Spin-density distribution in the [FeO2]-complex. Electron spin resonance of myoglobin single crystals.

X-irradiated oxymyoglobin (MbO2) exhibits electron spin resonance (ESR) spectra at 77 K due to two distinct [FeO2]-centers formed by electron addition to the dioxygen. In single crystals with 17O and 57Fe isotope enrichment in the heme-ligand complex, the full set of spectral parameters is analyzed for one of the centers (gmax = 2.23, gint = 2.13, gmin = 1.97; HOmax = 2.66 mT, HOint = 1.61 mT, HOmin = 0.57 mT; HFe max = 1.62 mT, HFe int = 0.57 mT, HFe min = 0.49 mT) and the iron-dioxygen spin-density distribution and bonding geometry is derived. The g-tensor is evaluated for the second species at 77 K (gmax = 2.25, gint = 2.11, gmin = 1.95). Both centers transform into secondary species at 180 K for which the g-tensor elements are analyzed (gmax = 2.31, gint = 2.18, gmin = 1.93; gmax = 2.35, gint = 2.21, gmin = 1.91).

An ENDOR study of human and bovine erythrocyte superoxide dismutase: 1H and 14N interactions.

Hyperfine interactions (1H and 14N) with the paramagnetic Cu(II)-site obtained from frozen solutions of human and bovine erythrocyte superoxide dismutase (superoxide:superoxide oxidoreductase, EC 1.15.1.1) as well as from their derivatives produced by anion binding (N3-, CN-) and by depletion of the Zn(II) site were studied using electron nuclear double resonance (ENDOR) spectroscopy at about 15 K. Both interactions were found to be identical in human and bovine erythrocyte superoxide dismutase. In all compounds, an anisotropic, exchangeable 1H interaction with a nearly constant coupling value (approximately 3 MHz along g perpendicular ) was observed which is due to either histidine NH- or water protons. Other proton interactions were tentatively assigned to H beta 1 of His-44, H delta 2 of His-46 and to H beta 2 of His-44. Depletion of the Zn(II) site did not alter appreciably the pattern of the proton interactions. The 14N couplings of the native specimen indicated equivalent coordination, whereas Zn(II) depletion and CN- addition were found to produce either some or drastic inequivalences, respectively. For N3- addition to either the native or the Zn(II)-depleted sample only minor effects on the respective 14N coupling pattern were observed.

The mechanism of vanadium action on selective K+-permeability in human erythrocytes.

Low concentrations of chelating agents such as EDTA prevent the air oxidation of vanadyl (VO2+, +4 oxidation state) to vanadate (VO3-, +5 oxidation state). Under these conditions, the ionophore A23187 mediates the rapid entry of vanadyl into human erythrocytes. In the presence of A23187, vanadyl at concentrations in excess of EDTA gives rise to a dramatic increase in K+ permeability, which is very similar to the Gardos Ca2+-induced K+ permeability increase with respect to ion selectivity, response to inhibitors, effects of pH, and stimulation by external K+. In ultrapure media with very low Ca2+, however, vanadyl has no effect on K+ permeability. These experiments suggest that Ca2+ is displaced from EDTA by vanadyl and then enters the cell via A23187 where it triggers the increase in K+ permeability. This hypothesis is confirmed by experiments demonstrating that vanadyl does displace Ca2+ from EDTA. Vanadate, an inhibitor of Ca2+-ATPase, causes a selective increase in K+ permeability in metabolically depleted cells, but the increase is abolished by low concentrations of EDTA, indicating that this effect is also due to entry of extracellular Ca2+. Earlier observations of effects of vanadyl and vanadate on erythrocyte K+ permeability can thus be explained on the basis of inhibition of the Ca2+ pump by vanadium, leading to an increase in intracellular Ca2+ concentration.

Xanthine dehydrogenase from Pseudomonas putida 86: specificity, oxidation-reduction potentials of its redox-active centers, and first EPR characterization.

