J. Phys. Chem. 1993,97, 1707-1711
1707
Spectroscopic Investigation of Differential Binding Modes of A- and A-Ru( bpy)z(p ~ z ) with ~ + Calf Thymus DNA Steven A. Tysoe, Robert J. Morgan, A. David Baker,' and Thomas C. Strekas' Department of Chemistry and Biochemistry, Queens College-City University of New York, Flushing, New York 11367 Received: October 6. 1992
The binding of enantiomers of Ru(bpy)2(ppz)2+ (bpy = 2,2'-bipyridine; ppz = 4,7-phenanthrolino[6,5-b]pyrazine) to calf thymus DNA is investigated using absorption, fluorescence and resonance enhanced Raman spectroscopies. Both isomers show absorption hypochromicity, steady-state fluorescence increase, reduced accessibility to an anionic quencher, and fluorescence lifetime increase associated with binding to B-form DNA, though the effect for the A isomer is always less. However, the maximal fluorescence polarization for both isomers is the same, indicating a similarly rigid binding to DNA. These findings are consistent with a major groove binding in which the ppz ligand is partially intercalated for both enantiomers. The bpy ligands are relatively small and not competent for inducing intercalative binding, but enantioselectivity is maintained by the differential interactions of these bpy ligands with the surface of the major groove as the ppz ligand partially intercalates.
The binding to DNA, RNA, and synthetic polynucleotides of Norden et al. have also studied*the binding of enantiomers of cationic tris-chelatesof ruthenium(I1) and rhodium(II1)in which Ru(phen)32+to DNA via linear and circular dichroism meathe ligands are bidentate diimines with aromatic ring structures surements in a flow system and concluded that both enantiomers bind within the major groove of B-form DNA, but that the A has been extensively studied'-'2J7J9in recent years. An interisomer, and not the A one, is bound in an intercalative fashion. calative mode of binding, in which a portion of one of the three chelated aromatic rings inserts between adjacent base pairs of Recently reported9 viscosity measurements for DNA in the presenceof each enantiomer of Ru(phen)32+indicate that neither the native DNA structure via major groove access, is a key the structure of DNA as a classical intercalator would be component of models which have been p r ~ p o s e d ~ , ~ * to ~ , * J ~ Jaffects ~ expected to. Thermodynamic parameters for the binding as a describe the binding of such complex ions to polynucleotides. In function of salt concentration also indicate that the binding is particular, intercalative major groove binding is consistent with largely electrostatic in nature. and provides a rational basis for the observation that enantioCleavage studies4 of DNA restriction fragments show that selectivity is a feature of the binding of many such complex ions individual enantiomersof Rh(~hen)z(phi)~+ (phi = 9,lO-phenanto these polynucleotides. threnequinone diimine) give rise to different cleavage patterns. Although numerous complexes of this type have been shown Also, the A isomer of Ru(bpy)2(pp~)~+ (ppz = 4,7-phenanthrolinoto bind enantioselectively, only a few reports have appeared3-sJ3 [6,5-b]pyrazine)has been shown13to promote cleavage of plasmid which detail the interactions of individual enantiomerswith DNA DNA in the presence of copper(II), peroxide, and a thiol, while or other polynucleotides, principally for R ~ ( p h e n ) ~(phen ~+ = the A isomer is almost completely ineffective in promoting 1,lO-phenanthroline)and trisphenanthrolinecomplexes of Rh3+, cleavage. C03+,N?+, and Cr3+. Barton et al. have studiedl-3Jv6 the binding We have reported" on the binding to DNA of complexes of of A- and A - R ~ ( p h e n ) ~to~ + various DNA's and homopolynuthe formula Ru(bpy)2L2+,where L is an aromatic diimine with cleotides, mainly poly(dG4C) in both B- and Z-forms. An additional functionalities, such as N donor atomsor charge bearing analysis of spectroscopic and photophysical evidence leads them sites (e.g. quaternized nitrogens). A feature of most such to the conclusion that the favored binding of the A isomer within complexes is the ineffectiveness of bpy in inducing intercalative the major groove of B-form DNA is explained by an intercalative binding to