The role of metal ion binding in generating 8-hydroxy-2'-deoxyguanosine from the nucleoside 2'-deoxyguanosine and the nucleotide 2'-deoxyguanosine-5'-monophosphate

The metal ions Cu(II), Fe(II), and Cr(III) were allowed to react with H(2)O(2) in the presence of either the mononucleoside 2'-deoxyguanosine (dG) or the mononucleotide 2'-deoxyguanosine-5'-monophosphate (dGMP). The percentage of reacted dG or dGMP that formed the oxidative damage marker 8-hydroxy-2'-deoxyguanosine (8-OH-dG) was monitored. Oxidative damage from reactions involving Cu(II) appear dependent on an interaction between copper and N7 on the guanine base. Any interactions involving the phosphate group have little additional effect on overall oxidative damage or 8-OH-dG production. Reactions involving Fe(II) seem very dependent on an interaction that may involve both N7 on the guanine base and the phosphate group. This interaction may slow oxidation of Fe(II) to Fe(III) in solution, keeping iron in a readily available form to undergo the Fenton reaction. Chromium(III) appears to interact with the phosphate group of dGMP, resulting in significant overall oxidative damage. However, production of 8-OH-dG appears to be very dependent on the ability of Cr(III) to interact with N7 on the guanine base, an interaction that seems to be weak for both the mononucleoside and mononucleotide.     

Pulse radiolytic study of the interaction of SO4.- with deoxynucleosides. Possible implications for direct energy deposition

Using the technique of pulse radiolysis it has been demonstrated that the interaction of SO4.- with deoxynucleosides (k approximately less than 2 X 10(8)-2.3 X 10(9) dm3 mol-1 s-1) in aqueous solution at pH 7.0 results in the formation of the corresponding one-electron oxidized radicals which either deprotonate or hydrate to yield OH adducts. Based upon the ease of oxidation of the deoxynucleosides, dG, dA, dC, dT, by SO4.-, the apparent redox potentials are in the order dG much greater than dA approximately equal to dC greater than dT. With the exception of deoxyuridine, the deoxynucleoside radicals produced on interaction with SO4.- have been shown to have oxidizing properties based upon the interactions with tetranitromethane and the nitroxyls, TMPN and NPPN. The deoxynucleoside radicals (dG, dA and dC) do not interact with oxygen (k less than 10(6) dm3 mol-1 s-1) in contrast to the interaction observed with the thymidine radical (k = 2.5 X 10(7) dm3 mol-1 s-1). The implications of these findings are presented in terms of the properties of the discussed radicals as relating to those of potential DNA base radicals (positive centres) produced by direct energy deposition within DNA. The use of SO4.- to mimic, to some extent, the effects of direct energy deposition in DNA may assist in our understanding of the resulting molecular processes relevant to radiobiological studies.     

A pulse radiolytic study of the interaction of nitroxyls with free-radical adducts of purines: consequences for radiosensitization

Using the technique of pulse radiolysis, it has been demonstrated that the radicals, produced on interaction of the hydroxyl radical with purine nucleotides/nucleosides, interact with the nitroxyls, TMPN and NPPN. It has been possible to discern the various interactions in terms of the known redox properties of the various OH-radical adducts of the purines based upon spectral and kinetic data. It has been confirmed that the properties of the radical produced on interaction of Br2-. with dGMP, based upon its subsequent interactions with nitroxyls, are quantitatively the same as those for the .OH-radical adduct of dGMP with oxidizing properties. The implications of these findings are presented in terms of the potential competition between nitroxyls and cellular radiation modifiers for the various DNA radicals with different redox potentials, and thereby assess the potential importance of the reactivity of the oxidizing-purine radicals towards nitroxyls in radiobiological studies.     

