The conformation and orientation of copper(II)-bleomycin intercalated with DNA

Biochimica et Biophysica A cta, 697 (1982) I 13-120 I 13 Elsevier Biomedical Press

BBA91053

THE CONFORMATION AND ORIENTATION OF COPPER(II)-BLEOMYCIN INTERCALATED WITH DNA

HOWARD SHIELDS a CHARLES McGLUMPHY a and PHILLIP J. HAMRICK, Jr. b

Department of Physics and I, Department of Chemistry, Wake Forest University, Winston-Salem, NC 27109 (U.S.A.)

(Received December 23rd, 1981)

Key words: Bleomycin; Cu(ll)-complex; DNA binding; ESR

EPR data are used to describe the conformation and identity of the atoms coordinated to Cu(II) in Cu(ll)-bleomycin bound to oriented DNA fibers. The fibers were slowly drawn from viscous solutions of Cu(ll)-bleomycin-DNA containing one Cu(II)-bleomycin to 200 basepairs. EPR measurements were made at room temperature and 90 K for different orientations of the external magnetic field with respect to the helical axes of the fibers. The g-values (g11=2.21, g ± = 2 . 0 4 ) and the hyperfine constant (All = 1 7 5 G ) are consistent with values expected for Cu(II) chelated to a square planar array of ligands. In the oriented fibers, the square planar arrays do not all have the same orientations with respect to the fiber axes. At room temperature the chelated ions have rotational freedom in which the normal to the planar array has almost complete freedom of rotation about axes perpendicular to the DNA fiber axes. The normal maintains an angle of 75 ° with respect to the axis, in the plane of the basepair, about which it rotates. Nine superhyperfine peaks on the high field side of the EPR spectrum were partially resolved. The number and splitting (12 G) of these superhypedine peaks indicate that four nitrogen atoms are chelated to Cu(II) in a square planar array. These data on Cu(II)-bleomycin bound to DNA give information on the orientation of the metal-containing portion of bleomycin which lies outside the double helix.

Introduction

Bleomycin is a mixture of closely related glyco- peptides isolated from Streptomyces verticillus as copper complexes that differ only in the sub- stituent on the bithiazole unit [1]. Metal-free bleomycin is an anti tumor antibiotic that is em- ployed for the treatment of squamous cell carcinomas, lymphomas and testicular carcinomas [2]. The interaction of bleomycin with cellular D N A is known to produce strand scission [3-5], and base removal [6].

Studies on the biological activity of bleomycin indicate that the metal-free bleomycin is activated through its binding of available metal ions, such as Cu(II) and Fe(II) [7-8]. Takahashi et al. [9] have

0167-4781/82/0000-0000/$02.75 ~ 1982 Elsevier Biomedical Press

H "OH

H N ~"- . . . . ~ ' " H

, , v oko3- Qj - bleomycin A,

Fig. l. Structure of Cu(II)-bleomycin A 2. Bleomycin B 2 has Section VII replaced by NH-(CH2)4-NH- C -NH 2.

II NH~

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suggested that the Cu(II)-bleomycin complex re- acts with organisms. However, other studies find that Cu(II) inhibits the bleomycin-induced scission of double-stranded DNA [10]. The DNA strand breaks are believed to be initiated by the interaction of 0 2 with bleomycin which has chelated Fe(II) [ 11].

A crystal structure for bleomycin with or without a chelated metal ion has not been determined. The structure of a Cu(II) polypeptide complex, called P-3A which is structurally similar to a fragment of bleomycin has been reported [ 12]. This structural study shows that the coordination atoms consist of an imidazole nitrogen, a pyrimidine nitrogen, a secondary amino group and a peptide amide nitrogen in an approximate planar array with a fifth nitrogen atom from a terminal amine in an axial position. The structure of Cu(II)- bleomycin based on this model is shown in Fig. 1. Iitake et al. [12] assume an identical Fe(II) site in bleomycin. Because of the differences between P- 3A and bleomycin, and the chelating characteristics of Cu(II) and Fe(II) this may not be a valid assumption. In fact, a spectroscopic study by Bere- man and Winkler [13] indicates that the four ligand atoms in the square plane may be three nitrogens and one oxygen. However, this study supports the structure proposed by Iitake et al.