Xanthine dehydrogenase (XDH) from Pseudomonas putida 86, which was induced 65-fold by growth on hypoxanthine, was purified to homogeneity. It catalyzes the oxidation of hypoxanthine, xanthine, purine, and some aromatic aldehydes, using NAD+ as the preferred electron acceptor. In the hypoxanthine:NAD+ assay, the specific activity of purified XDH was 26.7 U (mg protein)(-1). Its activity with ferricyanide and dioxygen was 58% and 4%, respectively, relative to the activity observed with NAD+. XDH from P. putida 86 consists of 91.0 kDa and 46.2 kDa subunits presumably forming an alpha4beta4 structure and contains the same set of redox-active centers as eukaryotic XDHs. After reduction of the enzyme with xanthine, electron paramagnetic resonance (EPR) signals of the neutral FAD semiquinone radical and the Mo(V) rapid signal were observed at 77 K. Resonances from FeSI and FeSII were detected at 15 K. Whereas the observable g factors for FeSII resemble those of other molybdenum hydroxylases, the FeSI center in contrast to most other known FeSI centers has nearly axial symmetry. The EPR features of the redox-active centers of P. putida XDH are very similar to those of eukaryotic XDHs/xanthine oxidases, suggesting that the environment of each center and their functionality are analogous in these enzymes. The midpoint potentials determined for the molybdenum, FeSI and FAD redox couples are close to each other and resemble those of the corresponding centers in eukaryotic XDHs.

Copper(II)-substituted horse liver alcohol dehydrogenase: structure of the minor species.

Oxygen treatment of horse liver alcohol dehydrogenase EE isozyme substituted with Cu(II) at the catalytic site leads to bleaching with concomitant reduction to Cu(I) of approximately 90% of total Cu(II). The Cu(II) of the remaining 'minor species' cannot be reduced nor does it interact with exogenous ligands, e.g. 2-mercaptoethanol, imidazole, pyrazole, or azide ions. The EPR spectrum is axial with a super-hyperfine splitting of 15.6 G indicating binding of one nitrogen atom to Cu(II). These data as well as the energies and intensities of the absorption and CD spectra suggest the Cu(II) ion of the minor species to be located in the catalytic site of HLADH in a position and geometry different from that of the major species.

EPR-spectroscopy of reduced oxyferrous-P450cam.

X-irradiation of the ternary complex of P450:substrate:O2 at 77 K produces a reduced intermediate by electron addition to the Fe:O2 complex which can be studied by EPR-spectroscopy. The EPR spectrum of the new species exhibits rhombic symmetry with g-factors of 2.27, 2.17 and 1.95, respectively. Increasing the temperature of the sample to 190 K results in loss of intensity of the intermediate signals. X-irradiation of oxymyo- and oxyhemoglobin produces similar EPR signals indicating that the added electron is resident on the Fe:O2 compleX (Kappl, R., et al. (1985) Biochim. Biophys. Acta 870, 20-30).

X-band ESEEM spectroscopy of 15N substituted native and inhibitor-bound superoxide dismutase. Hyperfine couplings with remote nitrogen of histidine ligands.

The hyperfine couplings of the remote nitrogens of histidine ligands are determined for the first time by an X-band ESEEM spectroscopy study of 15N-substituted superoxide dismutase (SOD). They show a significant difference between two groups of ligands with different orientation relative to the metal ion. The ESEEM spectra of 15N SOD with cyanide as an inhibitor containing 14N and 15N are also discussed. They allow some conclusions to be drawn about structural changes upon inhibitor binding and indicate the necessity of further multifrequency investigations.

Free radicals from oriented DNA fibers after irradiation at 77 K: continuous-wave and pulsed electron paramagnetic resonance spectroscopy from D2O-equilibrated specimens.

Combined continuous-wave and field-sweep electron spin echo spectroscopy was employed to unravel the components of the spectra in oriented DNA fibers equilibrated in 76% relative humidity of D2O vapor formed upon X irradiation at 77 K and stabilized at that temperature. Using DNAs with different counter-ions (Na+, Li+, Cs+), different base composition (calf thymus, Cl. perfringens), substituted thymine bases (deuterated thymine, 5-halouracil substitution) as well as the copolymer poly(A:U), nine different components were discerned. The spectra of two components, known before partially, were fully characterized. One of them should probably be associated with a cytosine anion, the other with a guanine cation, the latter connection having already been made in previous work. A third component, invoked previously from work with DNA containing deuterated thymine, was also fully characterized and could be assigned to a thymine anion. For one of the new components, an assignment to the allyl radical, probably on the thymine base, could be given on account of the characteristics of the spectra. Tentative assignments are given for three other components which involve an anion on the adenine and the guanine base, respectively, as well as an oxidation-derived species comprising a glycosidic nitrogen, perhaps on cytosine.

