DNA Binding Geometries of Ruthenium(II) Complexes with 1,10

J. Phys. Chem. B 1998, 102, 9583-9594

9583

DNA Binding Geometries of Ruthenium(II) Complexes with 1,10-Phenanthroline and 2,2′-Bipyridine Ligands Studied with Linear Dichroism Spectroscopy. Borderline Cases of Intercalation Per Lincoln* and Bengt Norde´ n Department of Physical Chemistry, Chalmers UniVersity of Technology, S-412 96 Gothenburg, Sweden ReceiVed: June 4, 1998; In Final Form: September 22, 1998

The DNA binding geometries for a series of enantiomerically pure substitution-inert ruthenium(II) complexes, containing 1,10-phenanthroline (phen) and 2,2′-bipyridine (bpy) as ligands, have been studied with linear dichroism (LD) spectroscopy. Based on flow LD and emission anisotropy (EA) spectra of the complexes in the presence of calf thymus DNA, their absorption spectra in the visible region have been resolved into three component spectra that are polarized nearly perpendicular to each other. Spectral similarities between the D3 symmetric [Ru(phen)3]2+ and C2 symmetric [Ru(phen)2dppz]2+ (dppz ) dipyrido[3,2-a:2′,3′-c]phenazine), in which dppz is known to be intercalated between the DNA basepairs, indicates a symmetry breaking in the former chromophore, bringing up the long debated question whether one of the three phen ligands of [Ru(phen)3]2+ may be intercalated. Indeed, the complete analysis of the flow LD spectra for [Ru(L)2Y]2+ complexes (L ) bpy or phen) reveals similar angular binding geometries whether Y ) dppz or phen. When Y ) phen, for both the ∆- and Λ-enantiomers this ligand is thus found oriented nearly perpendicular to the DNA helix axis as if intercalated and, similar to the orientation of dppz in dppz complexes, with a small but distinct clockwise rotation around the pseudo-dyad axis when viewed toward the minor groove (“positive roll”). A markedly less ordered binding observed when passing from [Ru(bpy)2phen]2+ to [Ru(bpy)3]2+ supports a stacking interaction of the phen ligand with the nucleobases. However, in contrast to the lengthening of DNA observed with [Ru(phen)2dppz]2+ and classical intercalators, the absence of lengthening with [Ru(phen)3]2+ indicates that although a phen may have stacking interaction with the nucleobases, it does not enter a fully opened intercalation pocket. Two alternative binding modes are discussed: semi-intercalation (only edge of the intercalation pocket opened) and quasi-intercalation (indenture of a basepair allowing stacking of adjacent bases with the intruding phen ligand). Fair agreement is found between the proposed binding geometry of [Ru(phen)3]2+ and previously reported 2D-NMR NOE data.

Introduction Chelate compounds of polycyclic heteroaromatics with transition metals form a class of DNA binding substances that has received considerable attention over the years, as they combine properties of traditional DNA interacting polycyclic aromatics with important photophysical and photochemical potentials offered by the metal into rigid structures spanning all three dimensions.1 A prototypical complex is the chiral [Ru(phen)3]2+ which can be resolved into inversion-stable enantiomers, ∆ and Λ (phen ) 1,10-phenanthroline).2 Early expectations that such complexes could be used for probing helical conformation of DNA, spurred by the observation that DNA binding shifted the enantiomer equilibrium of the inversion labile iron complex [Fe(phen)3]2+ to the right-handed helical ∆-configuration,3,4 have not been fulfilled, however. Both of the [Ru(phen)3]2+ enantiomers in fact promote the transition from left-handed helical Z-DNA to the normal right-handed helical B-DNA,5 and for the latter conformation the level of enantioselectivity is rather modest; at best ∆ binds to DNA with about twice the affinity of Λ.6,7 Besides binding affinity, the hydrodynamic behavior of DNA shows substantial differences between the enantiomers.6-9 The nature of the binding of [Ru(phen)3]2+ has been extensively studied and debated (reviewed in refs 8 and 10). It was early suggested that tris(phenanthroline) transition-metal complexes bind to DNA by intercalation of one phenanthroline

ligand.11 For [Ru(phen)3]2+ this notion finds support from an observed unwinding of supercoiled DNA12 and in NMR from a rather strong upfield shift of phenanthroline proton resonances upon binding to DNA oligomers.13,14 On the other hand, no decrease in the interresidue NOESY cross-peaks of the nucleobases, as would be expected for intercalation, was observed,14 and in contrast to classical intercalators such as ethidium, various techniques have failed to detect any significant lengthening of the DNA helix upon binding either enantiomer of [Ru(phen)3]2+.7,10 The [Ru(phen)3]2+ chromophore displays a strong orange color and a bright emission from a long-lived excited state, and spectrophotometric methods are well-suited to probe its binding to DNA. While titrations in linear dichroism6 or emission spectra8 indicate only one predominant binding mode for each enantiomer, measurements of luminescence emission lifetimes have given indications of heterogeneity.15,16 Barton et al. interpreted the curved Stern-Volmer plots obtained with the anionic quencher [Fe(CN)6]4- as each enantiomer having two binding modes, one intercalative and one groove bound, with ∆ preferring the former and Λ the latter mode.16 This interpretation has later been questioned and the curvature largely explained as an ionic strength effect.8 From a molecular modeling study, Haworth et al. suggested a “partially inserted” mode of binding from the major groove, in which one

10.1021/jp9824914 CCC: $15.00 © 1998 American Chemical Society Published on Web 10/30/1998