Some features of the interaction of bleomycin with DNA are known. Linear dichroism data indicate that the bithiazole end of the molecule inter- calates with the DNA bases [14]. However, no data are available on the geometrical conformation of .the portion of the molecule which chelates the metallic ions. In this study a novel application [15-16] of EPR is used to obtain information on the orientation of the Cu(II) ligands with respect to the helical axis of DNA.

The large anisotropy in the Cu(II) EPR spectra may be used to specify the orientation of the chelated ions in single crystals or ordered samples such as liquid crystals [17]. Ordering of the Cu(II)- bleomycin complex is achieved by drawing DNA fibers from a viscous gel containing a low concentration of Cu(II)-bleomycin. The data obtained in this study give information on the conformation and orientation of the metallic ion coordination sphere which is believed to lie outside the helical structure of the DNA polymer, whereas the optical

dichroic data provide information on the portion of the molecule believed to be inserted between basepairs.

Since very little physical data are available on the intercalation of bleomycin with DNA, and since no data exist on the orientation of the metal ion coordination site with respect to the structure of the DNA polymer, this study was undertaken to better define the intercalation of bleomycin with DNA and in particular to describe the spaciai orientation of the metal ion coordination site when the Cu(lI)-bleomycin complex is bound to DNA.

Materials and Methods

Oriented DNA fibers were prepared using the technique developed by Wyckoff [18]. Approx. 200 /~1 distilled H 2 0 were added to 50 mg dry DNA in a 2 ml vial 1 cm in diameter. After the DNA was dissolved, 100 /~1 3 . 8 . 1 0 - 3 M Cu(II)-bleomycin was slowly stirred into the DNA to give one Cu(II)-bleomycin to 200 basepairs. Water was allowed to evaporate at room temperature from the DNA through the open end of the vial. When the DNA was sufficiently viscous, a 2 mm diameter stirring rod was used to draw DNA fibers approx. 1/~m in diameter from the DNA in the vial. The end of the fiber was transferred from the rod to the edge of a thin rectangular quartz plate which was rotated at 4 rev . /min with a small electric motor in such a way that the fiber formed a large number of rectangular loops 0.25 × 4.6 mm in size (see Fig. 7b).

The relative humidity was adjusted to 95% by sealing the fiber loop in the top of a quartz tube containing a saturated solution of disodium phosphate. EPR spectra at different orientations of the external magnetic field, H, with respect to the rectangular loop could be observed at room temperature by rotating this assembly in the EPR cavity. Spectra were observed at 90 K by removing the DNA fibers (at 95% relative humidity) from the quartz tube and quickly freezing in the EPR cavity with a variable temperature accessory. The fibers on the quartz plate in the variable temperature insert could be oriented with respect to H.

Other samples with a different geometry were prepared by slowly pulling single DNA fibers 10- 20/~m in diameter by hand and then cutting them

into 1.5-cm lengths before they were inserted into a 1 mm internal diameter quartz capillary. The fibers were held in place with glue at one end of the capillary. The other end of the capillary was left open so that the relative humidity could be adjusted by sealing the sample in a closed tube containing a saturated salt solution.

The Cu(II)-bleomycin was a 1:1 complex prepared by adding CuC12 to clinical blenoxane (obtained from Bristol Laboratories) dissolved in distilled water. Blenoxane is a sulfate salt containing approx. 60% bleomycin A2, 30% bleomycin B 2 and 10% other bleomycins. The blenoxane mixture was used without further purification since we were especially interested in the interaction of clinical bleomycin with DNA. A sample of 65Cu(II)- bleomycin was prepared by adding 65CUO dissolved in nitric acid to 3 . 1 0 - 3 M bleomycin. The 65CUO was dissolved in hot nitric acid and allowed to cool before adding it to the bleomycin solution. Calf thymus DNA (No. 2168 Calbiochem) was used without purification for the preparation of

115

the DNA fibers. EPR measurements were made with an E-12 Varian spectrometer.