Mechanistic aspects of radiation-induced free radical formation in frozen aqueous solutions of DNA constituents: consequences for DNA?

Frozen aqueous solutions of 1 M thymidine-5'-monophosphate were X-irradiated 77 K. The free radicals formed were analyzed by electron spin resonance spectroscopy between 77 K and about 260 K and were shown to result nearly exclusively from electron reaction at 77 K forming the thymine base anion, which converts into the well known 5-thymyl radical upon annealing. Primary oxidation of the substrate was not detectable. A minority species denoted TOH., which appeared at about 200 K, was suggested to result from OH. addition to carbon C6 of the base, perhaps via intermediate oxidation involving H2O2 or from direct reaction of OH. with the base. Another minority species at 77 K up to about 150 K, which was strongly enhanced by H2O2, was shown to be the allyl radical formed by reaction of the OH. with the methyl group. Support for this was given from experiments using BeF2 glasses. The possible spectral features for the cation of dTMP were extracted from aqueous pastes of the Ca2+ salt at 77 K. The mechanistic aspects derived from the results are in conflict with previous assumptions and are discussed for DNA model compounds and DNA.

Free radicals trapped in methyl alpha-D-mannopyranoside X-irradiated at 77 K: an ESR/ENDOR study.

Four free radicals are trapped in methyl alpha-D-mannopyranoside X-irradiated and maintained at 77 K. All four have been identified, with high confidence levels, using ESR and ENDOR spectroscopy. One, an alkoxy radical located at O4, is characterized by a gmax of 2.059, an isotropic beta hydrogen hyperfine coupling (hfc) of 98 MHz, and small interactions due to gamma or delta hydrogens. The second, a secondary dioxyalkyl radical due to loss of hydrogen from C1 is characterized by one beta hfc with an isotropic component of 19.03 MHz. The third, a secondary hydroxyalkyl radical due to loss of hydrogen from C2 is characterized by two nonexchangeable hydrogens with isotropic beta interactions of 22.45 and 6.44 MHz and one exchangeable hydrogen with an isotropic beta interaction of 9.88 MHz. The fourth is a .CH2OR radical that is formed by the net loss of hydrogen from the methyl group.

An EPR study of the transfer and trapping of holes produced by radiation in guanine(thioguanine) hydrochloride single crystals.

Single crystals of guanine hydrochloride monohydrate, guanine hydrochloride dihydrate and anhydrous guanine dihydrochloride, doped with thioguanine, were irradiated with X and gamma rays. In all three systems the dominant radicals were associated with thioguanine. In the former two systems the stabilized species is the thiyl radical, formed by initial loss of an electron at some of the guanines in the crystal lattice, followed by hole migration to thioguanine and subsequent deprotonation of the radical formed. In the anhydrous guanine(thioguanine) dihydrochloride, that process is followed by acquisition of a chlorine ion. In the guanine hydrochloride monohydrate and guanine hydrochloride dihydrate lattices, systems of interacting closely spaced stacked bases and strings of chloride ions might support the migration of electrons and/or holes. In anhydrous guanine dihydrochloride, neither the bases nor the Cl- ions alone are capable of providing the means for the long-range electron, energy and spin transfer. It is the interchangeable sequence of the charged bases and the Cl- ions that makes the supporting strings or networks. The ultimate chlorination of the thioguanine-centered electron-loss radicals depends mainly on the availability of the Cl- ions and the space for their accommodation in the vicinity of the sulfur atom.

Spin transfer from protein to DNA in X-irradiated 'dry' and hydrated chromatin: an electron spin resonance investigation of spectral components between 77 K and room temperature.

