Multi-frequency high-field EPR studies on metal-substituted xylose isomerase.

The bacterial enzyme D-xylose isomerase (XI) catalyses the conversion of D-xylose to D-xylulose. Each subunit of the homotetrameric protein contains a bimetallic active centre requiring divalent metal ions such as Mg2+, Mn2+ or Co2+ for catalytic activity. We report here on XI in which the metal binding site 1 is specifically loaded with EPR active Mn2+, while binding site 2 is occupied by Co2+ or Cd2+, rendering a catalytically active or inactive species respectively. The Q-band (34 GHz) EPR spectra of these mixed-metal samples (Co2+/Mn2+ and Cd2+/Mn2+ XI) show a clear influence of the metal in site 2 on the Mn2+ EPR parameters. Likewise, a systematic increase of the zero field splitting parameters (zfs) of Mn2+ in site 1 upon incubation with the inhibitor xylitol or substrates for both mixed-metal samples is found. For Co2+/Mn2+ XI complexed with substrate, a drastic line broadening of the central -1/2 <--> +1/2 transition is observed in Q-band EPR, which was not amenable to analysis so far. By means of a multi-frequency approach at frequencies beyond Q-band, the relevant zfs parameters were derived from spectral simulations of EPR spectra measured at 94, 285 and 670 GHz. It is shown that parallel to the increase of the D-value its distribution also grows considerably in going from free Co2+/Mn2+ XI to the species complexed with inhibitor or substrate. For XI with bound substrate, D-values in the range of 70-90 mT and a distribution of about 30 mT were found from simulation trials. The large distribution in zfs values is thought to be correlated to the structural disorder induced by the shift of the metal ion of site 2 into a location necessary for the isomerisation reaction. The results are discussed with respect to high-resolution crystal data.

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.

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'.

Probing magnetic properties of the reduced [2Fe-2S] cluster of the ferredoxin from Arthrospira platensis by 1H ENDOR spectroscopy.

The 1H electron nuclear double resonance (ENDOR) spectra in frozen solutions of the reduced [2Fe-2S] cluster in ferredoxin from Arthrospira (Spirulina) platensis have been measured at low temperatures (5-20 K) and simulated using orientational selection methods. The analysis confirmed the existence of a single paramagnetic species with iron valence states II and III connected uniquely to the cluster irons. The experimental ENDOR spectra were fitted to a model including the spin distribution on the centre, the orientation of the g-matrix, and the isotropic and anisotropic hyperfine couplings of the nearest protons in the crystallographically determined structure. In order to partially simulate ENDOR line shapes, a statistical distribution of the corresponding torsion angles between the Fe(III) centre and one of the beta-CH2 protons was introduced. From the analysis, four of the larger hyperfine couplings found were assigned to the cysteine beta-protons near the Fe(III) ion of the cluster, with isotropic hyperfine couplings ranging from 1.6 to 4.1 MHz. The spin distribution on the two iron ions was estimated to be +1.85 for the Fe(III) ion and -0.9 for the Fe(II) ion. The Fe(III) ion was identified as being coordinated to the cysteine ligands Cys49 and Cys79, confirming previous NMR results. The direction of the g-tensor with respect to the cluster was deduced. The g1-g2 plane is parallel to the planes through each iron and its adjacent cysteine sulfurs; the g2-g3 plane is nearly perpendicular to the latter planes and deviates by 25 degrees from the FeSSFe plane. The g1 direction is dominated by the bonding geometry of Fe(II) and does not align with the Fe(II)-Fe(III) vector.

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.

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.

Kinetics and interactions of molybdenum and iron-sulfur centers in bacterial enzymes of the xanthine oxidase family: mechanistic implications.

