ESR Parameters for Cupric Bleomycin in the Mobilized State

Abstract

The configuration of the copper complex of the glycopeptide bleomycin, CuBlm, is presumed to be pyramidal square planar from a previous X-ray structural determination of a fragment of cupric bleomycin. This study presents evidence for a difference in the ESR parameters for cupric bleomycin in the liquid as opposed to the solid state. A decrease in A_iso for CuBlm in the liquid state can be directly surmised from the low frequency S-band spectrum for which three of the four cupric hyperfine lines are partially resolved. Computer simulated spectra infer that the absolute value of A_∥ increases about 100 MHz and the value of A_⊥ may change sign for CuBlm in the liquid state. Simulations using a rotational correlation time of about 250 psec. indicate that CuBLM may not be spherical in the liquid phase. The fastest component for anisotropic motion could dominate and account for the well resolved cupric hyperfine structure. Furthermore, it is argued from an analysis of the cupric hyperfine coupling constants that the CuBlm structure opens up at room temperature and that the cupric ion is displaced from the square plane.

Supported by NIH Grants Ca-22184 and RR-01008 and the University of Wisconsin-Milwaukee.     

Orientation of non-blue cupric complexes on DNA fibers.

Three different orientations of non-blue, type 2 cupric complexes on DNA fibers are obtained from EPR data. The cupric complex of bleomycin, CuBlm, binds as described previously (Shields, H., McGlumphy,C., and Hamrick, P., J., Jr. (1982) Biochim. Biophys. Acta 697, 113-120), except possibly with more restricted motion. The square plane of CuBlm makes an angle of about 65 degrees with the fiber axis. The tridentate complex 2-formylpyridine monothiosemicarbazonato Cu2+ binds with its planar structure perpendicular to the fiber axis. In contrast, other tridentate cupric complexes of tripeptides, CuGHK and CuGHG, bind with the square plane parallel to the fiber axis. The bound forms of Cu(GHK) and Cu(GHG) are determined mostly by the GH moiety in the complex; the contribution of lysine in defining the orientation of the copper moiety is minimal. Thus, the structure of the ligand determines the orientation of these complexes on DNA.     

Studies on the chemical reactivity of copper bleomycin

The copper(II) complex of the clinically used antitumor agent bleomycin (Blm) has cytotoxic as well as antitumor properties. To understand the relationship of the bleomycin ligand, copper bleomycin, and other possible metal complexes of this agent, kinetic studies of the formation of Cu(II)Blm, ligand substitution reactions of CuBlm with ethylenediaminetetraaletic acid, and the redox reaction of CuBlm with thiols have been completed and interpreted along with previous studies of the thermodynamic stability of Cu2+ with bleomycin. Cu(II)Bm is found to be kinetically and thermodynamically stable in ligand substitution processes and is only slowly reduced and dissociated by sulfhydryl reagents. The rate constant of reduction of the complex by 2-mercaptoethanol (2-ME) at pH 7.4 and 25 degrees C is 9.5 X 10(-3) M-1 sec-1, explaining the inhibition of Fe2+-dependent strand scission of DNA by Cu2+ in the presence of 2-ME. CuBlm forms in preference to Fe(II)Blm and cannot be reduced and dissociated rapidly enough by thiols to liberate Blm and form the reactive iron complex. In agreement with the observed chemical stability of CuBlm, it is also shown that the complex is stable in human plasma and in the presence of Ehrlich cells suspended in ascites fluid. Interestingly, little CuBlm enters these cells to carry out cytotoxic reactions. Finally, it is shown that both Cu2+ and Zn2+, at equivalent concentrations to Fe2+, effectively inhibit the strand scission of DNA by Fe(II)Blm plus oxygen. However, at substoichiometric amounts of Cu2+, the ferroxidase activity of Blm enables the drug to remain effective in the strand-scission reaction, despite the lowered Cu-free Blm/Fe2+ ratio. These results are discussed in light of the proposed mechanism of action of bleomycin.     

The manganese site of the photosynthetic oxygen-evolving complex probed by EPR spectroscopy of oriented photosystem II membranes: the g = 4 and g = 2 multiline signals.