PURPOSE: To investigate the potential total and relative free-radical transfer from histones to DNA in X-irradiated chromatin, by analysing the relative contributions of individual radicals from protein and from DNA. MATERIALS AND METHODS: Chromatin was isolated from calf thymus, freeze-dried and either used as such or after equilibration at 76% relative humidity. A mixture of histones was purchased. X-irradiation was performed at 77 K (liquid nitrogen). Data acquisition was on a Bruker ESP 380 ESR-spectrometer (X-band, 9.5 GHz) and at high magnetic fields (285 GHz, Y-band, GHMFL Grenoble). Data analysis involved computer treatment of spectra. RESULTS: Three components were isolated from an annealing series of histones and assigned to specific radicals (X-band). Chromatin revealed the existence of radicals from both DNA, as well as from the histone compartment. The presence of the oxidized guanine base, as well as the reduced cytosine and thymine bases from DNA at 77 K was confirmed by high-field ESR. Relative radical yields were determined by superposition of individual components from DNA and histones in order to give complete reconstructions of the experimental spectra of the annealing series. CONCLUSIONS: The relative yields of individual radicals in chromatin differ from those in histones or DNA, respectively. Their behaviour upon annealing is, on the other hand, not significantly changed. Since the total radical yield of DNA radicals is about two times higher in the chromatin complex than in pure DNA, the hypothesis of spin transfer from protein to DNA prior to radical stabilization at 77 K is substantiated.

Reactions of water radiolysis intermediates with pyrimidine and purine bases in aqueous BeF2 glasses: an EPR study.

Free radical formation by reaction of water radiolysis intermediates produced by X-irradiation in aqueous glasses containing 7 M BeF2 at 77 K with pyrimidine and purine constituents of DNA (10 mM) was studied by electron paramagnetic resonance (EPR) spectroscopy. Reactions of electrons and .OH radicals were observed; H. form a minority and their contribution was difficult to establish. The electrons form substrate radicals at 77 K, while .OH radicals, stabilized in the matrix at 77 K, become mobile at about 140K and in part react with solute molecules. The radicals formed by both reactions were characterized after isolation of the corresponding components by thermal annealing up to about 190 K and spectra simulation using literature parameters whenever possible. The spectra from cytosine gave strong evidence for heteroatom protonation following electron addition while those from adenine were somewhat less clear. The spectra of uracil and all methylated pyrimidines gave no evidence for the protonation state of the electron adduct. For the .OH radical, the reaction with uracil and cytosine was found to be addition to the 5,6-double bond, in line with studies using aqueous solutions. For all methylated pyrimidines, however, H-abstraction from the methyl group was dominant. .OH addition to adenine was found to take place at C2.

Matrix-isolation of H-induced free radicals from purines in acidic glasses.

H.(D.)-atoms produced by photo-oxidation (lambda = 254 nm) of Fe2+ in acidic glasses (6 mol dm-3 H2SO4/H2O or D2SO4/D2O) at 77K were allowed to react with the purine bases adenine, guanine, hypoxanthine and xanthine as well as with ribo- and deoxyribosides (-tides) of adenine and guanine by annealing to 110-130 K. The ensuing free radicals were studied by electron spin resonance (ESR) spectroscopy at 77 K. Individual radical species were assigned by simulation of patterns isolated from thermal annealing studies up to 180 K. It is shown that H.-atom reaction with the bases produces C2- and C8-addition radicals for adenine and C8-addition species for guanine. Guanine is also photo-oxidized directly in the glass, producing a cation or its deprotonated successor species. In the nucleosides (-tides) of both bases, H. atoms were found to abstract hydrogen from carbon sites C1', C2' or C3', and C5' for ribosides and C1', C2', and C5' for deoxyribosides (-tides), respectively.

Free radicals from X-irradiated 'dry' and hydrated lyophilized DNA as studied by electron spin resonance spectroscopy: analysis of spectral components between 77K and room temperature.

