Orientation of dioxygen bound to cobalt (II) bleomycin-DNA fibers.

THEJOURNAL OF BIOLOGICAL CHEMISTRY Vol. 264, No. 36, lssue of December 25, pp. 21478-21480,1989 0 1989 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U. S A.

Communication Orientation of Dioxygen Bound to Cobalt(I1) Bleomycin-DNA Fibers*

Experiments by Shields et al. ( 5 , 6) have pointed to the potential of ESR studiesof metallobleomycins associated with oriented DNA as a means to explore the geometry of the (Received for publication, August 11, 1989) metal binding site inrelation to thehelix axis of the polymer. With the recognition that 02-Co(II)-Blm bound to DNA at Makoto ChikiraS, William E. AntholineOl, and large DNA to drug ratios isrelatively stable, itbecame attracDavid H. Peteringl 11 tive to examine the interaction of this adduct with linearly From the $Departmentof Industrial Chemistry, Faculty of oriented DNA fibers (7). Thepresent communicationdeScience and Engineering, Chuo University, Tokyo 112, Japan, the §National Biomedical Electron Spin Resonance scribes our initial results and structural interpretation. Center and Department of Radiation Biology and Biophysics, Medical College of Wisconsin, Milwaukee, Wisconsin 53226, and the lDepartment of Chemistry, University of Wisconsin-Milwaukee, Milwaukee, Wisconsin 53201

The molecular oxygen adduct of Co(I1)-bleomycinis stable for long periods when bound to salmon sperm DNA at large ratios of polymer to drug. According to ESR studies of orientation of the paramagnetic complex associated with DNA fibers, the oxygen-oxygen bond is restricted to a plane perpendicular to the fiber axis. Thus, one can define three g values for the adduct 2.104, 2.016,and 2.000, one parallel to the fiber axis and two orthogonal to it. There is no change in orientation over the range of 77 K to ambient temperature. Furthermore, there is no difference in resultsat a series of relative humidities ranging from less than 76% where bulk DNA alone assumes an A conformation to 95% where it is primarily B DNA. A structural model is presented for the geometry of the metal binding domain of Oz-Co-bleomycin in relationship to the fiber axis of DNA.

The mechanism of DNA strand cleavage by bleomycin has been the subject of numerous studies(1).It isrecognized that the bleomycin molecule contains DNA binding and versatile metal complexation domains that play fundamental roles in this reaction. In the iron-dependent pathway of DNA damage, molecular oxygen is activated to attack the polymer backbone. After Fe(I1)-Blm’ binds to DNA, 0, coordinates to the iron site and is subsequently reduced to the level of peroxide ( 2 ) . This species uniquely abstracts a hydrogenfrom the4”carbon of deoxyribose to initiate strandscission (1). Although Co(I1)-Blm does not activate dioxygen to react with DNA, it does bind O2 (3). As such, it may serve as a model for 02-Fe(II)-Blm. Importantly, one can observe the ESR spectrum of this species (3). ESR spectroscopy shows that thesingle unpaired spin of 0,-Co(I1)-Blm resides largely on the oxygen ligand so that the electronic structureapproaches 0;-Co(II1)-Blm. The ESR spectrum of the oxygen adduct is altered upon binding to DNA, suggesting that it interacts with the DNA structure in some way (4).