The g = 4 and g = 2 multiline EPR signals arising from the Mn cluster of the photosynthetic oxygen-evolving complex (OEC) in the S2 state were studied in preparations of oriented photosystem II (PSII) membranes. The ammonia-modified forms of these two signals were also examined. The g = 4 signal obtained in oriented PSII membranes treated with NH4Cl at pH 7.5 displays at least 16 partially resolved Mn hyperfine transitions with a regular spacing of 36 G [Kim, D.H., Britt, R.D., Klein, M.P., & Sauer, K. (1990) J. Am. Chem. Soc. 112, 9389-9391]. The observation of this g = 4 "multiline signal" provides strong spectral evidence for a tetranuclear Mn origin for the g = 4 signal and is strongly suggestive of a model in which different spin state configurations of a single exchange-coupled Mn cluster give rise to the g = 4 and g = 2 multiline signals. A simulation shows the observed spectrum to be consistent with an S = 3/2 or S = 5/2 state of a tetranuclear Mn complex. The resolution of hyperfine structure on the NH3-modified g = 4 signal is strongly dependent on sample orientation, with no resolved hyperfine structure when the membrane normal is oriented perpendicular to the applied magnetic field. The dramatic NH3-induced changes in the g = 4 signal resolved in the spectra of oriented samples are suggestive that NH3 binding at the Cl- site of the OEC may represent direct coordination of NH3 to the Mn cluster.(ABSTRACT TRUNCATED AT 250 WORDS)     

Low frequency EPR of Pseudomonas aeruginosa azurin. Analysis of ligand superhyperfine structure from a type 1 copper site.

The type 1 copper in Pseudomonas aeruginosa azurin was studied by electron paramagnetic resonance (EPR) spectroscopy at low microwave frequencies. Partially resolved ligand hyperfine structure was observed in the perpendicular region of the spectra at both S-band (2.4 GHz) and L-band (1.1 GHz). A trial and error method, requiring several hundred simulations, has been used to simulate the low frequency EPR data and yield an optimum value of 30 MHz for ACUx, more than one half that previously reported. The fit between the simulated and experimental data is sensitive to changes in the Euler angles and, in particular, to the angle alpha which rotates the Cu A-tensor about the z-axis. Thus, the A- and g-tensors for copper in P. aeruginosa azurin do not appear to be coincident. A value for the Euler angle beta of at least 10 degrees does not disturb the fit between the simulated and experimental data. These studies demonstrate the advantage of evaluating EPR parameters from simulations at more than one frequency, especially at low frequencies where ligand superhyperfine structure may be resolved for type 1 copper.     

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

EPR data are used to describe the conformation and identity of the atoms coordinated to Cu(II) in Cu(II)-bleomycin bound to oriented DNA fibers. The fibers were slowly drawn from viscous solutions of Cu(II)-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 (g parallel = 2.21, g perpendicular = 2.04) and the hyperfine constant (A parallel = 175 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 degrees 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 superhyperfine 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 to double helix.     

Electron paramagnetic resonance Signal II in spinach chloroplasts. II. Alternative spectral forms and inhibitor effects on kinetics of Signal II in flashing light

Linewidth and hyperfine structure measurements of the EPR spectrum of Signal II in spinach chloroplasts show that the signal reflects two alternative states. One state is characterized by a 16-G linewidth and four partially resolved hyperfine components. The other state has 19 G linewidth and five partially resolved hyperfine components. It is possible to interconvert these two states by changing the ionic strength of the chloroplast suspension. Both states of Signal II show similar light-induced increases in dark-adapted chloroplasts and respond to 10-μs white light flashes with identical kinetics.

In chloroplasts at room temperature, Signal II dark decays to 50% of its total light-induced level in about 1 h. Single flashes increase the spin concentration in these aged chloroplasts but with decreased effectiveness compared with fresh, dark-adapted chloroplasts. Carbonyl cyanide-m-chlorophenylhydrazone (CCCP) decreases the decay time of Signal II from hours to seconds without appreciably altering the level of Signal II formed in saturating continuous light. However, both the formation time constant and the extent of Signal II increase stimulated by a single saturating flash are decreased in CCCP-treated chloroplasts.

These results are interpreted in terms of the model, proposed in the preceding paper, in which Signal II is generated by oxidation-reduction reactions on the water side of Photosystem II.     

Adduct formation between the cupric site of phenylalanine hydroxylase from Chromobacterium violaceum and 6,7-dimethyltetrahydropterin

The interaction of pterin-dependent phenylalanine hydroxylase from Chromobacterium violaceum with the cofactor analogue 5-deaza-6-methyltetrahydropterin and the cofactor 6,7-dimethyltetrahydropterin (DMPH4) has been investigated by multifrequency electron spin resonance (ESR) spectroscopy. 5-Deaza-6-methyltetrahydropterin, which lacks the N-5 nitrogen present in the pyrazine ring of DMPH4, binds tightly to the cupric form of the enzyme; however, no changes are observed in the ESR parameters of the copper center. In contrast, the binding of DMPH4 (or 6-methyltetrahydropterin) shifts the ESR parameters (g and A) associated with the cupric enzyme. In addition, superhyperfine transitions were resolved and assigned to hyperfine splitting from nitrogen ligands. ESR spectra of the enzyme recorded in the presence of [5-14N]DMPH4 or [5-15N]DMPH4 were computer simulated and found to be consistent with pterin serving as a direct donor ligand to the copper center through the N-5 position.     