DNA, combined with spectral resolvability of the binding mode of one of the phen ligands with the other two potential intercalating ligand (L). This combination enhances coordinated phens establishing a pattern of van der Waals contacts the ability to probe the interaction of the complex with DNA with atoms lining the major groove, providing a better "fit" for because one can spectrally resolve the effects of binding to DNA the A isomer. Similar binding of the A isomer within the major on the two ligand types. Several of the complexes we have studied groove is more sterically disfavored. In binding studies3 of the exhibit enantioselective binding to DNA, including the complex enantiomers of Ru(DIP)3Z+(DIP = 4,7-diphenylphenanthroline), with L = ppz, and L = Me2qpy2+(the dimethylquaternary form the same group concluded that binding of the A isomer of this of 2,2':4,4":4',4"'-quaterpyridyl), the latter of which, however, complex to the left-handedhelical Z-form poly(dGC) was favored shows no evidence suggestive of intercalative binding. We report via a similar (complementary)mechanism. NMR studies by the here our findings regarding the binding of individual A and A Barton group6 have not provided conclusive evidence of an isomers of Ru(bpy)*(ppz)Z+ to calf thymus DNA. Absorption intercalative major groove mode of binding when trisphenanand emission spectroscopy, including polarization measurements throline complexes of various metal ions interact with a selfand emission lifetimes, as well as resonance enhanced Raman complementary hexameric oligonucleotide. Rather, a nonvibrational spectroscopy,provide a basis for elucidating the binding intercalative minor groove mode of binding is observed. Similar modes of the two enantiomers with B-form DNA. findings have been reported7 in another study in which both enantiomersof R~(phen)3~+ interact with a self-complementary Experimental Section decanucleotide duplex. Here no absorption or fluorescence Materials. Ru(bpy)2(ppz)Cl2 was synthesized as described14 experimentswere reported for possible correlation of photophysical previously and resolvedi2 chromatographically on a DNA/ and NMR results. 0022-3654/93/2097- 1707$04.00/0
0 1993 American Chemical Society
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1708 The Journal of Physical Chemistry, Vol. 97, No. 8, 1993
hydroxylapatite column. Calf thymus DNA was purchased from Sigma Chemical Co. and purified via successive centrifugation and ethanol precipitation,followed by redissolving in buffer. Most measurements were performed in pH 7.2 Tris (0.0050M) with 0.050 M sodium chloride. All experiments were performed using air-saturated solutions. Instrumentation. UV-visible absorption measurements were made on an HP8452 diode array spectrophotometer. Raman spectra were recorded using an instrument previously described.I5 Raman frequenciesare f 1 cm-I. Relative intensitiesare reported as ratios of peak heights, measured from base line to maximum, and are reproducible to f 10%. Circular dichroism measurements were made with a Jasco S00C CD/ORD instrument. Fluorescence measurements were made using a Perkin-Elmer MPF-66 spectrometerinterfaced toa Perkin-Elmer Model 7000 computer. Polarization measurements were made using the polarization accessory, and time averaged over a period of 2 min. Each polarization measurement was an average of at least five trials, and complex concentrations between 5 and 20 pM showed a variation of less than 5% in polarization for a given P/Ru ratio. Values previously reported5 for R ~ ( p h e n ) ~with ~ + calf thymus DNA were reproducible using our equipment. Quenching experiments were performed using 10pM metal complex and the appropriate DNA concentration and were corrected for dilution of the metal complex and the DNA. Time-resolved emission spectroscopy was performed on a system previously described,16 utilizing the 355-nm line of a Nd:YAG laser as the excitation source. Oscilliscope traces were digitized by optical scanning of the photographic traces. Two components were resolvable in all traces when subjected to component stripping analysis, as described by Demas.20 Lifetimes were reproducible within f5% for the longer component and flO% for the shorter component.