9584 J. Phys. Chem. B, Vol. 102, No. 47, 1998 phenanthroline ligand is coplanar with the basepairs, closely facing but not opening the basepair stack.17 Linear dichroism (LD) is probably the optical spectroscopic technique that shows the most drastic differences between the two metal complex enantiomers upon DNA binding. Since the LD is directly related to the orientation of the chromophore relative to the preferred orientation axis (which in these systems is the DNA helix axis), this technique was early used to study DNA binding geometry of metal complexes.3,4 On basis of electric linear dichroism data and disregarding the spectral overlap of the E-derived transitions in the visible absorption bands, Yamagishi proposed the ∆-enantiomers of [Ru(phen)3]2+ and [Ru(bpy)3]2+ to bind by intercalation to calf thymus DNA (bpy ) 2,2′-bipyridine).18 By contrast, intercalation of Λ and groove binding of ∆ were suggested by Hiort et al. at the assumption of pure A2 polarization in the red tail of the long wavelength LD band of [Ru(phen)3]2+ enantiomers bound to flow oriented DNA.6 As we shall see, however, to obtain the correct binding geometry, a global analysis of the LD spectral features, including consideration of the band overlaps between different electronic transitions, is required. By a combination of LD and photoselection anisotropy, we recently resolved the entire absorption spectrum >300 nm of the [Ru(phen)2dppz]2+ complex when bound to DNA (dppz ) dipyrido[3,2-a:2′,3′-c]phenazine).19 Both enantiomers of this complex were concluded to have the dppz ligand intercalated and, furthermore, to show a similar, distinct deviation from an idealized B-DNA intercalative geometry, characterized by a clockwise roll of the intercalated dppz ligand around its 2-fold axis by approximately 10°. The spectral overlap between differently polarized transitions was found to be extensive over the whole range of wavelengths in the visible absorption spectrum. These results prompted us to reconsider our previous conclusions regarding the binding geometry of [Ru(phen)3]2+, based as they were on assumptions of idealized intercalation geometry and pure polarization in the red tail of the MLCT band.6 Here we report the decomposition of the isotropic absorption in the visible spectral range into appropriate transition moment components, by aid of linear dichroism and emission anisotropy, for both enantiomers of [Ru(phen)3]2+ when bound to calf thymus DNA. To further establish the role of the phenanthroline ligand for binding, data for the enantiomers of the [Ru(bpy)3]2+, [Ru(bpy)2phen]2+, [Ru(bpy)2dppz]2+, and the achiral trans-[Ru(phen)2(py)2]2+ complexes are also analyzed (py ) pyridine). The overall DNA binding geometries of ∆- and Λ-[Ru(phen)3]2+ are found to show strong similarities with those of the corresponding enantiomers of [Ru(phen)2dppz]2+, and evidence is found that the edge of the middle ring of one phenanthroline ligand is engaged in stacking interaction with the nucleobases. Good qualitative agreement with previous 2-D 1H NMR NOE data14 supports the concluded geometries for the respective enantiomers and indicates that this phenanthroline ligand is partly sandwiched between basepairs in a binding mode that does not involve full opening of an intercalation slot between adjacent basepairs. This stacking interaction can be either due to the wedging of the phen ligand into an opening at the edge of the basepair stack (semi-intercalation) or due to its insertion into the crevice created by the effective indenture of one basepair (quasi-intercalation). Experimental Section Chemicals. Since the DNA binding of ruthenium complexes with smaller ligands is weak and strongly ionic strength

Lincoln and Norde´n dependent,7,8,12c experiments were performed in a low salt buffer of 5 mM NaCl/1 mM sodium cacodylate of pH 7 in triple deionized (Milli-Q) water. Calf thymus DNA (Sigma) was dissolved in buffer to a concentration of about 3 mM nucleotides and filtered twice through a 0.8 µm Millipore filter. Racemic [Ru(phen)3]Cl2 and [Ru(bpy)2phen]Cl2 were synthesized by standard methods2,20,21 and resolved by recrystallization of arsenyltartrate salts (with arsenyltartrate prepared22 from natural L-tartaric acid for Λ and unnatural D-tartaric acid for ∆) from dimethyl sulfoxide/water23 until constant optical activity, as measured by CD, was obtained. Racemic [Ru(bpy)3]Cl2 was obtained from Sigma and resolved by fractional crystallization of the diastereomeric arsenyl-L-tartrate salts from water. Oxidation of optically pure [Ru(bpy)2phen]Cl2 enantiomer under Gillard conditions (H2SO4, HNO3, Br2)24 afforded ∆- or Λ-[Ru(bpy)2(1,10-phenanthroline-5,6-dione)]2+, isolated as the PF6 salt by substituting the perchlorate in the original description with NH4PF6. This quinone complex was condensed with ophenylenediamine in acetonitrile to afford ∆- and Λ-[Ru(bpy)2dppz]2+, analogous to the synthesis of the corresponding phen complex, and purified by chromatography on alumina with acetonitrile as eluent.23,24 The very light-sensitive complex trans-[Ru(phen)2(py)2](PF6)2 was prepared according to the literature25 and was always handled under subdued light conditions while in solution. The hexafluorophosphate salts of all complexes were recrystallized from acetonitrile/ethanol. In cases where the solubility in buffer of the hexafluorophosphate salt was insufficient, the readily soluble chloride salt was prepared by precipitating a solution of the hexafluorophosphate salt in acetone with tetra-n-butylammonium chloride. The UVvis spectra were in agreement with the literature. Concentrations were determined spectrophotometrically with the use of the following extinction coefficients (in units M-1 cm-1): ct DNA (per nucleotide): 6.600 (258 nm);26 [Ru(bpy)3]2+ 14.600 (452 nm);27 [Ru(bpy)2phen]2+ 16.000 (452 nm);12b [Ru(phen)3]2+ 19.000 (447 nm);27 [Ru(bpy)2dppz]2+ 16.100 (444 nm);28 trans[Ru(phen)2(py)2]2+ 12.000 (488 nm).25 Spectroscopy. Absorption, linear dichroism, and luminescence excitation spectra were measured with a spectral bandwidth of 2 nm for the incoming light. The spectra were recorded with one intensity reading per nanometer in the form of column matrices, denoted here by bold lowercase letters, e.g., aiso for an isotropic absorption spectrum and ld for a linear dichroism spectrum, with the wavelength dimension understood. The MATLAB software (The MathWorks, Inc.) was used for data treatment and analysis. (a) Isotropic Absorption. Isotropic absorption spectra were measured on a Cary 4 spectrophotometer in a 0.5 cm cell. The isotropic absorption spectrum will here be treated as a sum of absorption bands of different polarization, the bands of a common polarization i in the cromophore coordinate system being collected into a component spectrum pi:

aiso )

∑pi

(1)

(b) Emission Anisotropy. Samples of ruthenium complexDNA adducts (P/Ru ) 50, [DNA] ) 1 mM nucleotides) in sucrose-saturated buffer were prepared as previously described.19 Polarized excitation spectra i were recorded on a SPEX Fluorolog τ2 spectrofluorimeter in a liquid nitrogen cryostat (Oxford Instruments). The emission was measured at 590 nm, with a slit width of 15 nm, and calcite polarizers (Glan) were used for both the excitation and emission light. The anisotropy of the emitted light was measured as a function of excitation wavelength, and the emission anisotropy (ea) was calculated

DNA Binding Geometries of Ru(II) Complexes

J. Phys. Chem. B, Vol. 102, No. 47, 1998 9585

as29

ea ) (iVV - iVH(c))/(iVV + 2iVH(c))

(2)

where the first subscript indicates the position of the excitation polarizer and the second the position of the emission polarizer as vertical or horizontal. The photomultiplier-response-corrected excitation spectrum iVH(c) was calculated as iVH(c) ) iVH(iHV/ iHH). For an immobile chromophore, the anisotropy a is related to the angle χij between the absorbing transition dipole moment i and the emitting transition dipole moment j according to29

aij ) (1/5)(3 cos2 χij - 1)

(3)