For isoquinoline 1-oxidoreductase (IsoOr), the reaction mechanism under turnover conditions was studied by EPR spectroscopy using rapid-freeze methods. IsoOr displays several EPR-active Mo(V) species including the "very rapid" component found also in xanthine oxidase (XanOx). For IsoOr, unlike XanOx or quinoline 2-oxidoreductase (QuinOr), this species is stable for about 1 h in the absence of an oxidizing substrate [Canne, C., Stephan, I., Finsterbusch, J., Lingens, F., Kappl, R., Fetzner, S., and Hüttermann, J. (1997) Biochemistry 36, 9780-9790]. Under rapid-freeze conditions in the presence of ferricyanide the very rapid species behaves as a kinetically competent intermediate present only during steady-state turnover. To explain the persistence of the very rapid species in IsoOr in the absence of an added oxidant, extremely slow product dissociation is required. This new finding that oxidative conditions facilitate decay of the very rapid signal for IsoOr supports the mechanism of substrate turnover proposed by Lowe, Richards, and Bray [Lowe, D. J., Richards, R. L., and Bray, R. C. (1997) Biochem. Soc. Trans. 25, 774-778]. Additional stopped-flow data reveal that alternative catalytic cycles occur in IsoOr and show that the product dissociates after transfer of a single oxidizing equivalent from ferricyanide. In rapid-freeze measurements magnetic interactions of the very rapid Mo(V) species and the iron-sulfur center FeSI of IsoOr and QuinOr were observed, proving that FeSI is located close to the molybdopterin cofactor in the two proteins. This finding is used to relate the two different iron-sulfur centers of the aldehyde oxidoreductase structure with the EPR-detectable FeS species of the enzymes.

Flavonol 2,4-dioxygenase from Aspergillus niger DSM 821, a type 2 CuII-containing glycoprotein.

Flavonol 2,4-dioxygenase, which catalyzes the cleavage of quercetin to carbon monoxide and 2-protocatechuoyl-phloroglucinol carboxylic acid, was purified from culture filtrate of Aspergillus niger DSM 821 grown on rutin. It is a glycoprotein (46-54% carbohydrate) with N-linked oligo-mannose type glycan chains. The enzyme was resolved in SDS polyacrylamide gels in a diffuse protein band that corresponded to a molecular mass of 130-170 kDa. When purified flavonol 2,4-dioxygenase was heated, it dissociated into three peptides with apparent molecular masses of 63-67 kDa (L), 53-57 kDa (M), and 31-35 kDa (S), which occurred in a molar ratio of 1:1:1, suggesting a LMS structure. Crosslinking led to a 90-97 kDa species, concomitant with the decrease of staining intensity of the 63-67 kDa (L) and the 31-35 kDa (S) peptides. Analysis by matrix-assisted laser desorption/ionization-time of flight-MS showed peaks at m/z approximately 69 600, m/z approximately 51 700, and m/z approximately 26 500 which are presumed to represent the three peptides of flavonol 2,4-dioxygenase, and a broad peak at m/z approximately 96 300, which might correspond to the LS heterodimer as formed in the crosslinking reaction. Based on the estimated molecular mass of 148 kDa, 1 mol of enzyme contained 1.0-1.6 mol of copper. Ethylxanthate, which specifically reduces CuII to CuI ethylxanthate, is a potent inhibitor of flavonol 2,4-dioxygenase. Metal chelating agents (such as diethyldithiocarbamate, diphenylthiocarbazone) strongly inhibited the enzymatic activity, but inactivation was not accompanied by loss of copper. The EPR spectrum of flavonol 2,4-dioxygenase (as isolated) showed the characteristic parameters of a nonblue type 2 CuII protein. The Cu2+ is assumed to interact with four nitrogen ligands, and the CuII complex has a (distorted) square planar geometry.

The active site of purple acid phosphatase from sweet potatoes (Ipomoea batatas) metal content and spectroscopic characterization.