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Figure 1. Absorption spectra of Ru(bpy)2(ppz)2+(1 1 pM) in solution in the absence (-) and presence (- -) of calf thymus DNA: (top) A isomer; (bottom) A isomer. [DNA-phosphate]/[Ru] = 159, measured at 25 O C in 5 mM Tris, 50 mM NaCl buffer, pH 7.4.
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Hypocbromicity of MLCT Bands. One of the most immediate indicators17 of possible intercalative binding to DNA is hypochromicity associated with UV or visible transitions of an intercalating molecule. For the racemic Ru(bpy)2(ppz)2+ we have reported1I a pronounced hypochromicity upon binding to calfthymus DNA, of the metal to ligand charge-transfer (MLCT) transition at 475 nm which has been assigned as associated with the ppz ligand exclusively. The bpy associated band centered at 422 nm shows little or no hypochromicity. When the individual isomers are studied,at high DNA phosphate to ruthenium complex ratios (P/Ru) (Figure l), the A isomer shows a maximal hypochromicity which is approximatelydoublethat of the A isomer (top) for the MLCT band at 475 nm. In addition, the band shifts to -495 nm for the A isomer but to only -480 nm for the A isomer. In each case, the 422-nm MLCT band is little affected. Emission Enhancement. When each isomer binds to DNA, emission enhancement is observed. Figure 2 shows the (normalized) increase in emission enhancement, monitored at the emission maximum near 670475 nm. The emission for such complexes is associated with charge transfer from the lower energy ligand excited-state r* system, in this case the ppz, to the ruthenium(I1). For the A isomer, at a P/Ru ratio of 196X, the maximal emission enhancement upon binding to DNA is 17.OX compared to buffered solution, while for the A isomer it is 5.2X. Also, the limiting (Le. high P/Ru ratio) emission maximum is blue-shifted to 668 nm (from 690 nm) for the A isomer in the presence of DNA, but is less blue-shifted, to only 67 1 nm, for the A isomer. In acetonitrile solution, the complex emits with a maximum at 668 nm. Also evident in the room temperature emission spectra at high P/Ru ratio is the 371-cm-1 decrease in half-width (FWHM), from 1980 cm-l (buffer) to 1609 cm-l for the A isomer, but to only 1726 cm-l for the A isomer (254-cm-I decrease). Already, at a lower P/Ru ratio of l o x , the A isomer emission spectrum maximum and half-width is essentially the
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80 im im 0 [DNA-PI/[Ru] Figure 2. Normalized emission enhancement (intensities at A,), vs [DNA-phosphate]/(Ru] ratio for the AandA enantiomenof Ru(bpy)l( ~ p z ) ~ +All . samples excited with &, = 500 nm at 10.0-nm bandwidth. &,, = 660-690 nm. All samples measured at 20 OC in 5 mM Tris, 50 mM NaCl buffer, pH 7.4. ((A) A isomer; (0) A isomer). (open symbols: (A) A isomer; (0)A isomer).
0
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same as that observed at higher P/Ru ratio, but the A isomer shows a blue shift to only 675 nm, with a half-width of 1880cm-I (1OO-cm-I decrease). The normalized curves allow a comparison of the relative dependenceof the increase in emission enhancement on DNA phosphate to ruthenium ratio, and one can see from Figure 2 that the increase in enhancement is more evident at lower P/Ru ratios for the A isomer. This indicates qualitatively the higher binding constant for this isomer. A Scatchard analysis of the emission data, employing the McGhee and von Hippel equation, yields the following binding constants for the two isomers: 2.1 X 104 M-I (A)and 6.3 X IO3 M-I (A). The sitesize parameter giving the best fit in both cases was I = 3. Data fits for I = 4 were not significantly worse. These numbers compare favorably with our previously reported values of 6.0 X lo3 M-l (I = 3,4) for the racemic complexobtained by equilibriumdialysis methods. Emission Quenching. Quenching by ferrocyanide ion has been used3v5to infer an intercalative mode of binding for tris-chelates with DNA. The intercalated complex within the major groove
Ru(bpy)2(ppz)2+Binding with Calf Thymus DNA
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The Journal of Physical Chemistry, Vol. 97, No. 8, 1993 1709
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Figure 3. Quenching with Fe(CN)b4- for the A and A enantiomers of Ru(bpy)z(ppz)Z+at [DNA-phosphate]/[Ru] = 10 (filled symbols: (A) A isomer; ( 0 )A isomer) and [DNA-phosphate]/[Ru] = 43. Quenching of ra~-Ru(bpy)2(ppz)~+ by Fe(CN)b4- in the absence of DNA is shown for comparison (straight line). All samples were measured at 20 OC in 5 mM Tris, 50 mM NaCl buffer, pH 7.4.