PURPOSE: To investigate the number, spectroscopic signatures and chemical structures of free radicals from X-irradiated lyophilized DNA (dry and equilibrated at 76% relative humidity) between 77 K and room temperature by electron spin resonance (ESR) spectroscopy. MATERIALS AND METHODS: Samples were prepared by freeze drying DNA (sodium salt, salmon testes) in H2O or D2O and used as such ('dry' DNA) or after equilibration at 76% relative humidity. K3[Fe(CN)6] was co-lyophilized in some samples as an electron scavenger. X-irradiation was performed at 77 K (liquid nitrogen). Data acquisition was on a Bruker ESP 380 ESR-spectrometer (X-band, 9.5 GHz) and at high magnetic fields (245 GHz, Y-band; GHMFI, Grenoble, France). Data analysis involved computer treatment of spectra. RESULTS: There were 12 different radical components isolated from DNA in four different conditions (dry and after equilibration at 76% relative humidity in either H2O or D2O) with the additional help of high magnetic field ESR and the use of K3[Fe(CN)6] as an electron scavenger. Several components were detected at 77 K and were found to be common for both hydration conditions, although their spectral shape varied considerably. These involved reduced thymine and cytosine bases, the oxidized guanine base, probably a C1'-located sugar radical, a thymine allyl radical and a secondary thymine H-addition radical. For the reduced cytosine base the amino-protonated form was observed in H2O samples, which was only partially exchanged in the D2O samples. At high water content another species, perhaps due to a sugar radical, contributes in addition even at low temperatures. All radical components anneal out with temperature, with only small secondary reactions taking place. A peroxy radical and a sharp singlet, probably due to the deprotonated radical cation from guanine, come into the balance together with the secondary thymine radical. At high doses, a further sugar radical (perhaps at the C3'-position) was detected in dry DNA. The relative yields of the isolated patterns were determined by precise reconstruction of the experimental spectra. CONCLUSIONS: The comprehensive component delineation performed at 77 K and upon annealing to room temperature for lyophilized DNA showed a larger diversity and a higher variance of radicals at 77 K than discussed so far. Thermal annealing brings about only a few reactions to produce secondary species. Most components decay without paramagnetic successors.

Free radicals from lyophilized 'dry' DNA bombarded with heavy-ions as studied by electron spin resonance spectroscopy.

PURPOSE: To investigate the number and species of free radicals from dry DNA after bombardment with heavy-ions at low temperature in comparison with X-irradiation at 77 K by electron spin resonance (ESR) spectroscopy. MATERIALS AND METHODS: Solid DNA samples were either X-irradiated at 77 K as cylinder samples or heavy-ion-bombarded (LET range 1500-12400keV/microm) at the Gesellschaft für Schwerionenforschung (GSI Darmstadt) as very thin tablets. Data acquisition was on a Bruker ESP 380 ESR-spectrometer (X-band, 9.5 GHz). Data analysis involved computer treatment of spectra. RESULTS: Spectra from heavy-ion-bombarded samples were found to reveal the same free radical species with about the same relative contributions for most components as those from X-irradiated samples at comparable doses. Only two components, a thymine allyl radical and a Cl' deoxyribose located species, were enhanced after heavy-ion bombardment. Dose effects in both cases involved a higher relative amount of deoxyribose-derived free radicals. The analysis of G (total free radicals, taken from dose-response curves) were typically smaller than those for X-rays but showed no clear dependence on LET. CONCLUSIONS: The differing biological response to high-LET particle bombardment compared with low-LET irradiation is not strongly reflected by the chemical structure and total number of initial free radicals. It might rather derive from an inhomogeneous distribution of energy deposition (resulting in 'clustered damages') or from effects at higher chemical and biological levels (e.g. product formation and repair) which is not strongly apparent in the free radical characteristics obtained by ESR-spectroscopy.

Products from polycrystalline DNA constituents after X-irradiation and heavy-ion bombardment: formation of the 5,6-dihydroadduct in thymidine 5'-monophosphate and release of unaltered bases in nucleotides.

PURPOSE: The major products from polycrystalline purine and pyrimidine DNA nucleotides after low- and high-LET irradiation were investigated quantitatively by HPLC and 1H-NMR spectroscopy. MATERIALS AND METHODS: Solid nucleotide samples were either X-irradiated as cylindrical pellets or heavy-ion bombarded (LET range of 100-12,500 keV/microm) as very thin tablets at 300K. Product analysis was performed by HPLC and 1H-NMR. RESULTS: For TMP the 5,6-dihydroadduct was found to be formed as product of electron reaction. In addition, all four DNA nucleotides showed a radiation-induced base release, which is probably connected with the oxidative radiation action. The formation of the products was linear with dose up to 300 kGy for X-irradiation or 200 kGy for heavy-ion bombardment. The estimation of the radiation chemical yields revealed G-values of about 10(-7) mol x J(-1) and were typically smaller for irradiation with charged particles than those for X-rays. After heavy-ion bombardment the G-values first increased with increasing LET and decreased for very heavy ions. CONCLUSIONS: The yields for base release from both purine and pyrimidine nucleotides are comparable in magnitude. The 5,6-dihydroadduct from TMP is a major radiation induced product with larger yields than found for base release after X-irradiation and comparable yields after heavy-ion bombardment. The LET dependence of the G-values for base release in nucleotides is similar and resembles the double strand break formation in DNA. The observed similarity in the LET dependence of the G-values might derive from an inhomogeneous distribution of energy deposition resulting in 'clustered damage'.