Results

The EPR spectrum in Fig. 2A is of Cu(II)-DNA fibers (1 Cu/200 basepairs) oriented with the external magnetic field, H, parallel to the fiber axes. This spectrum is different from the spectrum of oriented or random Cu(II)-bleomycin-DNA complexes with the same Cu(II) concentration (Fig. 2B and C). The spectrum of randomly oriented Cu(II)-bleomycin-DNA (Fig. 2B), has the same characteristics as the spectrum reported earlier for a Cu(II)-bleomycin complex [19]. The noticeably different spectrum in Fig. 2C is for fibers on a quartz plate oriented in the EPR cavity with H parallel to the axes of the fibers. A comparison of Fig. 2B and C shows only a small peak in Fig. 2C at the position of the major peaks in Fig. 2A and B. The four hyperfine components in Fig. 2C are the parallel hyperfine components centered at the

Fig. 2. First derivative EPR spectra of Cu(II)-bleomycin-DNA at 90 K. A is of Cu(II)-DNA fibers oriented with the magnetic field parallel to the fiber axes, B of randomly oriented Cu(II)-bleomycin-DNA and C of Cu(II)-bleomycin-DNA fibers with the magnetic field parallel to the fiber axes.

116

• f , , ,

B . . . .

,/i E ~ J i

1-300 G A U S S ' ~

'k t ~<V t

i

/

/ / ) ,......_ _

,;

t

Fig. 3. First derivative EPR spectra observed at 90 K of Cu(II)- bleomycin-DNA fibers for the following orientations of the magnetic field with respect to the fiber axes: 0 ° in A, 22 ° in B, 45 ° in C, 67 ° in D and 90 ° in E.

gll field position. The EPR spectra from Cu(II) chelated to atoms in a square planar array are described in terms of parallel and perpendicular hyperfine components and g-values. The parallel or perpendicular components are observed when H is parallel or perpendicular, respectively, to the symmetry axis (the normal to the plane containing

A

- J \ \

\ I--- 40G ---I

/

200G --I

I 'l /

: I /

I '[ i i i ! i ', i I' <~' I ,i'

,I

Fig. 4. First derivative EPR spectra showing nitrogen superhyperfine structure for a 90 K glass of Cu(II)-bleomycin-DNA in 60% glycerine. B is the portion of A between the vertical markers.

Cu(II) and the square plane of ligating atoms). Spectra of Cu(II)-bleomycin-DNA fibers wound

on a quartz plate are shown in Fig. 3 for different orientation of the fibers with respect to H. The ratio of the long to short dimension in the rectangular loop of fibers is 18.4 to 1. Thus, when H is directed along the long dimension of the loop, Fig. 3A, 95% of the DNA is oriented with fiber axes parallel to the field and 5% of the DNA is oriented with axes perpendicular to the field. As the DNA loop is rotated in the cavity from this position to one with the field perpendicular to the long dimension, Fig. 3E, the percentages of the DNA with fiber axes parallel and perpendicular to H are reversed.

Parallel and perpendicular hyperfine components are present in the spectrum shown in Fig. 3A. However, the intensities of the parallel components are significantly greater than those of the perpendicular components. As the sample is rotated from this orientation with H parallel to 95% of the DNA fiber axes, the intensity of the perpendicular hyperfine components increases and there is a corresponding decrease in the intensity of the parallel hyperfine components.

The EPR spectra observed at room temperature for oriented fibers were weaker and not as well resolved in the high field region of the spectrum. Nevertheless, the resolution of the room temperature spectra was sufficient for making direct corre- lations between room temperature and 90 K transitions, and supports the assumption that the 90 K spectra may be used to describe room temperature structure.

The spectrum shown in Fig. 2A for oriented Cu(I I ) -DNA fibers prepared in the same way as the Cu(II)-bleomycin-DNA fibers does not con- tain Cu(II) hyperfine structure at the gH magnetic field value. The structure at the g± magnetic field value is different from that of Cu(II)-bleomycin- DNA. It is thus concluded that the Cu(lI) EPR spectra for Cu(I1)-bleomycin-DNA is for Cu(lI) chelated in bleomycin and not for Cu(lI) bound to DNA.