is less accessible to anionic quenchers in solution, as compared to surface or minor groove bound complex. Figure 3 shows the Stern-Volmer plots ( l o / l ) for racemic Ru(bpy)z(ppz)Z+in buffer and for each enantiomer in the presence of DNA at low (1OX) and high (43X) P/Ru ratios. Note that the A isomer is weakly quenched at both ratios, with little difference evident between the two CUNCS, and both vary strongly from the free solution curve. The A isomer, however, shows a more marked decrease in quenching at the higher P/Ru ratio, indicating that the typical binding site for the A isomer is less accessible to the ferrocyanide at these higher ratios, and indeed, may be qualitatively different than the typical binding site at lower P/Ru ratios. Indeed, the characteristic downward curvature of these plots has previously been attributed5 to the differential accessibilityof bound complex to the quencher due to a variety of binding sites on the DNA. Emission Polarization. When an emitting molecule becomes immobilized upon binding to DNA for a significant fraction of its emission lifetime, the polarization propertiesof emission excited by polarized light are indicative of that residence time, because of the relatively slow reorientation of the DNA itself when compared to the typical emission lifetimes of these ruthenium(11) tris-chelates (about 100-1000 ns). A tightly bound, intercalative binding mode, as compared to a surface bound mode, is e~pectedj-~ to result in more highly polarized emission. Emission polarization for the two enantiomers is presented in Figure 4 as a function of P/Ru ratio. It should be pointed out that the excitation wavelength dependenceof polarization of either isomer in the presence of DNA shows significant polarization only for excitation within the MLCT band associated with the ppz ligand, near 475-525 nm. Above a P/Ru of -3OX, both isomers show the same limiting value of -0.090, The A isomer, however, approaches this limitingvalue moreslowly. The A isomer already shows the maximum polarization value at a P/Ru of -5X. Emission Lifetimes. The above noted emission intensity increase in the presence of DNA is associated with an increase in emission lifetime when bound to DNA. Previous studies395 have associated the longer lifetime component of a biphasic emission decay curve seen for ruthenium(I1) complexes in the presence of DNA with an intercalated, major groove bound complex, and a shorter lifetime component with a surface bound complex. For Ru(bpy)2(ppz)2+ we also see biphasic emission decay curves (Figure 5 ) , which can be readily resolved into two components. The shorter lifetimecomponent for both enantiomers is similar to that of the complex in free solution ( 120-1 30 ns). The longer lifetime component is plotted in Figure 6 for each
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Time (ns) Figure 5. Natural logarithm of luminescence intensity versus time, for A(bottom)and A (top) isomersofRu(bpy)Z(pp@+at [DNA-phosphate]/ [Ru] = 33. Solid lines represent best fit for lifetimes indicated in figure (weighted as in parenthese) as described in the Experimental Section.
enantiomer as a function of P/Ru ratio. The limiting (high P/Ru ratio) value differs for each enantiomer. For the A isomer, a maximal value of -950 ns is obtained above P/Ru values of 20X. For the A isomer, a maximal value of 550 ns is obtained, but this is more slowly approached as the P/Ru ratio increases. The rise in these curves closely approximates the rise in the emissionpolarization curves of Figure 4. The polarization studies indicate a tightly bound complex at higher P/Ru values for both isomers, but the lifetime values (and the emission enhancements) distinguish a difference in degree (Le. a quantitative difference) between the modes of binding. What is also apparent from our analysis of the lifetime data is that the short component contribution is diminishedat higher P/Ru ratios for both isomers, suggesting that intercalation (which gives rise to the long component) is the preferred binding mode at low metal complex
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Tysoe et al.