Oxygen uptake during the gamma-irradiation of fatty acids

The radiation-induced oxidation of saturated and unsaturated fatty acids in aqueous solutions has been estimated by measurement of the continuous uptake of oxygen using an oxygen electrode. Chain reactions, initiated by HO radicals, are easily identified to be occurring in the case of unsaturated fatty acids. Other mild oxidation agents, namely (SCN)-.2, Br-.2 and N.3, are also found to be capable of oxidizing the polyunsaturated fatty acids. Evidence is presented that O-.2 may also initiate peroxidation. The oxidation of the polyunsaturated fatty acids is dependent on dose rate, fatty acid concentration, temperature and the presence of antioxidant and other protective agents. Kinetic studies of the reaction of (SCN)-.2 and Br-.2 with linoleic and linolenic acids have been carried out using pulse radiolysis. The bimolecular rate constants for both radical species with the lipids are approx 10(7) mol-1 dm3 s-1, below their critical micelle concentrations, and decrease at higher concentrations due to micelle formation.     

Kinetics of phosphate-mediated oxidation of ferrous iron and formation of 8-oxo-2'-deoxyguanosine in solutions of free 2'-deoxyguanosine and calf thymus DNA

Formation of 7,8-dihydro-8-oxo-2'-deoxyguanosine (8-oxo-dG) in solutions of free 2'-deoxyguanosine (dG) and calf thymus DNA (DNA) was compared for the diffusion-dependent and localised production of oxygen radicals from phosphate-mediated oxidation of ferrous iron (Fe2+) to ferric iron (Fe3+). The oxidation of Fe2+ to Fe3+ was followed at 304 nm at pH 7.2 under aerobic conditions. Given that the concentration of Fe2+ >or=phosphate concentration, the rate of Fe2+ oxidation was significantly higher in DNA-phosphate as compared for the same concentration of inorganic phosphate. Phosphate catalysed oxidation of ferrous ions in solutions of dG or DNA led through the production of reactive oxygen species to the formation of 8-oxo-dG. The yield of 8-oxo-dG in solutions of dG or DNA correlated positively with the inorganic-/DNA-phosphate concentrations as well as with the concentrations of ferrous ions added. The yield of 8-oxo-dG per unit oxidised Fe2+ were similar for dG and DNA; thus, it differed markedly from radiation-induced 8-oxo-dG, where the yield in DNA was several fold higher.For DNA in solution, the localisation of the phosphate ferrous iron complex relative to the target is an important factor for the yield of 8-oxo-dG. This was supported from the observation that the yield of 8-oxo-dG in solutions of dG was significantly increased over that in DNA only when Fe2+ was oxidised in a high excess of inorganic phosphate (50 mM) and from the lower protection of DNA damage by the radical scavenger (hydroxymethyl)aminomethane (Tris)-HCl.     

Superoxide and the production of oxidative DNA damage.

The conventional model of oxidative DNA damage posits a role for superoxide (O2-) as a reductant for iron, which subsequently generates a hydroxyl radical by transferring the electron to H2O2. The hydroxyl radical then attacks DNA. Indeed, mutants of Escherichia coli that lack superoxide dismutase (SOD) were 10-fold more vulnerable to DNA oxidation by H2O2 than were wild-type cells. Even the pace of DNA damage by endogenous oxidants was great enough that the SOD mutants could not tolerate air if enzymes that repair oxidative DNA lesions were inactive. However, DNA oxidation proceeds in SOD-proficient cells without the involvement of O2-, as evidenced by the failure of SOD overproduction or anaerobiosis to suppress damage by H2O2. Furthermore, the mechanism by which excess O2- causes damage was called into question when the hypersensitivity of SOD mutants to DNA damage persisted for at least 20 min after O2- had been dispelled through the imposition of anaerobiosis. That behavior contradicted the standard model, which requires that O2- be present to rereduce cellular iron during the period of exposure to H2O2. Evidently, DNA oxidation is driven by a reductant other than O2-, which leaves the mechanism of damage promotion by O2- unsettled. One possibility is that, through its well-established ability to leach iron from iron-sulfur clusters, O2- increases the amount of free iron that is available to catalyze hydroxyl radical production. Experiments with iron transport mutants confirmed that increases in free-iron concentration have the effect of accelerating DNA oxidation. Thus, O2- may be genotoxic only in doses that exceed those found in SOD-proficient cells, and in those limited circumstances it may promote DNA damage by increasing the amount of DNA-bound iron.     