Hyperfine structure resolved by 2 to 4 GHz EPR of cytochrome c oxidase.

Using low frequency 2 to 4 GHz EPR at 10 K, we have resolved previously unseen hyperfine structure associated with the EPR-detectable copper signal of cytochrome c oxidase. The observed hyperfine structure appears consistent with hyperfine coupling to copper; although to account for all of the observed structure, an additional magnetic interaction is required as well. This work points out the utility of the 2 to 4 GHz EPR technique for resolving electronic hyperfine structural information from copper and possibly other paramagnetic sites in biomolecules when random variation in electronic g values is a cause of EPR line-broadening.     

Proton nuclear magnetic resonance characterization of the oxidized intermediates of cytochrome c peroxidase

Oxidation of cytochrome c peroxidase with hydrogen peroxide to form the initial oxidized intermediate, cytochrome c peroxidase compound I, drastically alters the proton hyperfine nmr spectrum. In contrast to studies of horseradish peroxidase, where the spectrum of horseradish peroxidase compound I is similar to that of the native protein, cytochrome c peroxidase compound I exhibits only broad resonances near 17 and 30 ppm from 2,2-dimethyl-2-silapentane-5-sulfonate. No unique resonances attributable to cytochrome c peroxidase compound II could be identified. These results define the molecular conditions for which resolved hyperfine resonances of the iron(IV) states of heme proteins may be observed when the data presented here are compared with the data from horseradish peroxidase. Oxidation of cytochrome c peroxidase while it is complexed to ferricytochrome c reveals that the heme resonances of cytochrome c are not influenced by the oxidation state of cytochrome c peroxidase.     

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

EPR data are used to describe the conformation and identity of the atoms coordinated to Cu(II) in Cu(II)-bleomycin bound to oriented DNA fibers. The fibers were slowly drawn from viscous solutions of Cu(II)-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 ($\text{g}{}_{\mathrm{}}\text{=2.21}$, g_⊥ =2.04) and the hyperfine constant ($\text{A}{}_{\mathrm{}}\text{=175}\text{G}$) 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 superhyperfine 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.     

Physicochemical characterization of the four-iron-four-sulphide ferredoxin from Bacillus stearothermophilus.

1. A stable ferredoxin was prepared from Bacillus stearothermophilus and purified by chromatography on DEAE-cellulose and by electrophoresis. 2. The minimum molecular weight determined from the amino acid composition was about 7900 and this was in reasonable agreement with a value of 8500 determined by polyacrylamide-gel electrophoresis. The ferredoxin contained four iron atoms and four labile sulphide groups per molecule. 3. The optical absorption, optical-rotatory-dispersion and circular-dichroism spectra are typical of ferredoxins containing 4Fe-4S clusters. 4. Oxidation-reduction titrations, combined with electron-paramagnetic-resonance (e.p.r.) spectroscopy, showed that the protein has a mid-point potential, at pH8, of -280 +/- 10mV, and that only one electron-accepting paramagnetic species is present. 5. The e.p.r. spectrum of the reduced ferredoxin is more readily saturated with microwave power at low temperatures than those of the eight-iron ferredoxins, indicating that there is another mechanism of electron-spin relaxation in the latter. 6. Mossbauer spectra of both redox states were observed over a range of temperatures and in magnetic fields. At high temperatures (77 degrees K and above) both redox states appear as quadrupole-split doublets; in the reduced state two resolved doublets are seen, suggesting appreciable localization of the additional reducing electron. 7. The average chemical shift indicates formal valences of two Fe3+ and two Fe2+ in the oxidized state and three Fe2+ and one Fe3+ in the reduced state. However, the spectra indicate that there are differing degrees of electron delocalization over the iron atoms. 8. At low temperatures (4.2 degrees K) the oxidized form shows no hyperfine magnetic interaction, even in an applied magnetic field, evidence that the oxidized ferredoxin is in a non-magnetic state as a result of antiferromagnetic coupling between the iron atoms. 9. At 4.2 degrees K the reduced form shows a broad asymmetric pattern resulting from magnetic hyperfine interaction. This contrasts with the reduced ferredoxin of Clostridium pasteurianum, which shows a doublet, suggesting that in the latter there may be interaction between the two 4Fe-4S centres. 10. In large applied magnetic fields, positive and negative hyperfine fields are seen in the Mossbauer spectra of the reduced ferredoxin, evidence for antiferromagnetic coupling between the iron atoms in the 4Fe-4S centre. The high-field spectra of the reduced ferredoxin of B. stearothermophilus are similar to those of the reduced ferredoxin of C. pasteurianum.     