Purple acid phosphatase from sweet potatoes Ipomoea batatas (spPAP) has been purified to homogeneity and characterized using spectroscopic investigations. Matrix-assisted laser desorption/ionization mass spectrometry analysis revealed a molecular mass of approximately 112 kDa. The metal content was determined by X-ray fluorescence using synchrotron radiation. In contrast to previous studies it is shown that spPAP contains a Fe(III)-Zn(II) center in the active site as previously determined for the purple acid phosphatase from red kidney bean (kbPAP). Moreover, an alignment of the amino acid sequences suggests that the residues involved in metal-binding are identical in both plant PAPs. Tyrosine functions as one of the ligands for the chromophoric Fe(III). Low temperature EPR spectra of spPAP show a signal near g = 4.3, characteristic for high-spin Fe(III) in a rhombic environment. The Tyr-Fe(III) charge transfer transition and the EPR signal are both very sensitive to changes in pH. The pH dependency strongly suggests the presence of an ionizable group with a pKa of 4.7, arising from an aquo ligand coordinated to Fe(III). EPR and UV/visible studies of spPAP in the presence of the inhibitors phosphate or arsenate suggest that both anions bind to Fe(III) in the binuclear center replacing the coordinated water or hydroxide ligand necessary for hydrolysis. The conserved histidine residues of spPAP corresponding to His202 and His296 in kbPAP probably interact in catalysis.

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.

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.

A divalent metal site in the small subunit of the manganese-dependent ribonucleotide reductase of Corynebacterium ammoniagenes.

Based on its metallo-cofactor, the manganese-dependent ribonucleotide reductase (Mn-RRase) responsible for delivery of DNA precursors in the Mn-requiring Gram-positive bacterium Corynebacterium (formerly Brevibacterium) ammoniagenes ATCC 6872 is no longer considered as a simple analogue of the aerobic Fe-RRase of Escherichia coli but as the prototype of the class IV enzymes (1). Deliberate dissociation of the Mn-RRase holoenzyme and an improved sample preparation of the dimeric CA2 protein allowed further characterization of the inherent metallo-cofactor by Q-band electron paramagnetic resonance (EPR) spectroscopy. At 40 K, a distinct hyperfine sextet (I = 5/2,55Mn) pattern with a weak zero-field splitting was detected in the CA2 protein prepared from manganese-sufficient cells displaying high RRase activity as expected. This Q-band Mn(II) signal was absent in the apo-CA2 protein obtained from manganese-depleted cells devoid of this enzymatic activity. The presence of a mixed valence manganese cluster in the C. ammoniagenes RRase is excluded since no complex multiline EPR signals were detected in the CA2 protein even at very low (8 K) temperature. The observed Mn(II) spectrum indicates a protein-bound manganese which was modified in the presence of 5.7 mM p-methoxyphenol, but is insensitive toward 10 mM EDTA. Thus, the manganese appeared to be either strictly bound or buried within a hydrophobic pocket of the CA2 protein, inaccessible for EDTA.

Reconstitution and characterization of the polynuclear iron-sulfur cluster in pyruvate formate-lyase-activating enzyme. Molecular properties of the holoenzyme form.

The glycyl radical (Gly-734) contained in the active form of pyruvate formate-lyase (PFL) of Escherichia coli is generated by the S-adenosylmethionine-dependent pyruvate formate-lyase-activating enzyme (PFL activase). A 5'-deoxyadenosyl radical intermediate produced by the activase has been suggested as the species that abstracts the pro-S hydrogen of the glycine 734 residue in PFL (Frey, M., Rothe, M., Wagner, A. F. V., and Knappe, J. (1994) J. Biol. Chem. 269, 12432-12437). To enable mechanistic investigations of this system we have worked out a convenient large scale preparation of functionally competent PFL activase from its apoform. The previously inferred metallic cofactor was identified as redox-interconvertible polynuclear iron-sulfur cluster, most probably of the [4Fe-4S] type, according to UV-visible and EPR spectroscopic information. Cys --> Ser replacements by site-directed mutagenesis determined Cys-29, Cys-33, and Cys-36 to be essential to yield active holoenzyme. Gel filtration chromatography showed a monomeric structure (28 kDa) for both the apoenzyme and holoenzyme form. The iron-sulfur cluster complement proved to be a prerequisite for effective binding of adenosylmethionine, which induces a characteristic shift of the EPR signal shape of the reduced enzyme form ([4Fe-4S]+) from axial to rhombic symmetry.