The emission anisotropy over the absorption band originating from a single electronic (vibronic) transition is independent of excitation wavelength. However, the net anisotropy eaj, measured on an emisson band of j polarization, often shows an excitation wavelength dependence as a result of overlap of differently polarized transitions, being the average of the anisotropies of the individual transitions weighted by their respective absorptions:

eaj )

(4)

(c) Linear Dichroism. The linear dichroism (LD) spectra of the ruthenium complexes in the prescence of calf thymus DNA in buffer (1-2 mM as nucleotide phosphate, P/Ru ) 30 unless otherwise indicated) were measured on a Jasco J-500 spectrodichrometer equipped and used as described elsewhere.30 The DNA was oriented by a flow gradient of 1800 s-1 in a Couette cell with an outer rotating cylinder.31 Samples were run in pairs with or without 10 µM of the intercalating dye methylene blue as internal orientation reference.6,19 The linear dichroism is a differential absorption spectrum, obtained with light with the plane of polarization parallel or perpendicular to the macroscopic orientation direction of the system, and can be understood as a weighted sum of the differently polarized components of the isotropic absorption spectrum (eq 1):31

∑wipi

(5)

The reduced linear dichroism is formed by dividing the linear dichroism with the isotropic absorption of the sample:31

ldr ) ld/aiso )

[aiso(∆) aiso(Λ) ld(∆) ld(Λ)] )

[

1 0 wA(∆) 0 wA(Λ) 0 1 0 [pA(∆) pA(Λ) pBE pBA2] 1 1 wBE(∆) wBE(Λ) or

S ) EC

]

1 1 wBA2(∆) wBA2(Λ)

(8)

As long as the coefficient matrix C is nonsingular, the component spectra pi can be obtained by multiplying the experimental spectra with the inverse:

E ) SC-1

(9)

Results

∑aijpi/∑pi

ld ) aparallel - aperpendicular )

absorption spectra corresponding to the latter two directions can be taken to be common to an enantiomeric pair. Taking the experimental spectra and the component spectra p as columns of two matrices, S and E, eqs 1 and 5 can then be expressed as

∑wipi/∑pi

(6)

Similar to the emission anisotropy, the reduced linear dichroism over the absorption band originating from a single electronic (vibronic) transition is independent of wavelength. The expression for the weight wi, i.e., the reduced linear dichroism value for an electronic transition i, whose transition moment makes an angle θi to the DNA helix axis, is similar to that for the emission anisotropy:31

wi ) LDri ) S(3/2)(3 cos2 θi - 1) 0 e S e 1

(7)

The orientation factor S denotes the degree of orientation of the DNA in the flow field and was determined from the LDr of the 675 nm band of the intercalated reference methylene blue, assumed to be perpendiculary oriented relative to the DNA helix axis, i.e., S ) -LDr(675 nm)/1.5.19,32 (d) Resolution of Spectral Components. We here assume (vide infra) that the major electric transition moments of the trigonal ruthenium complexes in this study are polarized along either the A, the BE, or the BA2 direction and that the component

Isotropic Absorption and Linear Dichroism. Linear dichroism (LD) spectra for the various pairs of enantiomers of ruthenium tris-chelates [Ru(L)2Y]2+, with L ) phen or bpy and Y ) dppz or phen, bound to flow-oriented calf thymus DNA, are shown together with corresponding isotropic absorption spectra in Figure 1a-d. Parts e and f of Figure 1 show the corresponding spectra for the enantiomers of [Ru(bpy)3]2+ and the achiral trans-[Ru(phen)2(py)2]2+, respectively. All spectra are normalized to unit concentration, and LD is normalized to perfect DNA orientation (S ) 1 in eq 7). The value of S/S0, the orientation of the DNA helix with bound metal complex relative to that of free DNA under the same conditions, is given in Table 1. It can be seen that the ∆-enantiomers generally give lower values of S/S0 than the corresponding Λ-enantiomers and that complexes containing a large ligand, like dppz, generally tend to improve the DNA orientation while complexes with only smaller ligands generally impair it significantly. Reported DNA binding constants for [Ru(bpy)3]2+ and [Ru(bpy)2phen]2+ show that they are rather low in moderate ionic strength buffer (K < 103 M-1 in 50 mM NaCl).12c,33 To estimate the fraction of free metal complex under our low-salt conditions, samples with DNA and metal complex used for the spectroscopic measurements were titrated with salt, and the absorption changes were analyzed with Record-Manning theory (not shown).34 The fraction of free complex in the samples, and binding constants extrapolated to 50 mM NaCl, were found to be 5% for Λ-[Ru(bpy)3]2+ (K ) 400 M-1), 450 nm.19 A least-squares projection in this wavelength range of the LD spectra onto the EA data, weighted with the isotropic absorption, provided estimates for the apparent LDri values of the dominating A and BE transitions. For the [Ru(phen)3]2+ enantiomers, using an S value determined from the LD of intercalated methylene blue, the average of the most negative apparent LDri value was found to be -2.1 ( 0.2, which is close to the range of values found with the intercalating [Ru(phen)2dppz]2+and [Ru(phen)2bdppz]2+ complexes.19 Since an LDr value cannot be less than -1.5, the corresponding transitions were assumed to be perpendicular to the helix axis and LD spectra accordingly rescaled with a factor 1.5/2.1. LDr values more negative than the DNA bases are occasionally observed for intercalating dyes, indicating a corresponding inclination of the nucleobases (usually 15-20°) away from perpendicularity.31 The large negative amplitude of the apparent LDri value as estimated from EA could to some extent be due to depolarization of the emission also for [Ru(phen)3]2+, caused by incomplete binding to the DNA. Although the binding is practically quantitative for the present concentrations and ionic strength conditions at room temperature in solution, the effect of high sucrose concentration and low temperature could be expected to lower the binding affinity as effects of respectively hydrophobic interactions and potentially negative enthalpy of bindning.8,40 In saturated sucrose solution at -3 °C in the absence of DNA, the emission of [Ru(phen)3]2+ is almost completely depolarized due to its long excited-state lifetime (data not shown). Resolution of Absorption Matrix. The coefficient matrix C was calculated according to eqs 7 and 8 with trial values of θBE for ∆ and for Λ. θA was set to 90°, and θBA2 was taken to be equal to θBE ( 80° (+ for ∆ and - for Λ), 80° being the average between 90° (three unperturbed identical ligands) and 71° (two identical ligands uncoupled from the third). Since the BA2 transitions make relatively minor contributions to the