MATERIALSANDMETHODS

Method for Muking DNA Fiber-DNA fiher was prepared according to a literature method with minor modifications (8). Salmon sperm DNA (Sigma) was dissolved by stirring 100 mg of the polymer gently in 40 ml of 10 mM Tris-HC1 and 1 mM EDTA solution (pH 7.4-7.5) at 4 “C for about 24 h. After this, DNA was precipitated with ethanol. The precipitatewas collected with glass rodsand washed with a 60% aqueous ethanol solution. After being dried a t 4 “C, the DNA was dissolved in 40 ml of 10 mM NaCl solution and centrifuged at 30,000 X g for 90 min to remove undissolved material. The concentration of the DNA was monitored by the absorbance at260 nm ( t = 1.30 X lo4 M” cm”/base pair). To this DNA solution, 25 ml of free hleomycin (4 LLM in 10 mM NaCl solution) was added very slowly dropwise. Subsequently, 25 ml of 4 pM CoC1, were mixed with the solution to generate 02-Co-Blm bound toDNA. The pH of all the solutions was kept in the range 7.3-7.5. In the preparation, the ratio of base pairs to bleomycin was 50:l. This solution was ultracentrifuged at 260,000 X g for 16 h; the resulting DNA pellet was used to make fibers. A drop from a pellet was suspended anddried a t 4 “C between heads of the two toothpicks which were fixed 5 mm apart from each other. E S R Measurements-Thin DNA filaments (outer diameter, 10.4 mm) containing 02-Co-Blm were placed into parallel holes (inner diameter, 0.5 mm) made in a quartz rod, which was attached to a goniometer head for the ESR measurements. This setup permitted spectratobetakenat variousdefined orientations of filaments relative to the magnetic field, Ho, of the spectrometer. X-band ESR datawere obtained at theNational Biomedical ESR Center. A standard Bruker ERZOOD spectrometer and variable temperature unit were used. RESULTSANDDISCUSSION

Fig. 1 shows the EPR spectra of 0,-Co(I1)-Blm bound to salmon sperm DNA at 77 K. The spectral envelop is essentially that of 0; with the splittings deriving from the interaction of the unpaired electron spin with the nuclear spin of cobalt, I = 712, As noted by previous authors, spectraof these complexes ligated to DNA are better resolved than those of the unbound structures (4). Possibly this indicates that the complex adopts a more fixed conformation when attached to the polymer. Whereas thedioxygen complex in solution readily undergoes reaction to produce species of Co(II1)-Blm, the same form bound to DNA at a ratio of less than 1 cobalt/lO basepairs is stable for theextended periodnecessary to prepare linear orientedDNA fibers. The same EPR spectra of O,-Co(II)-Blm bound to oriented DNA can be seen both a t 77 K and at ambient temperature (Fig. 2, A and B ) . This fortuitous circumstance permits the * This work was supported by American Cancer SocietyGrant CH- analysis of the orientation of the oxygen ligand with respect 466 as well as National Institutes of Health Grants CA-22184 and to DNA at temperatures which approach those relevant to RR-01008. The costs of publication of this article were defrayed in the cellular activities of the drug. It also indicates that the part by the payment of page charges. This article must therefore he oxygen ligand is held in a conformation when interacting with hereby marked “aduertisement” in accordance with 18 U.S.C. Section DNA that is independentof temperature over this range. 1734 solely to indicate this fact. Fig. 2 summarizes the effect of systematic change in the 11 To whom correspondence should be addressed. orientation of the helix axis of DNA with respect to the The abbreviation used is: Blm, hleomycin.

