in between the two polynucleotides, to the extent that the decay over the first few hundreds of microseconds is less than in poly[dG-dC]poly[dG-dC] but more than in poly[dA-dT]poly[dA-

12028 J. Phys. Chem., Vol. 99, No. 31, 1995

dT], and the final decay to the base line occurs faster than in poly[dG-dC]poly[dG-dC] and slower than in poly [dA-dT]poly[dA-dT]. In the case of poly[dA-dT]poly[dA-dT] the decay may be fit by an initial first-order decay ( k = (5 f 1) x lo5 s-l) followed by a second-order decay. The presence of the secondorder component was verified by varying the beam intensity by a factor of 4, thus decreasing the concentration of oxidized ethidium. As expected, the first half-life of the decay increased, while the rate constant remained essentially unchanged at (1.5 f 0.3) x lo9 M-' s-l, assuming our value of 1.7 x lo3 m2 mol-' for the extinction coefficient of oxidized ethidium at 420 nm.I4 The decay for poly[dG-dC]poly[dG-dC], however, is more complex and has the features of a time dependent rate constant, in that similarly shaped decay curves are observed over several orders of magnitude of time. This is shown in Figure 2.

Atherton and Beaumont

H I

Discussion The decay kinetics in the case of poly[dA-dT]poly[dA-dT] may be explained if those Cu+ ions which escape rapid geminate recombination remain at the helix on which they were formed. There is now a competition between helix escape (kesc)and migration along the helix until an oxidized ethidium is encountered and recombination occurs ($). Since this takes place on individual helices, the kinetics will be first order with a rate constant k&S (k&S = k,,, kr). Those Cu+ ions which escape the helix must now recombine with helix-bound oxidized ethidium via second-order kinetics. A simple reaction scheme would be

+

CU+~,

+ EB+ 4 cu2+ + EB

followed by

cuff+ EB+ 5 cu2+ + EB where C U + ~and N Cu+f are copper ions bound to the polynucleotide and free in solution, respectively, and k, is the secondorder rate constant for recombination. Under the conditions of our experiments, taking the average molecular weight for our particular lot of poly[dA-dT]poly[dA-dT] as 750 000, we calculate that an average of around 0.7 oxidized ethidiums per helix take part in the second-order recombination. The measured rate constant of (1.5 f 0.3) x lo9 M-' s-' is somewhat less than diffusion controlled and may reflect the need for more than one helix encounter for recombination to occur. By extrapolating both the first- and second-order decay components to time zero, we calculate that the fraction of Cu+ ions which escape from the helix (Fesc)is 0.74. Since Fesc = ,&/(kr kesc),and kobs = kesc k,, we calculate that the rate constant for escape of Cu+ from a poly[dA-dT]poly[dA-dT] helix is (1.3 f 0.3) x lo5 s-I. Priitz et have measured a rate of dissociation of the Cu+ complex from poly[dA-dT]poly[dA-dT] of 0.15 s-l, apparently in contradiction to the present value. This value, however, represents the breakup of an already formed complex, whereas the present value reflects competition between the formation of the complex and helix escape. They also measured a value of ca. 7 x lo7 M-' s-I , expressed in terms of bases, for the reaction of Cu+ formed in the bulk solution with poly[dAdT]poly[dA-dT]. This is a factor of 10 lower than the rate

+

+

Figure 5. Schematic diagram showing the binding sites of ethidium and Cut to poly[dG-dC]poly[dG-dC].

constant for OH' radical reaction with DNA,I6 which is itself less than diffusion controlled. Thus, less than one in 10 encounters between poly[dA-dT]poly[dA-dT] and Cu+ results in the formation of a complex, and from our data these encounters last some 8 ,us. What remains unclear at present is why, in our experiment, a significant fraction of Cu+ which escapes from the helix does not become re-bound at sites remote from oxidized ethidium, resulting in absorptions stable on the time scale of seconds. The decay kinetics in the case of poly[dG-dC]poly[dG-dC] may be explained in terms of strong intrahelical binding of Cu+ to guanine, in agreement with Minchenkova and Ivan0v5 and Prtitz et a1.6h After the laser flash, intercalated ethidium in its excited singlet state is quenched by Cu2+, forming Cu+, and those Cu+ ions which escape rapid geminate recombination migrate along the helix and then bind within it, residing at the N7 of guanine in the plane of the G-C pair. This is in contrast to poly[dA-dT]poly[dA-dT], where Cu+ binds more weakly to the DNA surface without any internal binding.4 Figure 5 shows a schematic view of the binding sites of ethidium and Cu+ to poly[dG-dC]poly[dG-dC]. The Cu+ binding site is that suggested by Minchenkova and I v a n o ~The .~ binding constant of Cu+ to the G-C pair is very large, ca. lo9 M-',6b and we propose that Cu+ binds at a free G-C base pair soon after it is formed with an asymptotic distribution favoring small ethidium-Cu+ separation. Recombination now occurs via electron transfer over a series of well-defined distances, the shortest of which is 0.48 nm, increasing in steps of 0.32 nm, assuming a base pair separation of 0.32 nm for the B form of DNA. The assumption that the shortest distance for electron transfer would be 0.48 nm is based on the premise that Cu+ is bound in the plane of the base pair, ethidium is intercalated