DNA Binding Geometries of Ru(II) Complexes absorption spectrum, the uncertainity of this angle did not significantly affect the final geometries. The matrix S containing the isotropic absorption and the linear dichroism spectra (the latter rescaled with the same factor 1.5/2.1, see above), for the two enantiomers, was then multiplied with the inverse of the coefficient matrix C according to eq 9, and the resulting absorption component spectra were examined. The angles θBE were varied to make the BA2 intensity vanish at all wavelengths >450 nm, with the additional requirement that all absorption components be nonnegative within experimental uncertainity over the entire wavelength range. The angles obtained previously for [Ru(phen)2dppz]2+ 19 were used as initial guess values for [Ru(bpy)2dppz]2+. For [Ru(phen)3]2+ and [Ru(bpy)2phen]2+ initial values were obtained from the projection of LD upon EA as described above. For [Ru(bpy)3]2+ we assumed, on basis of the markedly reduced orientational degree, and in order to test the possibility of some intercalation, that the bulk of bound metal complex could be partitioned in one randomly oriented fraction with zero LD and one oriented fraction with θA ) 90°. Then we tried to optimize the similarity of the BA2 and BE components to those obtained for [Ru(bpy)2phen]2+. Due to the well-separated MLCT bands for charge transfer to phenanthroline and pyridine, the isotropic absorption and LD spectrum of trans[Ru(phen)2(py)2] could straightforwardly be resolved with a 2 × 2 coefficient matrix according to eq 9 (the TEM method41) into two absorption components of different polarization (data not shown). The crystal structure of this complex shows the phenanthroline ligands to be coplanar, but due to steric clash of the 2- and 9-hydrogens, slightly translated relative to each other and the 2-fold axis of the pyridines somewhat tilted from being normal to the phenanthroline plane.25 Assuming absorption intensity exclusively polarized in the direction metal to ligand, the angle of the phenanthroline C2 axis to the DNA helix axis was estimated to be 90° and the angle of the pyridine C2 axis to be 20 ( 10°. The consistency of the spectral resolutions is excellent: the respective BA2 and BE components of [Ru(phen)2dppz]2+and [Ru(phen)3]2+ are almost identical as shown in Figure 4a, despite the different overall shapes of the absorption and LD spectra of the two complexes. The same holds for L ) bpy (Figure 4b); also, the A components for [Ru(phen)3]2+ and [Ru(bpy)2phen]2+are seen to be very similar. By contrast, the A component of [Ru(bpy)3]2+ is quite different as shown in Figure 4c. The angular parameters θBE are summarized in Table 1 and show a surprisingly small range of freedom for each sense of chirality: 40-47° for the ∆- and 22-28° for the Λ-enantiomers. Binding Geometry and the Roll Angle. Since LD depends on the squared cosine of the angle between the transition moment and the orientation axis, additional data are required for the determination of the sign of the angle. For the [Ru(phen)2dppz]2+ complex, identification of a dppz short-axis polarized π f π* transition with negative LDr, together with unequivocal evidence that the dppz ligand is indeed pointing toward and not away from DNA, allowed us to assign unique angular orientations for the two enantiomers of this complex.19 In this way a deviation from an idealized B-DNA intercalated geometry was found, which may be described as a rotation of the intercalating ligand around its perpendiculary oriented 2-fold axis, denoted as a roll. The roll angle β is defined as a clockwise rotation (viewed from the ligand toward DNA) of the molecular plane of the intercalating ligand from the plane perpendicular to the helix axis. For ∆- and Λ-[Ru(phen)2dppz]2+ the roll angles +7 ( 2° and +13 ( 5° were found with calf thymus DNA (Table 1).19 For ∆- and Λ-[Ru(phen)2bdppz]2+

J. Phys. Chem. B, Vol. 102, No. 47, 1998 9589

a

b

c

Figure 4. Component spectra corresponding to the calculated angular parameters given in Table 1: (a) the BE (intense spectra) and BA2 polarized components of [Ru(phen)3]2+ and [Ru(phen)2dppz]2+; (b) polarized components of [Ru(phen)3]2+ (A), [Ru(bpy)2phen]2+ (A, BE, BA2), and [Ru(bpy)2dppz]2+ (BE, BA2); (c) the A, BE, and BA2 polarized components of [Ru(bpy)3]2+ and [Ru(bpy)2phen]2+.

very similar roll angles within a small range (∆, +5° to +12°; Λ, +7° to +10°) were found for calf thymus DNA and alternating poly[dG-dC] and poly[dA-dT] duplexes,19 as well as for the triplex poly[dT]-poly[dA]-poly[dT] (bdppz ) benzodipyrido[b:3,2-h:2′,3′-j]phenazine).42 The values for the roll angle (β ) 7° and β ) +11°) for ∆- and Λ-enantiomers of [Ru(bpy)2dppz]2+ nicely fit this picture, giving an average roll of +9 ( 4° for all three pairs of truly intercalating dppz and bdppz Ru complex enantiomers. However, also for the enantiomers of the two smaller, possibly nonintercalated, complexes [Ru(bpy)2phen]2+ and [Ru(phen)3]2+, one set of solutions fit into this narrow range of roll angle values. The angular geometry as determined by linear dichroism is thus

9590 J. Phys. Chem. B, Vol. 102, No. 47, 1998 completely consistent with intercalation for the latter two enantiomeric pairs, and even with [Ru(bpy)3]2+ the data can be interpreted this way for a certain fraction of the DNA-associated metal complex ions (Table 1). However, for these more symmetrical compounds we have no clear “indicator” transitions of unequivocal polarization like those for the dppz complexes, and therefore several alternative angular binding geometries result from the spectral analysis. We shall now examine them in turn to see whether other binding geometries offer plausible interpretation of data. With the information from EA that the E degeneracy is broken, we can unambiguously determine only the (unsigned) angle of the (pseudo) C3 axis of the enantiomers of the two complexes relative to the helix axis: |θC3| ) 44 ( 2° for ∆ and |θC3| ) 66 ( 2° for Λ. The C3 direction is orthogonal to both the A and the BE directions, which we can determine, although not yet assign which is which. Comparison of the emission anisotropy (Figure 3) with the difference absorption spectra [bound] - [free] for [Ru(bpy)2phen]2+ and [Ru(phen)3]2+ (Figure 2a) shows that the maximum in EA coincides with the maximum in DNA perturbation (red shift) of the spectrum at about 480 nm, suggesting that if the emission is A polarized, one ligand is facing the DNA (case 1), or, if the emission is BE polarized, two ligands are symmetrically facing the DNA (case 2). This conclusion is supported by the observation of a substantial decrease in the oxygen quenching rate constant for [Ru(bpy)3]2+ and [Ru(phen)3]2+ upon binding to DNA,15,43 indicating that the ligand(s) that has an anion radical character in the excited state is shielded by the DNA. Let us for a moment assume that case 2 holds, i.e., that the emission is BE polarized and the two ligands face the DNA in a relatively symmetric fashion. The assignment in Table 1 of θA and θBE has then to be reversed for these two compounds, i.e., θBE ) 90°, and the axis joining the two ligands (bpy or phen) that lie closest to the DNA should be oriented perpendicular to the helix axis. The third ligand has then to be tilted away from the DNA to point toward the bulk solution by an angle 22-47°. This arrangement clearly can be dismissed on electrostatic grounds, so the tentative assignment of the emission as A polarized (case 1) is confirmed. With this assignment, the 2-fold axis of the single ligand close to the DNA will be perpendicular to the helix axis, and from symmetry arguments this axis is likely to be more or less parallel to the pseudo-dyad symmetry axis of DNA.6 Two orientations, depending on what sign is chosen for θBE, are then possible for each enantiomer: they are characterized by different roll angles β (Table 1). The observation by 2D 1H NMR of intermolecular NOE contacts exclusively between oligomer protons located in the minor groove, and the phenanthroline protons for both enantiomers of [Ru(phen)3]2+, bound to the DNA duplex 5′d(CGCGATCGCG), strongly suggest the complex to be accommodated in the minor groove.14 However, neither of the roll angles obtained from the spectroscopical analysis are close to the range of values typically found for minor groove binders (β ) -40 to -50°),31 and therefore “slotting” one ligand along the groove can be excluded. For the ∆-enantiomers, β ) -80° would place the long axis of the innermost ligand (phen or bpy) almost parallel to the helix axis, and β ) +60° would place it nearly perpendicular to the direction of the groove for the Λ-enantiomers, both arrangements seeming very unlikely for steric reasons. This conclusion is further supported by the unambiguous interpretation of the LD spectrum of the steric mimic trans-[Ru(phen)2(py)2]2+, which clearly demonstrates that the phenanthroline ligands here are approximately coplanar to the basepairs. Although θBE ≈ +45° for the ∆-enantiomers