21478

Binding ofOriented Bleomycin Cobalt to

DNA

21479

is completely isolated fromg, and g,. Shields et al. (5, 6) have previously obtained ESR data for Cu(I1) and Fe(II1)-Blm and thendescribed the orientation of Cu(I1)-Blm andFe(II1)-Blm withrespect to DNAfibers drawn I I 7 from calf thymus DNA. According to their geometric model, 9-21 0 I I the low field g value of Fe(II1)-Blm, g,, makes anangle of 1530" with the fiber axis of DNA (6). In their scheme the data were simulated with a 26" cone of rotational freedom of g, about the average direction of g,. Similar analysis of Cu(I1)-Blm bound to DNA indicates FIG. 1. ESR spectrum of 0;-Co(II1)-Blm in the presence of gel phase DNA at -160 "C. ESR spectrometer conditions: micro- that like Fe(II1)-Blm,g, makes anangle of 15" with thefiber wave frequency 9.423 GHz; incident power, 24 milliwatts; modulation axis (5). However, rotational freedom of g, about the average value f80" wasneeded to model the experimental results. frequency, 100 kHz; time constant, 0.5 s; scan time, 200 s. Simulations provide good fits to ESR data taken when the fiber axis is parallel or perpendicular to themagnetic field. Our unpublished ESR data for Fe(II1)-Blm andCu(I1)-Blm bound to salmon sperm DNA fiber are very similar to those of Shields andco-workers, demonstrating that results are not dependent on thesource of DNA and that methodsof preparation of fibers in the two studies yield comparable information. Preliminary simulations suggest that good fits can be obtained withmore restricted motionof Cu-Blm. In anycase, 0,-Co-Blm bound to DNA displays the least motion of the three paramagnetic centers. The following model for the dioxygen binding site of 02Co(I1)-Blm-DNA is proposed on the basisof current results, the conformationof binding of the Fe(II1)- and Cu(I1)- centers in metallobleomycin-DNA, and the bent geometry of dioxygen J A I bound toCo(I1) complexes (Fig. 3) (9). It is assumed the Blm binds to metals with a 5-coordinate geometry in which the primary amine is the axial ligand and the secondary amine, pyrimidine nitrogen, peptide nitrogen, and imidazole nitrogen 9-2.1 0 from the histidine form a pyramidal square planar configuration (11).It is also assumed that the angle between the fiber axis and the axis parallel to the bond for the axial ligand for 506 \ -I Co-Blm is similar to theangle in Cu-Blm and Fe-Blm bound to DNA fibers, about 25" (5, 6). If a reasonable angle for the binding of O2 to cobalt is 125" as described for one form of FIG. 2. ESR spectra of 0;-Co(II1)-Blm bound to oriented g value definedby the oxygen-oxygen DNA fibers with magnetic field parallel to the fiber at 02Co-Mb (lo), then the -140 "C ( A ) and at 27 "C (A') and with the magnetic field internuclear axis lies in a plane within 10" of being perpenperpendicular to the fiber axis at -100 "C ( B ) and at 20 "C dicular to the fiber axis. The g, value is parallel to the fiber (B'). Spectrometer conditions given in Fig. 1, except the incident axis and the thirdg value (gmaX) is parallel to the coordination power is 10 milliwatts. plane. A recent model for the binding of the derivative deglycomagnetic field, Ho, of theESRspectrometer.To a good Co(III)-BlmA2 to DNA places the square planeof the square approximation, one of the g values, g,, is coincident with the pyramidal binding sitefor Co(II1) approximately parallel not fiber axis (Fig. 2 A ) . When the fiber axis is rotated until it is nearly perpendicular to the axis of B-DNA (12). Upon binding perpendicular toHo, the spectrum reveals two other g values, to Co(I1)Blm in this conformation, dioxygen could also be g, and g,. Although previous studies have used g,, as the axis oriented perpendicular to the fiber axis, but g,,, (gz)would colinear with the 0-0 internuclear axis (9), g,, the g value not be parallel to the squareplane. closest to the g value of the free electron, lies along the 0-0 In order to explain the restricted conformation of bound internuclear axis in oxyproto-Co-myoglobin (10). For 02Co- dioxygen, itappearsthatthe ligand must be inintimate Blm, both axes g, and g, are perpendicular to the fiber axis. Therefore, the assignment of either g value to the oxygenoxygen bond orients the structure perpendicular to thefiber axis. The resolvedgvalues are 2.104,2.016,2.000. The respective splittings along the g axis are 20, 13, and 11 G, but the g and A axes are not expected to be colinear. These numbers are consistent with those reported for O,-Co(II)-Blm in frozen solution, g values of 2.106 and 2.004 and A values of 18.9 and 11.5 G (3). The rotational freedom of dioxygen about the Co-0 bond can be determined by assessing the admixture of lines from g, and the g,-g, plane when the fiber axis is parallel and perpendicular to Ho. It is evident from Fig. 2 that there is FIG. 3. Schematic for geometry of fiber axis (vertical line) little rotational freedom for in these orthogonal directions g, with respect to molecular axes for 0;-Co(II1)-Blm. "