Reactions between Copper Ions and Ethidium Bromide

J. Phys. Chem., Vol. 99, No. 31, 1995 12029 Acknowledgment. The authors are grateful for the award of a NATO grant (RG0185-88) to assist with these studies. Laser flash photolysis and fluorescence experiments were carried out at the Center for Fast Kinetics Research (CFKR) at the University of Texas at Austin, supported jointly by the Biotechnology Research Technology Program of the Division of Research Resources of NIH (RR 00886) and the University of Texas at Austin. Pulse radiolysis was carried out at the Paterson Institute for Cancer Research.

n &

Q)

Y

W

t -

0.7

0.9

1.1

1.3

1.5

r/nm Figure 6. Plot of ln(k,,) vs separation distance for reverse electron transfer reaction between Cu+ and oxidized ethidium in poly[dGdC]p~ly[dG-dC].

between base pairs, and Cu+ is sterically hindered from binding to a base adjacent to ethidium. It is given by 1.5 times the base pair separation. The first-order rate constant, kt,for electron transfer between spatially and orientationally fixed reactants may be described as a function of the electronic matrix element, Franck-Condon factor, and the free energy of reaction.” For many systems the distance dependence of the electronic matrix element is such that the rate constant may be written as

where the preexponential factor, v, includes the free energy and the Franck-Condon factor, p is a constant that depends on the overlap of the wave functions of the donor and acceptor, and r is the donor-acceptor separation. It follows that for our system a distributed kinetic scheme applies where

+

where the summation extends over r = 0.48 n(0.32) nm for n = l , 2 , 3 ,.... Our data for the decay of oxidized ethidium in poly[dGdC]poly[dG-dC] are well fit to three exponentials with a nonzero base line. The nonzero base line may be due to reverse electron transfer involving Cu+ bound at greater distance, Cu+ which escapes the helix, or a combination; however, attempts to fit further exponentials were unsuccessful. The preexponential factors indicate relative populations of 0.55, 0.31, and 0.14 as the separation increases and support our assumption of a distribution which favors small separation. If we assume that the three rate constants correspond to reverse electron transfer with the distribution of distances discussed above, then a plot of In&) vs r should be linear with a slope of ,5. Such a lot is shown in Figure 6 and gives a p value of 0.73 f 0.05 These values are essentially the same as those measured for the forward electron transfer from ethidium bromide to N,”dimethyl-2,7-diazapyrinium dichloride or acridine orange, where both reactants are intercalated within DNA,’J and are similar to the values obtained for modified proteins.’*

1-l.