Poorly resolved nitrogen hyperfine structure is evident in the g± magnetic field region when the Cu(lI) of Cu(II)-bleomycin-DNA is the 65Cu iso- tope. The nitrogen hyperfine components were best resolved in a sample of 65Cu(II)-bleomycin-

117

DNA mixed with 80% glycerine to form a good glass (Fig. 4).

Discussion

The observed EPR spectra for the Cu(II)- bleomycin-DNA fibers are typical of spectra observed for Cu(II) chelated in molecules of biological significance [20]. The spin-hamiltonian for the Cu 2+ ion is given as [21]

~ s -- f l H " g" S + S . D . S + h S . A . I - g N f l ~ H . I

(1)

In this equation, fl is the Bohr magneton, H the external magnetic field, g the g tensor, S the electronic spin, D the zero-field splitting, h Planck's constant, A the hyperfine tensor, I the nuclear spin, gr~ the nuclear g-value, and flr~ the nuclear magneton. Energy levels described by this hamiltonian in the absence of zero-field splitting are

E = g f l H m s + K M I m ~ + higher order terms. (2)

--2,,2 2 COS 2 2 2 - 2 h ~1±~± sin20 Here K 2 - - n ~,gll + (3)

g2 g2

a n d g2 : g~l c ° s 2 0 + g 2 s in20 , (4)

M I 3 / 2

- - " 3 i 2

- - 1 / 2 ~ -I/2

ICt~ Z£EttRN SPLITTING HYPt[RFIN£ SPLITTING UNR£SOLV~D

H F~qRN.LEL HYP£RFIN£ SPLITTING SYtINETRY RXZS H ~ I C U L J t R

SYIIIIETRY FtXI5

Fig. 5. A Cu(II) energy level diagram. Four resolved hyperfine transitions are expected when H is parallel to the symmetry axis, since A is 175 G and the line width is 30 G. The transitions coalesce into an unresolved transition when H is perpendicular to the symmetry axis, since A ± is less than the 30 G line width.

where m s and M t are respectively the electronic and nuclear magnetic quantum numbers, and 0 the angle between the direction of H and the symmetry axis (the normal to the plane containing the Cu 2÷ ion and the square planar array of ligands). In the expression for the hyperfine parameter K, A~t is the parallel hyperfine constant, A± is the perpendicular hyperfine constant, gll the parallel g-value, and g± the perpendicular g-value. The Cu 2+ ion is in a 2D electronic state which splits into two Zeeman energy levels, the first term in the energy equation with m s = - 1/2. The energy levels are further split by a hyperfine interaction, the second term in the energy equation with M I = ± 3 / 2 , - 1 / 2 . This hyperfine splitting is shown in Fig. 5 along with EPR transitions, and the result- ing spectra for H parallel and perpendicular to the fiber axes.

In biomolecules the Cu(II) is normally chelated with four ligands in a square planar configuration with the Cu(II) at the center of the square. This central Cu(II) may chelate with one or two more distant atoms in sites on a symmetry axis normal to and passing through the center of the square planar configuration [22]. X-Ray diffraction data for a fragment of the Cu(II)-bleomycin molecule [12], tend to indicate that the ligands to Cu(II) in bleomycin are nitrogens; however an unequivocal identification of the ligands has not previously been made. The EPR g-values and hyperfine constant for the Cu(II) resonance are consistent with a square array of nitrogen ligands [22]. The four Cu(II) hyperfine transitions ( I for Cu(II) is 3/2) on the low field side of the randomly oriented fibers in Fig. 2B are known to be transitions observed when the orientation of the symmetry axis is parallel to H. The more intense transitions on the high field side occur when H is perpendicular to the symmetry axis.