Metabolism and elimination of the endogenous DNA adduct, 3-(2-deoxy-beta-D-erythropentofuranosyl)-pyrimido[1,2-alpha]purine-10(3H)-one, in the rat

Endogenously occurring damage to DNA is a contributing factor to the onset of several genetic diseases, including cancer. Monitoring urinary levels of DNA adducts is one approach to assess genomic exposure to endogenous damage. However, metabolism and alternative routes of elimination have not been considered as factors that may limit the detection of DNA adducts in urine. We recently demonstrated that the peroxidation-derived deoxyguanosine adduct, 3-(2-deoxy-beta-D-erythropentofuranosyl)-pyrimido[1,2-alpha]purine-10(3H)-one (M1dG), is subject to enzymatic oxidation in vivo resulting in the formation of a major metabolite, 6-oxo-M1dG. Based on the administration of [14C]M1dG (22 microCi/kg) to Sprague-Dawley rats (n=4), we now report that 6-oxo-M1dG is the principal metabolite of M1dG in vivo representing 45% of the total administered dose. When [14C]6-oxo-M1dG was administered to Sprague-Dawley rats, 6-oxo-M1dG was recovered unchanged (>97% stability). These studies also revealed that M1dG and 6-oxo-M1dG are subject to biliary elimination. Additionally, both M1dG and 6-oxo-M1dG exhibited a long residence time following administration (>48 h), and the major species observed in urine at late collections was 6-oxo-M1dG.     

Evidence for a radical mechanism of halogenation of monochlorodimedone catalyzed by chloroperoxidase

A radical species of monochlorodimedone has been characterized by its high reactivity with molecular O2. Horseradish peroxidase greatly accelerated O2 uptake by acidic solutions of this substrate; the enzymatic reaction required exogenous H2O2 only with freshly prepared substrate solutions, and the total substrate oxidized was equal to the sum of H2O2 added and O2 consumed. However, with excess Br- and horseradish peroxidase, or high Br- or Cl- and chloroperoxidase, a 1:1 stoichiometry between H2O2 and substrate was observed. In the absence of halide, the stoichiometry of the chloroperoxidase-catalyzed oxidation of monochlorodimedone changed to two molecules of the organic donor per H2O2. Moreover, in the absence of halide, at substrate:H2O2 ratios greater than 2.0, chloroperoxidase catalyzed significant O2 uptake; this enzyme-dependent autoxidation of monochlorodimedone also occurred in the presence of Cl- or Br-, when H2O2 was limiting. These data, and recent evidence from this laboratory for free hypohalous acid as the first product of chloroperoxidase-catalyzed halide oxidation [B. W. Griffin (1983) Biochem. Biophys. Res. Commun. 116, 873-879], strongly support a mixed enzymatic/nonenzymatic radical chain process as the mechanism for halogenation of monochlorodimedone by chloroperoxidase. Both horseradish peroxidase and chloroperoxidase can catalyze either bromination or oxidation of this substrate, depending on the experimental conditions. Implications of these results for the mechanism of HOCl formation catalyzed by chloroperoxidase are considered.     

Oxidative damage to mitochondrial DNA is inversely related to maximum life span in the heart and brain of mammals.