References and Notes (1) (a) Wacker, W. E. C.; Vallee, B. L. J . Biol. Chem. 1969,234,3257. (b) Bryan, S. E.; Vizard, D. L.; Beary, D. A,; La Biche, R. A.: Hardy, K. J. Nucleic Acids Res. 1981, 9, 5811. (2) (a) Lewis, C. E.; Laemmli, U. K. Cell 1982, 29, 171. (b) Lewis, C. E.; Lebkowski, J. S.; Daly, A. K.; Laemmli, U. K. J . Cell. Sci. 1984, I, 103. (c) Cramp, W. A.; George, A. M.; Kahn,H.; Yatvin, M. B. In Free Radicals, Metals Ions and Biopolymers; Beaumont, P. C., Deeble, D. J., Parsons, B. J., Rice-Evans, C., Eds.; Richelieu: London, 1989; pp 127141. (3) (a) Goldstein, S.; Czapski, G.J . Free Rad. Biol. Med. 1986, 2, 3. (b) Priitz, W. A. In Free Radicals, Metal Ions and Biopolymers; Beaumont, P. C., Deeble, D. J., Parsons, B. J., Rice-Evans, C., Eds.; Richelieu: London, 1989; pp 117-126. (4) (a) Izatt, R. M.; Christensen, J. J.; Rytting, J. H. Chem. Rev. 1971, 71, 439. (b) Eichhorn, G. L. In Inorganic Biochemistry; Eichhorn, G. L., Ed.; Elsevier: Amsterdam, 1975; pp 1210-1243. (c) Liebe, D. C.; Stuehr, J. E. Biopolymers 1972, 11, 145. (d) Albiser, G.;Premilat, S. J . Biomol. Struct. Dyn. 1985, 2, 745. (e) Eichhom, G. L. In Advances in Inorganic Biochemistry 3: Metal Ions in Genetic Information Transfer: Eichhom, G. L., Marzilli, L. G., Eds.; ElsevierNorth Holland: Amsterdam, 1981; pp 2-47. ( 5 ) Minchenkova, L. E.; Ivanov, V. I. Biopolymers 1967, 5, 615. (6) (a) Stoewe, R.; Priitz, W. A. Free Rad. Biol. Med. 1987, 3, 97. (b) Priitz, W. A.; Butler, J.; Land, E. J. In?. J . Radiat. Biol. 1990, 58, 215. (7) (a) Fielden, E. M.; Lillicrap, S. L.; Robins, A. B. Radiat. Res. 1971, 48, 421. (b) Whillans, D. W. Biochim. Biophys. Acta 1975, 414, 193. (c) van Lith, D.; Warman, J. M.; de Haas, M. P.; Hummel, A. J . Chem. Soc., Faraday Trans. I 1986, 82, 2933. (d) van Lith, D.; Eden, J.; Warman, J. M.; Hummel, A. J . Chem. Soc., Faraday Trans. I 1986, 82, 2945. (e) Fromherz, P.; Rieger, B. J . Am. Chem. SOC.1986, 108, 5361. (0 Barton, J. K.; Kumar, C. V.; Turro, N. J. J . Am. Chem. SOC.1986, 108, 6391. (g) Davis, L. M.; Harvey, J. D.; Baguley, B. C. Chem.-Biol. Interact. 1987, 62, 45. (h) Purugganan, M. D.; Kumar, C. V.; Turro, N. J.; Barton, J. K. Science 1988, 241, 1645. (i) HouCe-Levin, C.; Gardks-Albert, M.; Rouscilles, A.; Ferradini, C.; Hickel, B. Biochemistry 1991,30, 8216. (i) Brun, A. M.; Harriman, A. J. Am. Chem. SOC.1992,114,3656. (k) Murphy, C. J.; Arkin, M. R.; Ghatlia, N. D.; Bossman, S.; Turro, N. J.: Barton, J. K. Proc. Natl. Acad. Sci. U.S.A. 1994, 91, 5315. (8) Pethig, R. Dielectric and Electronic Properties of Biological Materials; Wiley: Chichester, 1979. (9) Dee, D.; Baur, M. E. J. Chem. Phys. 1974, 60, 541. (10) Suhai, S. Biopolymers 1974, 13, 1739. (11) (a) Atherton, S. J.; Beaumont, P. C. J. Phys. Chem. 1986,90,2252. (b) Atherton, S . J.; Beaumont, P. C. In Free Radicals, Metal Ions and Biopolymers: Beaumont, P. C., Deeble, D. J., Parsons, B. J., Rice-Evans, C., Eds.; Richelieu: London, 1989; pp 93-116. (12) LePecq, J.-B.; Paoletti, C. J. Mol. B i d . 1967, 27, 87. (13) Kelly, J. M.; van der Putten, W. J. M.; McConnell, D. J. Photochem. Photobiol. 1987, 49, 167. (14) Atherton, S. J.; Beaumont, P. C. Radiat. Phys. Chem. 1990, 36, 819. (15) Atherton, S. J.; Beaumont, P. C. J . Phys. Chem. 1987, 91, 3993. (16) Buxton, G. V.; Greenstock, C. V.; Helman, W. P.; Ross, A. B. J . Phys. Chem. Re$ Data 1988, 17, 513. (17) Marcus, R. A.; Sutin, N. Biochim. Biophys. Acta 1985, 811, 265. ( 18) Jacobs, B. A.; Mauk, M. R.; Funk, W. D.; MacGillivray, R. T. A,; Mauk, A. G.; Grey, H. B. J . Am. Chem. SOC. 1991, 113, 4390. JF943333P