\V I

1

21480

Binding Bleonof Cobalt

nycin to OrientedDNA

contact with the DNA structure. These results add to the preferentially with the C-4' carbon hydrogen bond of DNA to initiate DNA strand cleavage. growing list of experiments which suggest that the metal binding domain of bleomycin interacts with the DNA strucAcknowledgment-We appreciate the useful discussions with Dr. ture (13).These include the perturbationof the ESR spectrum Hideo Kon (National Instituteof Diabetes and Digestive and Kidney of NO-Fe(I1)-Blm and the conversion of high spin Fe(II1)- Diseases, National Institutesof Health). Blm to thelow spin form upon binding to DNA (2, 14). The tertiary structure of the DNA double helix changes REFERENCES withthe humidity andthecounterions of thephosphate 1. Stubbe, J., and Kozarich, J. W. (1987) Chem. Reu. 87,1107-1136 backbones. It hasbeen shown that DNA-sodium salt fibers at 2. Burger, R. M., Peisach, J., and Horwitz, S. B. (1981) J. Biol. Chem. 256,11636-11644 98% relative humidity assume the B-form conformation and under 79% humidity, the A-form (8).Under our experimental 3. Sugiura, Y. (1980) J.Am. Chem. SOC.102,5216-5221 4. Gamier-Suillerot, A,, Albertini, J.-P., and Tosi, L. (1981) conditions, of 60% humidity, therefore, the DNA should be Biochem. Biophys. Res. Commun. 102,499-506 in the Aconformation. Measurements taken at arelative 5 . Shields, H., McGlumphy, C., andHamrick,P. J., Jr. (1982) humidity of 76%,using the vapor pressure of saturated Biochim. Biophys. Acta 6 9 7 , 113-120 6. Shields. H.. and McGlumahv. aqueous NaCl as the standard, gave rise to spectra identical . " . C. (1984) . . Biochim. Bio~hvs. . - Acta 800,'277-281 to thosein Fig. 2. At 95% humidity, the line shapes and their 7. Albertini. J. P.. and Garnier-Suillerot., A. (1982) . . Biochemistrv dependence on orientation remainedunchanged, though ESR 21,6777-6782 signal intensity had decreased. According to this analysis, 8. Shindo, H., Woolen, J . B., Pheiffer, B. J., and Zimmerman, S. B. orientation of the oxygen adduct is not sensitive to the con(1980) Biochemistry 19,518-526 9. Jones, R. D., Summerville, D. A., and Basolo, F. (1979) Chem. formation of the bulk of the DNA. Reu. 7 9 , 139-179 In this experiment bleomycin initially binds to solution DNA, which is B-form in the absence of drug; the adduct is 10. Hori, H., Ikeda-Saito, M., and Yonetani, T. (1982) J. Biol. Chem. 257,3636-3642 examined under conditions inwhich DNA fibers adopt either 11. Iitaka, Y., Nakamura, H., Nakatani, T., Muraoka, Y., Fujii, A., an A- or B-form. One recognizes that once the drug binds to Takita, T., and Umezawa, H. (1978) J. Antibiot. 31,1070-1072 12. Kuwahara. J., and Supiura. DNA at least local and, perhaps, extended conformational . Y. (1988) Proc. Natl. Acad. Sci. U. S. A. 85,2459-2463 changes may occur in the polymer (15). Nevertheless, if 02Fe(I1)-Blm adopts a similar highly constrained conformation 13. Petering.D. H.. Bvrnes. R. W.. and Antholine. W. E. (1990) ~hemT:~iol.ziter&t., in press for the boundoxygen relative to DNA, then the additionof a 14. Antholine, W. E., and Petering, D. H.(1979) Biochem. Biophys. secondelectron to the adduct may generate the activated Res. Commun. 91,528-533 to react 15. Levy, M. J., and Hecht,S. M. (1989) Biochemistry 27,2647-2650 intermediatewiththe precisespecifiedgeometry '