High-field (94-GHz) EPR spectroscopy on the S(2) multiline signal of Photosystem II

The multiline signal of the S(2) state in Photosystem II was measured both in frozen-solution and single-crystal preparations from the cyanobacterium Thermosynechococcus elongatus. The frozen-solution EPR spectrum shows a gaussian-like line shape without any resolution of Mn hyperfine couplings. This line shape can be understood on the basis of the single-crystal spectra, where a strong orientation dependence of partially resolved hyperfine structures appears. Simulation of the frozen-solution spectrum on the basis of Mn hyperfine couplings taken from published pulse-ENDOR data yields a fully rhombic g-matrix for the multiline signal with principal components 1.997, 1.970, and 1.965. The resulting isotropic g-value g(iso)=1.977 is surprisingly small compared to other manganese complexes containing manganese ions in the formal oxidation states three and four.     

Inhibition of ascorbate oxidation by urate

Cupric sulfate is reduced by ascorbate to the cuprous ion. The cuprous ion is then oxidized back to the cupric form by oxygen. A steady state concentration of the cupric ion is thus established to maintain a continuous oxidation of ascorbate in the presence of a trace amount of copper. In the presence of urate there is an instantaneous oxidation of ascorbate by the cupric ion. However, urate complexes with the cuprous ion and thus reduces the steady state concentration of the cupric ion. This decrease in cupric ion concentration interrupts ascorbate oxidation. The interaction of urate and cuprous ion was documented by analysis of uv absorption spectrum and the isolation of urate-Cu⁺ by high-pressure liquid chromatography.     

Bleomycin-metal interaction: ferrous iron-initiated chemiluminescence

Chemiluminescence often accompanies the spontaneous degradation of intermediates in an electronically excited state. The interaction of iron with bleomycin results in the activation of bleomycin to a reactive intermediate which can alter DNA or undergo self-inactivation. This report demonstrates that the interaction of ferrous iron with bleomycin results in chemiluminescence, that this response is iron-specific and that the presence of DNA prevents the generation of chemiluminescence. These observations suggest that the activated bleomycin intermediate may be in an electronically excited state.     

Oxygenated iron bleomycin. A short-lived intermediate in the reaction of ferrous bleomycin with O2.

The reaction of Fe(II) . bleomycin with O2 to yield Fe(III) . bleomycin has been resolved into two kinetic events by stopped-flow spectrophotometry. The first event is first order with respect to both bleomycin and O2 and may be regarded as a second order reaction (k = 6.1 x 10(3) M-1s-1 at 2 degrees C). The first product has no EPR spectrum. The optical spectrum resembles those of Fe(II) . bleomycin complexes with CO, NO, and ethyl isocyanide. We propose that the first product is an Fe(II) . bleomycin . O2 complex. The second kinetic event is first order with respect to the first accumulated product (k = 0.11 s-1 at 2 degrees C) and independent of oxygen concentration. The product of this reaction is indistinguishable from Fe(III) . bleomycin by optical and EPR spectroscopy.     

Sensitivity of biological objects to the effects of the geomagnetic field]

It is shown on the basis of calculations of energy sublevels of the hyperfine structure that the effect of the geomagnetic field upon the impurity atoms in the volume of living cells should be considered in relation to the value of geomagnetic field induction pulses delta B. When delta B > or = 10 pT and the dielectric constant epsilon > or = 10, magnetodipole transitions between sublevels of the hyperfine structure within one term are possible in impurity atoms in their 2P-state. During magnetic storms with delta B > or = 100 nT magnetodipole or magnetoquadrupole forced transitions from 2P1/2 and 2P3/2 states to 2S1/2 metastable state are possible in the resonant zones formed by intersection of hyperfine energy sublevels of the corresponding excited levels.     

Reaction of FeBlm with DNA: Fe(II)Blm-NO.

The structure of the iron bleomycin nitric oxide complex is altered in the presence of calf thymus DNA as determined from epr studies. This altered structure predominates for one iron bleomycin nitric oxide molecule per coil of the DNA helix. In the absence of nitric oxide, as the pH is lowered, iron bleomycin dissociates in two steps, supporting the hypothesis that in-plane nitrogens may be easily perturbed.     

Effect of bleomycin on lipid peroxides, glutathione peroxidase and collagenase in cultured lung fibroblasts

Bleomycin-Cu(II) complex tended to increase the lipid peroxide level in cultured lung fibroblasts, though neither free bleomycin nor free cupric ion increased the level. Simultaneous addition of DL-alpha-tocopherol decreased the level significantly. Bleomycin-Cu(II) complex decreased glutathione peroxidase activity remarkably, though free bleomycin reduced the activity only slightly. Collagenase activity was not decreased but rather increased by both free bleomycin and bleomycin-Cu(II) complex. Accordingly, the accumulation of collagen induced by bleomycin could be explained not by a decrease in collagenase activity, but by the occurrence of cross-linking of collagen due to the increased lipid peroxides.