Comparative EPR and redox studies of three prokaryotic enzymes of the xanthine oxidase family: quinoline 2-oxidoreductase, quinaldine 4-oxidase, and isoquinoline 1-oxidoreductase.

For three prokaryotic enzymes of the xanthine oxidase family, namely quinoline 2-oxidoreductase, quinaldine 4-oxidase, and isoquinoline 1-oxidoreductase, the electron transfer centers were investigated by electron paramagnetic resonance. The enzymes are containing a molybdenum-molybdopterin cytosine dinucleotide cofactor, two distinct [2Fe-2S] clusters and, apart from isoquinoline 1-oxidoreductase, a flavin adenine dinucleotide. The latter cofactor yields two different organic radical signals in quinoline 2-oxidoreductase and quinaldine 4-oxidase, typical for the neutral and anionic form, respectively. A "rapid" Mo(V) species is present in all enzymes with small differences in magnetic parameters. From spectra simulation of 95Mo-substituted quinoline 2-oxidoreductase, a deviation of 25 degrees between the maximal g and 95Mo-hyperfine tensor component was derived. The very rapid Mo(V) species was detected in small amounts upon reduction with substrates in quinoline 2-oxidoreductase and quinaldine 4-oxidase, but showed a different kinetic behavior with considerable EPR intensities in isoquinoline 1-oxidoreductase. The FeSI and FeSII centers produced different signals in all three enzymes and, in case of isoquinoline 1-oxidoreductase, revealed a dipolar interaction, from which a maximum distance of 15 A between FeSI and FeSII was estimated. The midpoint potentials of the FeS centers were surprisingly different and determined for FeSI/FeSII with -155/-195 mV in quinoline 2-oxidoreductase, -250/-70 mV in quinaldine 4-oxidase, and +65/+10 mV in isoquinoline 1-oxidoreductase. The slopes of the fitting curves for the Nernst equation are indicative for nonideal behavior. Only in quinoline 2-oxidoreductase, an averaged midpoint potential of the molybdenum redox pairs of about -390 mV could be determined. Both of the other enzymes did not produce Mo(V) signals in redox titration experiments, probably because of direct reduction of Mo(VI) to Mo(IV) in the presence of dithionite.

Electron paramagnetic resonance of D-xylose isomerase: evidence for metal ion movement induced by binding of cyclic substrates and inhibitors.

The interactions of substrates and inhibitors with the Mn2+ ions in the binuclear active center of D-xylose isomerase (XylI) were investigated by EPR spectroscopy at X- and Q-band frequencies. The metal binding site 1 (A site) was specifically occupied with Mn2+ ions by blocking the high-affinity metal binding site 2 (B-site) either with Co2+ ions, resulting in a catalytically active enzyme, or with Cd2+ or Pb2+ ions yielding an inactive enzyme species. Incubation of both the Co2+/Mn2+- and the Cd2+/Mn2+-XylI with the acyclic inhibitor xylitol revealed EPR spectra with well-resolved hyperfine patterns, but with increased zero field splitting (zfs) parameter D compared to the spectra without inhibitor. D was estimated by spectral simulation of the central --1/2<-->1/2 fine structure transition. D values of 33 and 50 mT were obtained for the Co2+/Mn2+-XylI and the Cd2+/Mn2+-XylI samples, respectively. These results indicate direct interaction of the xylitol with the Mn2+ in the A-site. More drastic changes are observed with the substrates D-xylose and D-glucose and with the cyclic inhibitors 5-thio-alpha-D-glucose and 2-desoxy-D-glucose. For Cd2+/Mn2+-XylI, the EPR spectra with substrates and cyclic inhibitors are similar to each other but different from the spectra with the acylic inhibitor xylitol. They exhibit well-resolved line patterns with a relative large zero field splitting, which was estimated to be in the range of D = 65-85 mT in the various complexes. Binding of substrates or of cyclic inhibitors to the Co2+/ Mn2+-XylI yields EPR spectra without resolved hyperfine interactions, indicative of dipolar interaction between the two paramagnetic metal ions. This can be explained with a decrease in the metal-metal distance. Furthermore, the EPR data strongly suggest that the corresponding metal ion movement is induced by binding of the cyclic conformation of either substrates or cyclic inhibitors and not by binding of the extended form of the sugars.