Free radicals from polycrystalline pyrimidines and purines upon heavy ion bombardment at low temperatures: an electron spin resonance study.

The formation of free radicals in polycrystalline samples of the DNA constituents thymine, cytosine, and adenosine after bombardment with heavy ions at about 100 K was investigated by electron spin resonance (ESR) spectroscopy. Spectra were observed at 77 K after irradiation at 100 K, upon annealing to 300 K and after storage at 300 K. Individual radical patterns were isolated from the spectra by computer manipulation and assigned to structures by powder-simulations based on literature data. The spectra of thymine contain an allyl radical, the octet pattern of the 5-thymyl radical and contributions of the 6-yl radical formed by net hydrogen gain at carbon C5. The latter species is also present in cytosine which in addition displays the pattern due to H-addition at the carboxyl oxygen C2. Adenosine exhibits two H-addition radicals, one at C2, the other at C8. Additionally, the spectra of all DNA subunits studied contain as a radical component a Gaussian singlet of about 0.9 mT line width. The spectra obtained at low temperature already contain the secondary radicals but exhibit a large linewidth. This feature is attributed to dipolar coupling caused by radicals in close proximity.

Influence of electron scavengers on the radical formation in thymidine-5'-monophosphate and DNA in frozen aqueous solution and glasses.

Structural and quantitative effects of different electron scavenger concentrations on the free radical formation in the nucleotide thymidine-5'-monophosphate (TMP) and in deoxyribonucleic acid (DNA) after X-irradiation in frozen aqueous solution and glasses (BeF2/H2O for TMP and LiCl/H2O for DNA) at 77K are investigated. At the highest concentration used (100 mmol dm-3) about 80% (TMP) and 70% (DNA) of the radicals are scavenged compared with the control in both matrices. In TMP, allyl radicals form the major population of radicals left unscavenged at 77 K. These are shown to transform into a quintet pattern upon annealing (> or = 220 K). Analysis of various substances for quintet formation shows that a sugar-group and a C4-carbonyl group are necessary structural prerequisites. For DNA three components can be extracted from spectra obtained with different scavenger concentration in frozen solutions. There are two components in LiCl glasses, which are comparable with two of the three in frozen aqueous solution. Their potential origin is discussed in comparison with nucleotide spectra.

Free radicals from irradiated lyophilized DNA: influence of water of hydration.

Lyophilized DNA equilibrated with water vapour at various relative humidities (0-95% H2O or D2O) was X-irradiated at 77 K and analysed for free radicals by electron paramagnetic resonance (EPR) spectroscopy in the temperature range 77-280 K. Analysis of spectra according to variation in humidity, microwave power and temperature generally yielded a doublet and a triplet spectrum at 77 K. The doublet partially converted into the 5-thymyl radical (TH.). DNA containing deuterated thymine (dTDNA) revealed that the doublet of 'normal' DNA should be composed of two similar doublets, one of which should be assigned to the thymine anion, the other possibly the cytosine anion. The triplet signal was more stable and could be related to the guanine cation or its deprotonated successor. Several other patterns were detected, among them an allyl radical in highly aquated DNA (95% humidity). Other features occurred either predominantly or exclusively in DNA equilibrated above 66% relative humidity and were ascribed to an influence of the secondary structure. Among them were components possibly indicating H- or D-addition to the cytosine base, a reaction also derivable for dTDNA. Quantitative analysis of the total radical yield and the relative TH. concentration revealed that one cause of the dominance of the latter is its thermal stability vs. other species. The total radical concentration increases with target size up to 76% relative humidity, the hydration water forming an integral part of the DNA. At 95% relative humidity OH. radicals are formed in addition to DNA radicals, showing that ice and hydrated DNA have separated into discrete targets.