Lincoln and Norde´n would be consistent with the symmetrical insertion of two ligands into the groove, in the excited state the ligand anion radical would protrude into the bulk solution, which is inconsistent with the observed large decrease in the rate of oxygen quenching.15,43 We can thus conclude that a “dppz-like” angular geometry for both [Ru(bpy)2phen]2+ and [Ru(phen)3]2+ bound to DNA, with positive roll angles of 9-13°, is by far the more probable of the binding geometries consistent with the LD and EA data. The similarity of the B absorption components between Y ) dppz and Y ) phen in both the L ) phen and L ) bpy series also strongly suggests that the phen ligand is the innermost ligand when [Ru(bpy)2phen]2+ is bound to DNA, a conclusion further supported by the similarity of the A absorption components between [Ru(bpy)2phen]2+ and [Ru(phen)3]2+ in contrast to the quite dissimilar A component of [Ru(bpy)3]2+. Discussion A Single Binding Geometry. Very similar LD spectra of [Ru(phen)3]2+ with [poly(dA-dT)]2 and [poly(dG-dC)]2, as compared to that of calf thymus DNA at a wide range of binding ratios,6 suggest that the unique binding geometry for each enantiomer is a general feature, essentially independent of DNA sequence and complex binding density, as found previously for the dppz and bdppz complexes.19,23,42 The biexponential luminescence decays reported for ∆- and Λ-[Ru(phen)3]2+ bound to calf thymus DNA under low-salt conditions,15 where the binding can be assumed to be practically quantitative, are thus likely to arise from complexes with the same binding geometry but differing environments. Sources of environmental heterogeneity could be for example the sequence heterogeneity of natural DNA or the presence or absence of bound complex neighbors.23 However, the emission anisotropy will mainly reflect the properties of the longer-lived species, which accounts for >80% of the steady-state luminescence intensity of ∆-[Ru(phen)3]2+ bound to calf thymus DNA.15 Binding Geometry Consistent with Intercalation of One Phen Ligand. The present results clarify several aspects of the debated question of the possibly intercalative DNA binding of the ∆- and Λ-enantiomers of [Ru(phen)3]2+. To begin with, our earlier evidence6 that the ∆-enantiomer could not be intercalated, based on the angle θC3 between the C3 axis of the complex and the DNA helix axis, and the assumption of pure A2 polarization in the red tail of the MLCT band, is invalid since that polarization assignment was incorrect. We calculate instead from the present results θC3 ) 43° for ∆- and θC3 ) 64° for Λ-[Ru(phen)3]2+. These values practically coincide with θC3 values found for the indisputably intercalating dppz and bdppz ruthenium complexes. However, the most important conclusions come from the other two angles that the detailed LD analysis provides. Thus, the resolution of the total absorption envelope and LD analysis of both the A and BE transition moment directions show nearly the same orientations for a given enantiomeric form of [Ru(L)2Y]2+ in the DNA complex, irrespective of whether Y ) phen or dppz or L ) phen or bpy, represented by the two structures modeled for ∆- and Λ-[Ru(phen)3]2+ in Figure 5. The protection of the excited-state ligand anion radical from oxygen quenching indicates that this ligand is directed inward the DNA, as is also supported by the observation of coincidence of the spectral perturbation of the MLCT band upon DNA binding and the observation of maximum emission anisotropy. In the case of [Ru(bpy)2phen]2+, examination of the resolved polarized component spectra suggests that also in this case the innermost ligand is the phenanthroline. Thus, the binding geometry of [Ru(phen)3]2+

DNA Binding Geometries of Ru(II) Complexes

J. Phys. Chem. B, Vol. 102, No. 47, 1998 9591 TABLE 2: Free Energies of Ruthenium Complexes Binding to DNA ∆G°/kJ mol-1 a compound

racemate

[Ru(bpy)3 [Ru(bpy)2phen]2+ [Ru(phen)3]2+

-15.9b,c

[Ru(bpy)2dppz]2+ [Ru(phen)2dppz]2+

-37.1b

]2+

∆

Λ

-22.3e

-14.6d -18.9d -22.4d -22.6e

-36.5f

-34.9f

-15.9c -20.7b

Standard free energy values refer to calf thymus DNA at 20 °C in 50 mM NaCl, 5 mM Tris buffer, pH 7.1, unless otherwise noted. b 50 mM potassium phosphate buffer, pH 7, calculated from values of ref 33. c Calculated from values of ref 12c. d This work, extrapolated to 50 mM NaCl. e Calculated from values of ref 7. f Calculated from values of ref 40. a

Figure 5. Schematic model of the proposed binding geometry for [Ru(phen)3]2+: ∆-enantiomer (left) and Λ-enantiomer (right), bound to [5′d(CGCGATCGCG)]2. The sugar protons that give strong intermolecular NOE cross-peaks14 to metal complex protons are highlighted in black together with their corresponding carbon atoms. These NOE contacts are (the number of the phenanthroline proton in parentheses, the numbering scheme employed is shown below) for ∆ [H4′-C7 (2)] and for Λ [H4′-C7 (5), H4′-T6 (5, 4), H1′-A5 (3, 2)].14 The models were generated by energy minimization with the Amber force field of the appropriately docked complexes using the HYPERCHEM software package (Hypercube Inc.).