The almost complete absence of the perpendicular transitions and the presence of typical parallel transitions in Fig. 2C indicate that the plane of the ligands chelated to Cu(II) in the Cu(II)-bleomycin- DNA is preferentially oriented with the symmetry axis normal to the plane having a higher probabil- ity of being parallel to the fiber axis. A rectangular loop of fibers was used to provide additional experimental evidence for this conclusion. The spectra shown in Fig. 3A through 3E show the varia-

118

tion in intensities of the parallel ( H parallel to symmetry axis of the Cu(II) complex) and perpendicular ( H perpendicular to the symmetry axis of the Cu(II) complex) transitions as the sample of fibers is rotated from a position in which H is parallel ( 6 = 0 ) to 95% of the fiber axes to a position where H is perpendicular (O = 90) to 95% of the fiber axes.

According to EPR theory for transition metal ions [21], if all the Cu(II) complexes have their symmetry axes in the same direction, the four parallel transitions should smoothly transform into the perpendicular transitions as the magnetic field, with respect to the symmetry axis of the chelated complex, is rotated from a parallel to perpendicular orientation. This angular dependence o f the hyperfine splitting and the g-value is given by the Eqns. 3 and 4. The angular variation of the g-value and hyperfine constant could not be determined from the spectra in Fig. 3. The partial resolution of the transitions (they look more like transitions in a randomly oriented sample than in a completely

A

" / '1 J

Fig. 6. A comparison of simulated and experimental spectra of Cu(II)-bleomycin-DNA. The dashed curves are the simulated spectra. A is of randomly oriented Cu(II)-bleomycin-DNA, B of Cu(II)-bleomycin-DNA fibers with the magnetic field parallel to the fiber axes, and C of Cu(II)-bleomycin-DNA fibers with the magnetic field oriented perpendicular to the fiber axes.

7 X

(a) (b)

Fig. 7. The orientation of the coordination sphere of the Cu(lI)- bleomycin complex relative to the fiber axis. Diagram a shows the orientation of the normal (vector N) to the plane of the ligands relative to the fiber axis, Z, and the plane of the basepairs XY. Diagram b shows the X, Y, and Z coordinates with respect to the fibers on the quartz plate which was used to orient the fibers in the EPR cavity.

ordered sample such as a single crystal) and the increase or decrease in intensity at parallel and perpendicular hyperfine field values, as the orientation of the sample is changed, indicate that the alignment of the symmetry axis of the Cu(II)- bleomycin complex with respect to the DNA fiber is not completely along the DNA fiber axis as a cursory analysis of the data would indicate.

In order to describe the spatial orientation of the Cu(II)-bleomycin complex with respect to the DNA fiber axis, spectra for the magnetic field parallel and perpendicular to the fiber axis were simulated (Fig. 6). The constants for the simulated spectra were chosen as those which gave the best simulated spectrum for a randomly oriented Cu(II)-bleomycin-DNA sample (Fig. 6A). The contants used to simulate the spectra are gll = 2.21; g ±=2 .0 4 ; All = 175G; half-line widths in both the parallel and perpendicular directions are 30 G, and a Gaussian line shape is used. Simulation of spectra from randomly oriented square planar complexes is well understood and the constants can be assigned with confidence. The best fit of simulated spectra with experimental spectra was obtained for the orientation of Cu(II)-bleomycin shown in Fig. 7. The symmetry axis (normal to the square planar array of ligands with Cu(II) at the center of the plane) makes an angle of 75 ° with the X-axis which lies in the plane of the basepairs.

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The Cu(II)-bleomycin molecule when bound to DNA has a large amount of rotational freedom about the X-axis in the plane of the basepairs. Spectra in Fig. 6B and C were simulated by allow- ing the symmetry axis to rotate through an angle of ± 80 ° about the X-axis. This rotation carries the symmetry axis out of the X Z plane and almost into the X Y plane. The rotation angle is measured with respect to the fiber axis and it has a value of zero when the symmetry axis is in the X Z plane. The 75 ° angle with respect to the X-axis in t-he plane of the basepairs remains fixed and each angular position in the ± 80 ° range about X is considered to be equally probably. When the Cu(II)-bleomycin-DNA sample freezes, the symmetry axis of the Cu(II)-bleomycin complex is spacially distributed through this ±80 ° range about the axis in the plane of the basepairs. In this model the symmetry axis remains almost perpendicular (75 ° ) to the X-axis in the plane of the basepairs for all orientations of the symmetry axis with respect to the fiber axis. The deviation of the angles in the model is approx. ± 5 ° since changes of 5 ° in the orientation of the symmetry axis with respect to the X-axis or the range of rotation about the X-axis gave significantly different simulated spectra.