DNA damage is considered of paramount importance in aging. Among causes of this damage, free radical attack, particularly from mitochondrial origin, is receiving special attention. If oxidative damage to DNA is involved in aging, long-lived animals (which age slowly) should show lower levels of markers of this kind of damage than short-lived ones. However, this possibility has not heretofore been investigated. In this study, steady-state levels of 8-oxo-7, 8-dihydro-2'-deoxyguanosine (8-oxodG) referred to deoxyguanosine (dG) were measured by high performance liquid chromatography (HPLC) in the mitochondrial (mtDNA) and nuclear (nDNA) DNA from the heart of eight and the brain of six mammalian species ranging in maximum life span (MLSP) from 3.5 to 46 years. Exactly the same digestion of DNA to deoxynucleosides and HPLC protocols was used for mtDNA and nDNA. Significantly higher (three- to ninefold) 8-oxodG/dG values were found in mtDNA than in nDNA in all the species studied in both tissues. 8-oxodG/dG in nDNA did not correlate with MLSP across species either in the heart (r=-0.68; P<0.06) or brain (r = 0.53; P<0.27). However, 8-oxodG/dG in mtDNA was inversely correlated with MLSP both in heart (r=-0.92; P<0.001) and brain (r=-0.88; P<0.016) tissues following the power function y = a(.)x(b), where y is 8-oxodG/dG and x is the MLSP. This agrees with the consistent observation that mitochondrial free radical generation is also lower in long-lived than in short-lived species. The results obtained agree with the notion that oxygen radicals of mitochondrial origin oxidatively damage mtDNA in a way related to the aging rate of each species.-Barja, G., Herrero, A. Oxidative damage to mitochondrial DNA is inversely related to maximum life span in the heart and brain of mammals.     

Protection or sensitization by thiols or ascorbate in irradiated solutions of DNA or deoxyguanosine.

The initial aim of this study was to investigate how charge and other chemical properties of some radical scavengers influence the radiation-induced formation of 7,8-dihydro-8-oxo-2'-deoxyguanosine (8-oxo-dG) in two model systems. The target molecule, deoxyguanosine (dG), was either organized in the DNA-helix form or present as a free nucleoside in an aerated aqueous phosphate buffer. Samples were irradiated with 137Cs gamma rays, alone or in the presence of different thiols, alcohols or ascorbate with net charges from -1 to +1. The formation of 8-oxo-dG was assayed with reverse-phase HPLC coupled to an electrochemical detector. In the absence of radical scavengers, the radiation-induced formation of 8-oxo-dG in DNA was extensive, and the ratio for formation of 8-oxo-dG was 20-fold higher for DNA compared to dG. The yields of 8-oxo-dG in DNA and dG were 7.7 x 10(-3) micromol J(-1) and 3.8 x 10(-4) micromol J(-1), respectively. Yield-dose plots showed that the efficiency of the positively charged thiol cysteamine to counteract the radiation-induced formation of 8-oxo-dG in DNA was significantly (P < 0.001) greater compared to the uncharged or negatively charged thiols. Uncharged thiols were significantly (0.001 < P < 0.05) more effective in protecting DNA compared to negatively charged thiols. In contrast to the protection against oxidative damage provided by thiols and ascorbate when they were present during irradiation of DNA, the formation of 8-oxo-dG was significantly increased when these compounds were present during irradiation of dG in solution. Compared to the irradiated control, the increase was 11- to 116-fold for thiols and ascorbate, respectively. The enhanced oxidative damage of dG observed in the presence of ascorbate or thiols suggests that secondarily formed radicals from thiols or ascorbate may react with dG, or that transformation of different primary sites of damage on dG to 8-oxo-dG is enhanced.     

A kinetic study on the interaction of deprotonated purine radical cations with amino acids and model peptides

By use of pulse radiolysis techniques, the radical cations of purine nucleotides have been successfully produced by the SO4- ion oxidation. Time-resolved spectroscopic evidence is provided that the one-electron-oxidized radicals of dAMP and dGMP can be efficiently repaired by aromatic amino acids (including tyrosine and tryptophan) via electron transfer reaction. As a model peptide, Arg-Tyr-AcOH was also investigated with regard to its interaction with deprotonated purine radical cations. The rate constants of the electron transfer reactions were determined to be (1 approximately 5) x 10(8) dm(3) mol(-1) s(-1). These results suggest that the aromatic amino acids in DNA-associated proteins may play some role in electron transfer reactions through DNA.     