as well as of [Ru(bpy)2phen]2+ seems to be perfectly consistent with the intercalation of one phen ligand for both the ∆- and Λ-enantiomers. This result does not constitute a proof for intercalation, though, since LD reflects only to the angular orientation of the chromophore relative to the DNA helix axis and does not provide any information about the distance between the chromophore and the DNA. The positive roll angles, as well as the overall binding geometries, are thus found to be general features of the [Ru(L)2Y]2+ complexes irrespective of DNA sequence, enantiomeric form. or whether Y is phen, dppz, or bdppz or L is bpy or phen.19,23,42 Although the origin of the positive roll angle could be the rotation of the complex alone, enforced by steric interactions of the ancillary L ligands with the groove walls, the generality of the phenomenon suggests that the roll rather reflects a corresponding local (induced by binding) or global (inherent) tilt of the DNA bases. Neither X-ray crystal nor NMR data on B-form DNA have revealed a positive tilt of the basepairs as a general feature inherent of the normal DNA structure. However, the X-ray structure of actinomycin-D, bound from the minor groove into the center of a DNA oligomer, indicates a significant positive roll of the intercalated phenoxazone moiety.44 An interesting observation is that the disposition in the groove of the two cyclic peptides of the drug bears a striking resemblance to the disposition of the two anxillary ligands L for the ∆-enantiomers in the model proposed here. Semi-Intercalation or Quasi-Intercalation? Most experimental evidence suggests that the trigonal ruthenium complexes are preferentially located in the minor groove,14,42,45,46 although

there have been suggestions about major groove binding, too.13,16,17,47 The free energy of binding to DNA for [Ru(phen)3]2+ is dominated by electrostatic contributions.7,8 The positioning of the center of charge of this bulky cation at the position with the most electronegative potential, i.e., the mouth of the minor groove, is thus likely to be an important determinant for the binding geometry. Molecular modeling suggests that, in order to accomplish this, not only has the minor groove to be widened but the steric clash of the inner ligand(s) with the nucleobases has to be resolved. A binding orientation in which one ligand is coplanar with the nucleobases and confronted with the floor of the minor groove, as concluded from the present spectroscopic results, would require a conformational change of the basepair stack. Indeed, comparing the magnitude of the linear dichroism of [Ru(bpy)3]2+ with that of [Ru(bpy)2phen]2+ shows that a dramatic increase in the orientation of the chromophore is accompanying the exchange of one bpy ligand for phen. Thus, the observed similarities in binding geometries between the same enantiomers of [Ru(bpy)2phen]2+ and [Ru(bpy)2dppz]2+ is probably not just a coincidence of the preferred orientation of the two ancillary bpy ligands and the preferred orientation of the intercalated dppz ligand, but determined largely by the phen ligand or rather by the -CHdCH- part of the phenanthroline B-ring, being forced into coplanarity with the nucleobases. The finding that the perturbation of the MLCT band of the [Ru(L)2Y]2+ complexes upon binding to DNA is of comparable magnitude for Y ) phen or dppz (Figure 2) taken together with the virtually identical angular orientation (Table 1) suggests that the interactions of the Ru(L)22+ moiety with the groove is also similar in the two series and that differences in binding thermodynamics should reflect mainly the differences in the interactions of the Y ligand. The DNA binding free energies for some of the complexes at moderate (50 mM NaCl) ionic strength are given in Table 2. Upon passing from [Ru(bpy)3]2+ to [Ru(phen)3]2+, ∆(∆G°)I ) -6 ( 1 kJ mol-1, while on passing from [Ru(bpy)3]2+ to [Ru(bpy)2dppz]2+ or [Ru(phen)2dppz]2+, ∆(∆G°)II ) -20 ( 1 kJ mol-1. If the stacking interaction of the inner phen ligand were to account for the whole of ∆(∆G°)I, the entropic contribution from the 3-fold symmetry, -RT(ln 3) ) -2.7 kJ mol-1, has to be subtracted to yield the free energy contribution for the stacking of one phenanthroline to -3 ( 1 kJ mol-1, which is close to the value calculated with the extrapolated binding constant for Λ-[Ru(bpy)2phen]2+ obtained in this study. This small value, compared to the free energy of intercalation of the dppz ligand of -20 ( 1 kJ mol-1, suggests a rather limited stacking interaction of the phenanthroline ligand with the nucleobases. Also supporting the notion of a small

9592 J. Phys. Chem. B, Vol. 102, No. 47, 1998

Figure 6. Definition of the concepts of intercalation (a), semiintercalation (b), and quasi-intercalation (c).

degree of stacking interaction are fluorescence energy-transfer experiments that show contact energy transfer from the DNA nucleobases to both of the enantiomers of [Ru(phen)2dppz]2+ but not to any detectable extent to any of the enantiomers of [Ru(phen)3]2+.40 The most simple explanation of the similarity in roll angles observed would be that the conformation adopted by the DNA is the same whether Y ) dppz or phen, i.e., that the phen complexes are intercalated with only the edge of the phenanthroline B-ring inserted into an intercalation pocket virtually identical to that formed with dppz. This situation is schematically depicted in Figure 6a. However, several observations indicate that the DNA conformation is differently affected by the binding of complexes with Y ) phen compared to dppz. In the present work, we note that the orientation of the DNA in the flow field (S/S0 < 1) is lowered upon binding of complexes with Y ) phen at low binding densities while it is marginally increased when Y ) dppz (S/S0 > 1). The classical model of intercalation predicts the DNA to be lengthened by the insertion of a planar aromatic moiety into the basepair stack. In contrast to the prototypical intercalator ethidium, or in particular to either enantiomer of [Ru(phen)2dppz]2+, [Ru(phen)3]2+ does not increase the viscosity of solutions of short DNA rods (the viscosity actually decreasing with the ∆-enantiomer), indicating that there is no lengthening of DNA.7,40 Further, the contour length of DNA as recently measured with scanning force microscopy (SFM) is not changed upon binding of any of the [Ru(phen)3]2+ enantiomers, while ethidium and [Ru(tpy)(dppz)(H2O)]2+ gave the contour length increase theoretically expected for intercalative binding.10 These observations indicate that neither of the enantiomers of [Ru(phen)3]2+ induce a classical intercalation pocket, in contrast to [Ru(phen)2dppz]2+, which clearly can be considered a classical DNA intercalator. With regard to the high degree of freedom and variability in DNA conformation, in the absence of data of higher structural resolution, we can but speculate about the way the base pair stack could adapt to the intrusion of the phenanthroline edge. The effects of binding of the ∆-enantiomer of [Ru(phen)3]2+ on DNA orientation,6 on viscosity of short rodlike DNA,7,8 and on the electrophoretic properties of DNA,9 which all indicate an increase in the DNA flexibility, have been proposed to be due to a kink, or bend, in the resulting DNA adduct.7,9 As suggested by Kapicak and Gabbay,48 a bend of the helix axis of DNA is expected to arise in the case of partial intercalation due to the collapse of the classical intercalation pocket. For ∆-[Ru(phen)3]2+, the decreased DNA viscosity was interpreted as the edge of the phenanthroline B-ring wedging an opening between the basepairs upon binding.7 We denote this binding