This model is consistent with the chemical structure of bleomycin. If on intercalation the bithiazole rings are inserted between basepairs, the chelated portion of the molecule would be outside the sugar phosphate backbone of the DNA helical structure. This portion of the bleomycin molecule, Fig. 1 (I, II and III), outside the helical structure can rotate about bonds in the central part of the molecule, Fig. 1 (IV and V). The lack of complete rotation about an axis perpendicular to the fiber axes may be due to the structure of bleomycin itself or its interaction with the outside contours of the DNA.

The portion of Fig. 4A between the vertical lines is shown in Fig. 4B with an enlarged magnetic field scale. The spectrum in Fig. 4B has nine poorly resolved superhyperfine peaks which are attributed to four ligand nitrogens in a square planar array complexed to Cu(II) at the center of the ligands. Nine superhyperfine peaks with intensity ratios of 1:4:10:16:19:16:10:4:1 are expected from four equally interacting nitrogen

nuclei. However, the ligand nitrogens are from different chemical groups and probably have slightly different coupling constants. The superhy- per fine peaks were observed in a frozen glass of 65Cu(II)-bleomycin-DNA and glycerine. The best resolution was observed from samples containing 50-80% glycerine. Surprisingly, superhyperfine structure could not be resolved in the oriented samples. This lack of resolution is attributed to the poor glasses which form when Cu(II)-bleomycin- DNA with a high water content is frozen.

The magnetic field splitting between the superhyperfine peaks is 12 G. Bereman and Winkler [13] reported a seven peak superhyperfine spectrum of Cu(II)-bleomycin in a glycerine glass, and concluded that the interacting ligands in the square planar array are three nitrogens and one oxygen. The spectrum in Fig. 4B has outside peaks or inflections on the high and low field sides which were not visible in the spectrum reported by Bere- man and Winkler. The nine component superhyperfine structure substantiates the previously pos- tulated square planar structure (Fig. 1), containing four nitrogen ligands [8,18,23], but it does not identify the chemical groups contributing the nitrogen ligands. The proposed planar conformation with coordination to four nitrogens is by pyrimidine N 1, imidazole N 1, the histidine amide nitrogen, and the secondary amine nitrogen atoms. Axial coordination by the adjacent primary amine nitrogen is also assumed.

Conclusions

Changes in EPR spectra as a function of the direction of the external magnetic field, H, with respect to the helical axes of the Cu(II)-bleomycin- DNA fibers show that the Cu(II)-bleomycin complex is bound to or intercalated with DNA. The EPR data as a function of orientation give information on the structure and spatial orientation of the portion of the bleomycin molecule which chelates with the Cu(II) ion. This part of the bleomycin molecule lies outside of the DNA helical structure since the bithiazole rings are known to intercalate with the basepairs [14]. The EPR data indicate that the conformation of Cu(II)- bleomycin has the Cu(II) ion chelated with four atoms in a square plane with the Cu(II) ion at its

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center. The superhyperfine structure of the Cu(II) resonance identifies the square planar array of atoms as four nitrogens. The normal to the chelated plane makes an angle of 75 ° with an axis which lies in the plane of the basepairs. The chelated plane has almost complete rotational freedom about this axis always keeping a 75 ° angle between the normal to the chelated plane and the axis in the plane of the basepairs.

Acknowledgements

We thank the Research Corporation for finan- cial support and Bristol Laboratories for the blenoxane. The help and support given by Frank- lin Hutchinson of Yale University is acknowl- edged with gratitude by H.S. who started this study at Yale while on sabbatical leave. At Yale, the work was supported in part by Grant CA17938 from the National Cancer Institute, DHEW. We also thank Arthur Brill for a computer program (originally written by John Venable, Jr.) which we modified for the simulations in Fig. 6.

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The conformation and orientation of copper(II)-bleomycin intercalated with DNA