DNA and 2'-deoxyguanosine damage in the horseradish-peroxidase-catalyzed autoxidation of aldehydes: the search for the oxidizing species

The horseradish-peroxidase(HRP)-catalyzed aerobic oxidation of aldehydes, in particular isobutanal, was used for the oxidative damage of DNA. In isolated calf-thymus DNA, the enzymatic oxidation of isobutanal led to 7,8-dihydro-8-oxoguanine (8-oxoGua) in up to 1.3% yield and appreciable single-strand breaks in supercoiled pBR 322 DNA. For the nucleoside dG, significant amounts of the guanidine-releasing products oxazolone and oxoimidazolidine have been detected, but 7,8-dihydro-8-oxo-2'-deoxyguanosine (8-oxodG) was not obtained. Only enolizable aldehydes are effective, molecular oxygen is essential, and radical scavengers inhibit efficiently the oxidation. Comparative experiments with 3,3,4,4-tetramethyl-1,2-dioxetane (TMD) revealed that triplet-excited acetone does not play a significant role in this enzymatic DNA oxidation. 2-Hydroperoxy-2-methylpropanal, an intermediate in the HRP-catalyzed aerobic oxidation of isobutanal, does not contribute directly in the observed dG conversion. However, the peroxyl radical derived from the 2-hydroperoxy-2-methylpropanal appears to be active as oxidant because model studies with a structurally related peroxyl radical, produced by HRP-catalyzed one-electron oxidation of 3-hydroperoxy-3-methyl-2-butanone, causes both dG conversion and DNA strand breaks, but to a moderate extent. The active oxidant, as established by control experiments, is the peroxyisobutyric acid, that is efficiently formed through the HRP-catalyzed autoxidation of isobutanal. Still more effective is the acylperoxyl radical, conveniently generated from the peracid by one-electron oxidation by HRP.     

A formation mechanism for 8-hydroxy-2'-deoxyguanosine mediated by peroxidized 2'-deoxythymidine

The oxidative formation of 8-hydroxy-2'-deoxyguanosine (8-OHdG) in DNA is closely associated with the induction of degenerative diseases, including cancer. However, the oxidant species participating in the formation of 8-OHdG has yet to be fully clarified. On the basis that peroxyl radicals are a strong candidate for this species, we employed 2,2'-azobis(2-amidinopropane) (AAPH) as a peroxyl radical generator. Exposure of calf thymus DNA to AAPH formed 8-OHdG, but the exposure of 2'-deoxyguanosine (dG) alone did not. From the exposure of various combinations of nucleotides, 8-OHdG was formed only in the presence of dG and thymidine (dT). A mix of dG with an oxidation product of dT, 5-(hydroperoxymethyl)-2'-deoxyuridine, produced 8-OHdG, but the amount formed was small. In contrast, 8-OHdG was produced abundantly by the addition of dG to peroxidized dT with AAPH. Thus, the formation of 8-OHdG was mediated by the peroxidized dT. Instead of artificial AAPH, endogenous peroxyl radicals are known to be lipid peroxides, which are probably the oxidant species for 8-OHdG formation mediated by thymidine in vivo.     

Kinetics of phosphate-mediated oxidation of ferrous iron and formation of 8-oxo-2′-deoxyguanosine in solutions of free 2′-deoxyguanosine and calf thymus DNA