Lincoln and Norde´n model for the phen complexes semi-intercalation to distinguish it from the partial insertion between fully separated basepairs, as illustrated in Figure 6b. However, also ∆-[Ru(phen)2dppz]2+ gave an indication of increased DNA flexibility or bending as measured by gel electrophoresis (Gisselfa¨lt, unpublished results), and we find always a slightly smaller degree of flow orientation of DNA (S/S0) for ∆- compared to Λ-enantiomers (Table 1), which suggests that also the ancillary ligands L somehow are involved in the bending of DNA. Thus, the kink inferred for the ∆-enantiomer of [Ru(phen)3]2+ cannot alone be taken as evidence for semi-intercalation. An alternative mechanism to be considered to explain the coplanar alignment of the phenanthroline ligand with the nucleobases could be that the edge of the phenanthroline B-ring pushes one base pair toward the major groove and thus achieves some stacking overlap with the neighboring base pairs. This binding mode, which we shall denote as quasi-intercalation, would not require separation of bases but is expected give a strong aligning effect of the entering aromatic plane by steric interaction with the two basepairs above and below the laterally displaced basepair even for small degrees of displacement and stacking overlap (Figure 6c). This aligning mechanism could further offer an explanation to the observation that [Ru(bpy)3]2+ has an average angular binding geometry consistent with the same binding geometry as [Ru(phen)3]2+ for the fraction of the complexes (30-40%) that are nonrandomly oriented (Table 1). The binding of the ∆- and Λ-[Ru(phen)3]2+ enantiomers to the DNA duplex 5′-d(CGCGATCGCG) has been studied with 2D 1H NMR.14 Both enantiomers of this complex were found to be in the fast exchange regime at room temperature with a preferred site of occupancy at the central A-T basepairs in the minor groove, in contrast to the intermediate exchange found with the more strongly binding [Ru(phen)2dppz]2+ complex.14 The ∆- and Λ-enantiomers of [Ru(phen)3]2+ were found to show distinctly different patterns of strong intermolecular NOE crosspeaks between phenanthroline and sugar protons, whereas between the phenanthroline and nucleobase protons similar intermolecular NOE cross-peak patterns were found. Examination of a simple molecular model, constructed to be consistent with the optical spectroscopy data, shows that the diastereomeric arrangement of the two outer phenanthrolines offers a good qualitative explanation for the differences in the cross-peak patterns observed for the two enantiomers (Figure 5). The only intermolecular NOE cross-peaks found between phenanthroline and nucleobase protons were between H2-A(5) and phenanthroline H2 (medium intensity) and H3 (low intensity). With the angular binding geometry of the complex concluded from the present spectroscopic analysis, this proximity of the protons is only possible with an at least partial stacking of an adenine on top of the innermost phenanthroline ligand. This arrangement is further supported by the fact that H2-A(5) is the proton of the oligomer that experiences by far the largest upfield shift upon binding of both enantiomers. Correspondingly, phenanthroline H5 is the most upfield shifted proton in the two enantiomers. When taking into account the fast exchange of the D3 symmetric complex,13 the magnitude of the upfield shift observed for the ∆-enantiomer (0.3 ppm) is well in accord with the largest upfield shifts found for the intercalated dpq ligand in ∆-[Ru(phen)2dpq]2+, a complex which also binds from the minor groove (dpq ) dipyrido[3,2-f:2′,3′-h]quinoxaline).45 Although the NMR data for [Ru(phen)3]2+ are in fact consistent with a stacking interaction of the edge of the innermost phenanthroline with the surrounding nucleobases, this interaction, however, does not lead to the formation of a classical

DNA Binding Geometries of Ru(II) Complexes intercalation pocket as indicated by the finding that sequential NOE cross-peaks between H6/H8 protons on G4, A5, T6, and C7 of the DNA oligomer are still observed, even at high ruthenium/oligomer ratios.14 On the other hand, the lack of any detectable nuclear Overhauser effect (NOE) between the nucleobase imino protons and the phenanthroline protons was taken as evidence against any (partial) intercalation.14 However, in view of the fast exchange rate observed for the imino protons together with the fast exchange and 3-fold symmetry of the metal complex, it seems likely that these NOE’s, if present, would be of rather small magnitude. Conclusions Using linear dichroism and emission anisotropy, the electronic absorption spectra for the DNA-bound ∆- and Λ-enantiomers of a series of ruthenium complexes related to [Ru(phen)3]2+ have been resolved, each into three nearly orthogonally polarized components, that compare well with the results obtained previously for the classically intercalating [Ru(phen)2dppz]2+ and [Ru(phen)2bdppz]2+ complexes. The angular parameters describing the binding geometries for complexes containing a phenanthroline ligand are concluded to be almost identical to those of the intercalating complexes. From the binding geometric features, and also the orientability of DNA itself by shear forces, in complexes in which chelate ligands have been systematically varied, the following has been learnt about the binding of these complexes: 1. Both ∆- and Λ-[Ru(phen)3]2+ appear to give angular binding geometries similar to those for the corresponding enantiomers of [Ru(phen)2dppz]2+ where dppz is fully intercalated. It is concluded that one phen chelate ligand of [Ru(phen)3]2+ is facing the DNA base pairs and is nearly parallel to them as if intercalated. However, the binding is not a classical intercalation between completely separated basepairs but rather semi-intercalation (wedging an opening between adjacent basepairs) or quasi-intercalation (indenture of one basepair). 2. Comparison between [Ru(bpy)2phen]2+, which shows a binding geometry similar to [Ru(phen)3]2+, and [Ru(bpy)3]2+, which shows a more unordered or averaged binding geometry, indicates that the ability to have stacking interactions with DNA bases drops markedly when passing from phen to bpy, i.e., when removing the protruding -CHdCH- group of the phenanthroline moiety. Still a certain similarity in the preferred angular geometries remains, suggesting that bpy also has a tendency to align itself parallel with the basepairs. 3. The chromophores of ∆- and Λ-enantiomers of [Ru(L)2Y]2+, whether chelate ligand Y is classically intercalated or only semi- or quasi-intercalated, in all cases show a diastereomeric relationship versus the DNA which is characterized by a positive θBE value (∼+45°) for ∆ and the two L ligands pointing along the minor groove and a negative θBE value (∼-25°) for Λ and the two L ligands pointing toward the groove walls. Generally, the ∆-enantiomer is found to impair the orientation of DNA, as seen by a smaller S/S0 ratio, compared to Λ, possibly arising from a static or dynamic bending of the helix, the effect being most pronounced with Y ) phen. 4. For all [Ru(L)2Y]2+complexes, fully intercalated as well as semi- or quasi-intercalated, both the ∆- and Λ-enantiomer display a characteristic clockwise roll of about +10°. The fact that sign and magnitude of the roll appear insensitive not only to the coordination handedness and choice of the two ancillary chelate ligands L (bpy or phen) but also to the size of inner ligand Y (dppz or phen) indicates that the roll is not primarily