Release of unaltered bases from polycrystalline pyrimidine DNA constituents after X-irradiation and bombardment with heavy ions.

PURPOSE: The radiation-induced release of unaltered bases from the lyophilized pyrimidine DNA constituents TMP, dCMP, CMP and dCyd was determined quantitatively in order to study explicitly the direct radiation effect. MATERIALS AND METHODS: X-irradiation under an air or a nitrogen gas atmosphere and heavy ion bombardment in the beam vacuum were employed at 300 K. The release of free bases was investigated using HPLC and 1H-NMR. Dose-yield curves for free radicals of X-irradiated TMP and dCMP samples were determined by EPR spectroscopy. RESULTS: As one of the main products, the release of unaltered bases is linear with dose up to 360 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 J-1. The heavy ion bombardment and X-irradiation under nitrogen effected a lower yield for base release than that for X-irradiation under air. For X-irradiation at sufficiently high doses about one order of magnitude difference in yields between bases released and free radicals formed was found. CONCLUSIONS: Compared with related findings as described in the literature, the G values from DNA are in the same order of magnitude, but from nucleosides in frozen aqueous solutions the yields of free bases are several orders of magnitude smaller.

Nature of the chlorine-centred paramagnetic species in irradiated crystals of cytosine hydrochloride doped with thiocytosine.

It has been demonstrated that the paramagnetic species formed in crystals of cytosine hydrochloride doped with thiocytosine are most likely the radicals formed by association of thiocytosine and chlorine. The unpaired electron is positively identified on the Cl atom and on at least one of the pyrimidine atoms, and the data are consistent with the formation of the 'three-electron' S-Cl sigma sigma * bonds. The spin density derived from the measured 35Cl and 14N coupling tensors compare reasonably well with the MO calculations for the 'in-plane' conformations of three hypothetical radicals formed by initial ionization of either thiocytosine or Cl- followed by their reaction. Two distinct and related centres, A and B, probably represent two different conformations of chemically the same radical in the crystal lattice of cytosine-HCL.

Formation of C1' located sugar radicals from X-irradiated cytosine nucleosides and -tides in BeF2 glasses and frozen aqueous solutions.

Free radical formation in nucleosides and nucleotides containing cytosine as base was studied after X-irradiation at 77 K of samples prepared in frozen aqueous BeF2 glasses and in frozen aqueous solutions by means of electron spin resonance (ESR) spectroscopy. By comparison with radical patterns from the cytosine base and from 1-CH3-cytosine, by using specifically base-deuterated nucleosides and by comparison between the 2'-deoxy- and the ribonucleotide it could be demonstrated that a radical at the C1'-position of the sugar was formed in nucleosides and nucleotides in both matrices. Quantitative analysis showed that in the BeF2 glass an initial population of about 10% of substrate located species due to this radical was present at 77 K and developed to about 25% after decay of the .OH (.OD) radicals at about 140 K. This was taken as proof that at least part of these radicals were formed from .OH radicals. In frozen aqueous solutions about 20% of C1' located sugar radicals were found to be present at 77 K, the population remaining roughly constant with increasing temperature to 140 K. The mechanistic findings of these unexpected results are discussed in terms of mobile .OH radicals and/or hole transfer in the glass and in the glassy regions of the frozen aqueous solutions.