Formation of 7,8-dihydro-8-oxo-2′-deoxyguanosine (8-oxo-dG) in solutions of free 2′-deoxyguanosine (dG) and calf thymus DNA (DNA) was compared for the diffusion-dependent and localised production of oxygen radicals from phosphate-mediated oxidation of ferrous iron (Fe²⁺) to ferric iron (Fe³⁺). The oxidation of Fe²⁺ to Fe³⁺ was followed at 304 nm at pH 7.2 under aerobic conditions. Given that the concentration of Fe²⁺ ≥phosphate concentration, the rate of Fe²⁺ oxidation was significantly higher in DNA-phosphate as compared for the same concentration of inorganic phosphate. Phosphate catalysed oxidation of ferrous ions in solutions of dG or DNA led through the production of reactive oxygen species to the formation of 8-oxo-dG. The yield of 8-oxo-dG in solutions of dG or DNA correlated positively with the inorganic-/DNA-phosphate concentrations as well as with the concentrations of ferrous ions added. The yield of 8-oxo-dG per unit oxidised Fe²⁺ were similar for dG and DNA; thus, it differed markedly from radiation-induced 8-oxo-dG, where the yield in DNA was several fold higher.For DNA in solution, the localisation of the phosphate ferrous iron complex relative to the target is an important factor for the yield of 8-oxo-dG. This was supported from the observation that the yield of 8-oxo-dG in solutions of dG was significantly increased over that in DNA only when Fe²⁺ was oxidised in a high excess of inorganic phosphate (50 mM) and from the lower protection of DNA damage by the radical scavenger (hydroxymethyl)aminomethane (Tris)–HCl.     

Mechanisms of nickel carcinogenesis. Interaction of Ni(II) with 2'-deoxynucleosides and 2'-deoxynucleotides

Interactions of Ni(II) with the base moieties of 2'-deoxynucleosides and 2'-deoxynucleotides were studied by means of UV difference spectroscopy in order to elucidate the mechanisms of site-specific enhancement by Ni(II) of DNA base oxidation with active oxygen species, observed previously (Kasprzak et al., Cancer Res., 49 (1989) 5964; Carcinogenesis, 11 (1990) 647). The interactions were generally weak and could be quantitated only at pH 7.2-7.9. The resulting coordination binding of Ni(II) was stronger with the purine derivatives, especially these of guanine, than with pyrimidine derivatives. Also, Ni(II) interacted more strongly with the bases of 2'-deoxynucleotides than with the bases of 2'-deoxynucleosides. The apparent stability constants for the interactions calculated with the use of a non-linear regression method, equalled 102 +/- 14, 159 +/- 30 and 290 +/- 70 M-1 for Ni(II) coordinated by 5'dAMP, 5'dADP and 5'dATP, respectively, and 305 +/- 73, 191 +/- 54, and 270 +/- 28 M-1 for 5'dGMP, 5'dGDP and 5'dGTP, respectively. Stability constant for the dG Ni(II) interaction was 39 +/- 7 M-1. Interactions of Ni(II) with the bases of dA, dC, dT and the dC- and dT- mono-, di- and tri-phosphates were too weak for meaningful quantitation. The strongest relative Ni(II) interaction with dG may explain high sensitivity of the dG site at the DNA molecule to Ni(II)-mediated oxidation observed in vitro and in vivo. The present results contrast with Ni(II)-directed site specific cleavage of DNA with H2O2 that occurs preferentially at the pyrimidine bases (Kawanishi et al., Carcinogenesis, 10 (1989) 2231).     

Assays for 8-hydroxy-2'-deoxyguanosine: a biomarker of in vivo oxidative DNA damage.

HPLC with electrochemical detection (HPLC-EC) is a highly sensitive and a selective method for detecting 8-hydroxy-2'-deoxyguanosine (oh8dG), a biomarker of oxidative DNA damage that is formed from hydroxyl radical attack of guanine residues in DNA. We propose that the noninvasive measurement of oh8dG in urine can be used to estimate in vivo oxidative damage. Application of this assay to urine samples obtained from rats of different ages and various species provide examples of the utility of this assay. The measurement of steady-state levels of oh8dG in DNA combined with the urinary excretion rates of oh8dG and oh8Gua, offer a powerful approach for estimating oxidative DNA damage and its repair. This method will be useful for studies designed to investigate the relationship of oxidative stress in DNA damage and the role of this damage in aging and cancer.     