1710 The Journal of Physical Chemistry, Vol. 97, No. 8, 1993
With the buffer spectrum being selected as a reference, the A and A isomer spectra are more similar to one another than to the
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[DNA-PI/ [RuI Figure 6. Luminescence lifetime of the long component emission of the Ru(bpy)2(ppz)2+ A and A enantiomersvs [DNA-phosphate]/ [Ru]ratio. All emission profiles were averaged over at least 100 laser pulses and measured at 20 O C with 355-nm excitation. Luminescence was monitored at 680 nm with 8.0-nm bandwidth. ((A) A isomer; (0)A isomer). All samples measured in 5 mM Tris, 50 mM NaCl buffer, pH 7.4.
concentrations (high P/Ru), and surface (or minor groove) binding modes become appreciable only when all possible sites of intercalation are occupied, at low P/Ru ratios. ResonanceRaman Spectroscopy. Resonance enhanced Raman vibrational spectra for racemic Ru(bpy)z(ppz)2+ in buffer and the A and A isomers in the presence of calf thymus DNA (P/Ru = 41) using 488-nm Argon ion excitation are displayed in Figure 7. This excitation is utilized to maximize the resonance enhancement of the ppz ligand, which would be the only ligand expected to intercalate. Instrumental conditions are identical for the three spectra. Because the emission is enhanced for the complex in the presence of DNA, the signal to noise ratio is correspondingly smaller, in particular for the A isomer. Nevertheless, subtle differences are evident in the resonance enhanced spectra. In particular, the relative intensities of prominent bands vary from one spectrum to another, but no frequency changes of more than a wavenumber are observed. I
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buffer spectrum. In particular, note the 1271-cm-1shoulder on the 1256-cm-' band in the buffer spectrum. Itsintensity relative to the main peak is 0.33 in the buffer spectrum and drops to 0.50 in the A isomer spectrum and a bit lower in the A isomer spectrum. Also, a shoulder on the low frequency side of the 1491-cm-' peak in the buffer spectrum is not clearly observed in either the A or A isomer spectra. Likewise, the 1533-cm-l band decreases in intensity relative to the 1491-cm-l band in both DNA spectra (ratio of 1533 to 1491 cm-I in buffer spectrum is 1.35, drops to 1.21 for A + DNA and 0.95 for A + DNA). Several more minor bands (e.g. 1214 cm-I, 1578 cm-I) seem to be nearly absent in the A isomer spectrum. Overall, the pattern is consistent with the hypochromicity observed in the visible spectrum of both the A and A isomers in the presence of DNA. The corresponding Raman hypochromic effect would be expected to affect the intensities of only that part of the chromophore which is most strongly perturbed via the intercalative interaction with DNA base pairs. Although our knowledge of the detailed nature of the normal mode structure of the ppz ligand is not detailed enough to yield specificstructural information,it is encouragingtoobserve the differential intensity effects. Furthermore, the Raman evidence indicates that the interaction with DNA for the two isomers must be quite similar at higher P/Ru ratios.
Discussion If one examines the spectroscopic and photophysical evidence presented for either of the enantiomers of Ru(bpy)*(ppz)*+when bound to DNA, a strong case can be madel' that a mode of binding involving a variable degree of intercalation of the ppz ligand, probably within the major groove of B-form DNA, is important for each, particularly at high P/Ru ratios. In the presence of DNA, each enantiomer shows hypochromicity only in the visible MLCT absorption maximum associated with the ppz ligand, a substantial increase in emission intensity and blue shift of emission maximum, reduced quenching by ferrocyanide, and a long lifetime component of a biphasic emission decay. The I
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AWavenumber (cm-') Figure 7. Resonance Raman spectrum of the R~(bpy)2(ppz)~+ A and A enantiomers at [DNA-phosphate]/[Ru] = 41 using 488-nm Ar+ excitation and 4.0-cm-1 spectral bandwidth. [Ru] = 45 p M for all spectra: (top) A + DNA; (middle) A + DNA; (bottom) rac-Ru(bpy)2(ppz)2+ in buffer. All samples measured in 5 mM Tris, 50 mM NaCl buffer, pH 7.4.