J. Phys. Chem. B, Vol. 102, No. 47, 1998 9593 an effect of the interaction of the L ligands with the groove but instead a result of the interaction of the Y ligand with the basepairs. References and Notes (1) (a) Norde´n, B.; Lincoln, P.; Åkerman, B.; Tuite, E. DNA Interactions with Substitution-Inert Transition Metal Ion Complexes. Met. Ions Biol. Sys. 33; Marcel Dekker: New York, 1996; pp 177-252. (b) Stemp, E. D. A.; Barton, J. K. Electron-Transfer Between Metal-Complexes Bound to DNA-Is DNA a Wire? Ibid., pp 325-365. (c) Johann, T. W.; Barton, J. K. Philos. Trans. R. Soc. London A 1996, 354, 299-324. (d) Sigman, D. S.; Mazumder, A.; Perrin, D. M. Chem. ReV. 1993, 93, 2295. (2) Dwyer, F. P.; Gyarfas, E. C. J. Proc. R. Soc. N.S.W. 1949, 170173. (3) Norde´n, B.; Tjerneld, F. FEBS Lett. 1976, 67, 368-370. (4) Ha¨rd, T.; Norde´n, B. Biopolymers 1986, 81, 1961-1965. (5) Ha¨rd, T.; Hiort, C.; Norde´n, B. J. Biomol. Struct. Dyn. 1987, 5, 89. (6) Hiort, C.; Norde´n, B.; Rodger, A. J. Am. Chem. Soc. 1990, 112, 1971-1982. (7) Satyanarayana, S.; Dabrowiak, J. C.; Chaires, J. B. Biochemistry 1992, 31, 9319-9324. (8) Satyanarayana, S.; Dabrowiak, J. C.; Chaires, J. B. Biochemistry 1993, 32, 2573-2584. (9) Gisselfa¨lt, K. Lic. Thesis, Chalmers University of Technology, Gothenburg, 1997. (10) Coury, J. E.; Anderson, J. R.; McFail-Isom, L.; Williams, L. D.; Bottomley, L. A. J. Am. Chem. Soc. 1997, 119, 3792-3796. (11) Barton, J. K.; Dannenberg, J. J.; Raphael, A. L. J. Am. Chem. Soc. 1982, 104, 4967-4969. (12) (a) Barton, J. K.; Danishefsky, A. T.; Goldberg, J. M. J. Am. Chem. Soc. 1984, 106, 2172-2176. (b) Kelly, J. M.; Tossi, A. B.; McConnell, D. J.; OhUigı´n, C. Nucl. Acids Res. 1985, 13, 6017. (c) Pyle, A. M.; Rehmann, J. P.; Meshoyrer, R.; Kumar, C. V.; Turro, N. J.; Barton, J. K. J. Am. Chem. Soc. 1989, 111, 3051-3058. (13) (a) Rehmann, J. P.; Barton, J. K. Biochemistry 1990, 29, 17011709. (b) Rehmann, J. P.; Barton, J. K. Biochemistry 1990, 29, 17101717. (14) Eriksson, M.; Leijon, M.; Hiort, C.; Norde´n, B.; Gra¨slund, A. Biochemistry 1994, 33, 5031-5040. (15) Brodkorb, A. Dipl. Thesis, Trinity College, Dublin, 1995. (16) (a) Barton, J. K.; Goldberg, 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. (17) Haworth, I. S.; Elcock, A. H.; Freeman, J.; Rodger, A.; Richards, W. G. J. Biomol. Struct. Dyn. 1991, 9, 23-44. (18) Yamagishi, A. J. Phys. Chem. 1984, 88, 5709-5713. (19) Lincoln, P.; Broo, A.; Norde´n, B. J. Am. Chem. Soc. 1996, 118, 2644-2653. (20) Giordano, P. J.; Bock, C. R.; Wrighton, M. S. J. Am. Chem. Soc. 1978, 100, 6960. (21) Krause, R. A. Inorg. Chim. Acta 1977, 22, 209. (22) Trifonov, A.; Elenkowa, N. Z. Phys. Chem. (Leipzig) 1955, 205, 124. (23) Hiort, C.; Lincoln, P.; Norde´n, B. J. Am. Chem. Soc. 1993, 115, 3488-3454. (24) (a) Gillard, R. D.; Hill, R. E. E.; Maskill, R. J. Chem. Soc. A 1970, 1447. (b) Fees, J.; Kaim, W.; Moscherosch, M.; Matheis, W.; Klı´ma, J.; Krejcı´k, M.; Za´lisˇ, S. Inorg. Chem. 1993, 32, 166-174. (25) Bonneson, P.; Walsh, J. L.; Pennington, W. T.; Cordes, A. W.; Durham, B. Inorg. Chem. 1983, 22, 1761-1765. (26) Felsenfeld, G.; Hirschman, S. Z. J. Mol. Biol. 1965, 13, 409-419. (27) Lin, C.-T.; Bo¨ttcher, W.; Chou, M.; Creutz, C.; Sutin, N. J. Am. Chem. Soc. 1976, 98, 6536-6544. (28) Chambron, J.-C.; Sauvage, J.-P. Chem. Phys. Lett. 1991, 182, 603607. (29) Cantor, C. R.; Schimmel, P. R. Biophysical Chemistry. Part II; W. H. Freeman and Co.: San Fransisco, 1980. (30) (a) Norde´n, B.; Davidsson, Å. Tetrahedron Lett. 1972, 30, 3093. (b) Norde´n, B.; Tjerneld, F. Chem. Phys. Lett. 1977, 50, 508. (c) Norde´n, B.; Seth, S. Appl. Spectrosc. 1985, 39, 647. (31) (a) Norde´n, B.; Kubista, M.; Kurucsev, T. Q. ReV. Biophys. 1992, 25, 51. (b) Norde´n, B.; Kurucsev, T. J. Mol. Recognit. 1994, 7, 141. (32) Tuite, E.; Norde´n, B. J. Am. Chem. Soc. 1994, 116, 7548. (33) Kalsbeck, W. A.; Thorp, H. H. J. Am. Chem. Soc. 1993, 115, 71467151. (34) (a) Record, M. T.; Anderson, C. F.; Lohman, T. M. Q. ReV. Biophys. 1978, 11, 103. (b) Manning, G. S. Q. ReV. Biophys. 1978, 11, 179. (35) Broo, A.; Lincoln, P. Inorg. Chem. 1997, 36, 2544-2553.

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