Ru(bpy)Z(ppz)Z+ Binding with Calf Thymus DNA rwnance Raman spectra of the individual enantiomers both show similar Raman hypochromicity effects when bound to DNA. Perhaps most significantly, the emission polarization values for both isomers are the same at high P/Ru ratios, indicating a similar degreeof rigidity with a fixed orientation for the two enantiomers bound to DNA. It is further notable that these limiting ratios are achieved at relatively low P/Ru ratios (SX for the A isomer and 30X for the A isomer). All of these measurements are consistent with similar measurements reported for individual enantiomers for Ru(phen)3z+and Ru(DIP)j2+which have been interpreted as substantiating an intercalative mode of binding via major groove accessto B-form DNA. For these twocomplexes, however, intercalative binding of the A isomer is almost always favored over similar binding of the A isomer, even at high P/Ru ratios. The only exception is in binding of Ru(phen)jz+ to poly[d(AT)], where binding either was not enantioselective for the A isomer (based on polarization data) or even favored the A isomer (based on equilibrium dialysis data) at P/Ru ratios of (20-40) x . The clearest measurable (Le. quantitative) difference between the intercalative binding modes for the enantiomers of Ru(bpy)2(ppz)2+is evident from the emission enhancements (and spectral parameters) and emission lifetimes at high P Ru ratios. The A isomer shows an emission enhancement ( 5 . X) which is substantial, but only about I / 3 that of the A isomer (17.0X). The lifetime of the intercalatively bound A isomer is likewise significantly longer than in solution, but the lifetime of the A isomer is close to double that of the A isomer when both are intercalatively bound. Our findings are wholly consistent with the basic model proposed by Barton et al. for binding of tris-chelates of this type to DNA. For this particular complex, partial intercalation of a single bidentate aromatic diimine ligand (ppz) provides a significant part of the driving force for binding to B-form double stranded DNA, and van der Waals interactionswith the additional two (ancillary) bpy ligands result in a measurable degree of difference between the A and A isomers in binding within the major groove of B-form DNA. The spectroscopic and photophysical evidence indicates that the ppz ligand is more deeply inserted between DNA base pairs for the A complex than for the A one. Additional, surface bound or minor groove bound modes (both electrostatic and/or hydrophobic) are of lesser importance. Because 2,2’-bipyridine is the ancillary ligand for our complex, significant intercalation of these ligands is precluded and only the ppz ligand intercalates. Compared to 1,lo-phenanthroline, and certainly DIP, the bpy’s provide significantly less steric resistance to partial intercalation of the ppz ligand for either enantiomer. Thus, it seems that a mode of binding involving partial intercalation of ppz is highly favorable for both isomers at higher P/Ru ratios. However, chiral discrimination is still significant at P/Ru ratios of -2OX or less, because a quantitative preference for binding of the A isomer over the A isomer is still preserved via differential van der Waals interactions of the ancillary bpy’s of the two enantiomers within the DNA major groove. Our previously reported CD results, and the column chromatographic separation using DNA adsorbed on hydroxylapatite, are entirely consistent with the emission polarization
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The Journal of Physical Chemistry, Vol. 97, No. 8, 1993 1711
data, which indicate a maximal preference for the A isomer over the A-isomer of about (65:35) at a P/Ru ratio of 2.5X. Energy minimization calculations for Ru(phen)s*+binding to various polynucleotideshave indicatedI9that major groove binding with partial insertion (or intercalation) of one of the phen ligands is a favored mode of binding for both enantiomers. For the A isomer, another mode of binding, with two ligands within the major groove, but not inserted between bases, is only slightly less favored. We feel our results for Ru(bpy)*(ppz)2+are consistent with these calculations. Our report” of enantioselective cleavage of plasmid DNA utilizing a OP/Cu-like system can be interpreted in light of the above. Cleavage was significant only in the presence of the A-isomer of R~(bpy)2(ppz)~+ for experimentscarried out at P/Ru ratios of lox, where intercalative binding of the A isomer is favored over that of the A isomer (-60:40). More importantly, polarization studies show that the A isomer is almost completely intercalated within the major groove at this P/Ru ratio, while the A isomer is only 60-70% intercalated and is therefore able to deliver the active copper species to other sites, including most probably the minor groove sites determined18 to be effective in cleaving DNA in the Sigman OP/Cu system. Acknowledgment. The authors thank the PSC-CUNY Faculty Research Awards program for support of this research.
References and Notes (1) Pyle, A. M.; Barton, J. K. In Progress in Inorganic Chemistry: Bioinorgunic Chemistry; Lippard, S.J., Ed.; John Wiley & Sons: New York, 1990; Vol. 38, pp 413-475. (2) Barton. J. K. Science 1986. 233. 727-734. (3) Friedman, A. E.; Kumar, C. V.;Turro, N. J.; Barton, J. K. Nucleic Acids Res. 1991, 19, 2595-2602. (4) Pyle, A. M.; Mor& T.; Barton, J. K. J. Am. Chem. Soc. 1990, 112. 9432-9434. ( 5 ) (a) Barton, J. K.;Goldbcrg, J. M.; Kumar, C. V.; Turro, N. J. J . Am. Chem. Soc. 1986,108,2081-2088. (b) Kumar. C. V.; Barton, J. K.; Turro, N. J. J. Am. Chem. Soc. 1985, 107, 5518-5523. (6) (a) Rehmann, J. P.; Barton, J. K. Biochemistry 1990, 29, 17011709. (b) Rehmann, J. P.; Barton, J. K. Biochemistry 1990,29, 171G1717. (7) Eriksson, M.; Lejon, M.; Hiort, C.; Norden, B.; Graslund, A. J . Am. Chem. SOC.1992, 114,4933-4934. (8) Hiort, C.; Norden, B.; Rodger, R. J . Am. Chem. Soc. 1990, 112, 197 1-1 982. (9) Satyanarayana, S.; Dabrowiak, J. C.; Chaires, J. B. Biochemistry 1992.31, 9319-9324. (10) Tossi, A. B.; Kelly, J. M. Phorochem. Photobiol. 1989,5, 545-556. (11) Morgan, R. J.; Chatterjee, S.;Baker, A. D.; Strekas, T. C. Inorg. Chem. 1991.30, 2687-2692. (12) Baker, A. D.; Morgan, R. J.;Strekas, T. C. J. Am. Chem.Soc.1991, 113, 1411-1412. (13) Baker, A. D.; Morgan, R. J.; Strekas, T. C. J . Chem. Soc., Chem. Commun. 1992, 1099-1100. (14) Fuchs, Y.; Lofters, S.;Dieter, T.; Shi, W.; Morgan, R.; Strekas, T. C.; Gafney, H. D.; Baker, A. D. J . Am. Chem. SOC.1987,109,2691-2697. (15) Strekas, T. C.; Diamandopoulos, P. J . Phys. Chem. 1990,9419861991. (16) Strekas,T.C.;Gafney,H.D.;Tysoe,S.A.;Thummel,R.P.;Lefoulon, F. Inorg. Chem. 1989, 28, 2964-2967. (17) Long, E. C.; Barton, J. K. Acc. Chem. Res. 1990, 23, 273-279. (18) Sigman, D. S.;Chen, C. B. Annu. Rev. Biochem. 1990.59,207-236. (19) Haworth, I. S.;Elcock, A. H.; Freeman, J.; Rodger, A.; Richards, W. G. J . Biomol. Struct. Dyn. 1991, 9, 23-44. (20) Demas, J. N. Excired State Liferime Meusuremenrs; Academic Press: New York, 1983.