The lipocalin alpha1-microglobulin has radical scavenging activity

The lipocalin alpha(1)-microglobulin (alpha(1)m) is a 26-kDa glycoprotein present in plasma and in interstitial fluids of all tissues. The protein was recently shown to have reductase properties, reducing heme-proteins and other substrates, and was also reported to be involved in binding and scavenging of heme and tryptophan metabolites. To investigate its possible role as a reductant of organic radicals, we have studied the interaction of alpha(1)m with the synthetic radical, 2,2'-azino-bis-(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS radical). The lipocalin readily reacted with the ABTS radical forming reduced ABTS. The apparent rate constant for this reaction was 6.3 +/- 2.5 x 10(3) M(-1) s(-1). A second reaction product with an intense purple color and an absorbance maximum at 550 nm was formed at a similar rate. This was shown by liquid chromatography/mass spectrometry to be derived from covalent attachment of a portion of ABTS radical to tyrosine residues on alpha(1)m. The relative yields of reduced ABTS and the purple ABTS derivative bound to alpha(1)m were approximately 2:1. Both reactions were dependent on the thiolate group of the cysteine residue in position 34 of the alpha(1)m polypeptide. Our results indicate that alpha(1)m is involved in a sequential reduction of ABTS radicals followed by trapping of these radicals by covalent attachment. In combination with the reported physiological properties of the protein, our results suggest that alpha(1)m may be a radical reductant and scavenger in vivo.     

Coordination mode and oxidation susceptibility of nickel(II) complexes with 2'-deoxyguanosine 5'-monophosphate and l-histidine

The formation of binary and ternary complexes of Ni(II) with two biologically relevant molecules, 2'-deoxyguanosine 5'-monophosphate (dGMP) and l-histidine (histidine or His) was characterized by potentiometry and UV-visible spectroscopy. For dGMP, the mononuclear complexes with stoichiometries NiH(2)L(+), NiHL and NiL(-) were found. In the mixed system the ternary complexes NiH(2)LA, NiHLA(-) and NiLA(2-) were detected. In binary systems, the Ni(II) ion coordinates to dGMP through the N-7 atom of its purine ring and indirectly through a water molecule bonded to the phosphate group, while in ternary complexes Ni(II) is bonded to all three histidine donors and directly to the phosphate group of dGMP. Both binary and ternary complexes are susceptible to oxidation by H(2)O(2), with the increased formation of 8-oxo-dGMP in the ternary system. The toxicological relevance of these findings stems from possible disturbance by the major biological Ni(II)-His complex of the nucleotide pools homeostasis through the formation of ternary species and oxidation promotion, as well as from 8-oxo-dGMP capacity to inhibit enzymatic elimination of promutagenic oxidized nucleotides from such pools.     

Kinetic studies of reactions of iron(IV)-oxo porphyrin radical cations with organic reductants

Iron(IV)-oxo porphyrin radical cations are observed intermediates in peroxidase and catalase enzymes, where they are known as Compound I species, and the putative oxidizing species in cytochrome P450 enzymes. In this work, we report kinetic studies of reactions of iron(IV)-oxo porphyrin radical cations that can be compared to reactions of other metal-oxo species. The iron(IV)-oxo radical cations studied were those produced from 5,10,15,20-tetramesitylporphryinato-iron(III) perchlorate (1), 5,10,15,20-tetramesitylporphryinato-iron(III) chloride (2), both in CH(3)CN solvent, and that from 5,10,15,20-tetrakis(pentafluorophenyl)porphyrinato-iron(III) perchlorate (3) in CH(2)Cl(2) solvent. The substrates studied were alkenes and activated hydrocarbons diphenylmethane and ethylbenzene. For a given organic reductant, various iron(IV)-oxo porphyrin radical cations react in a relatively narrow kinetic range; typically the second-order rate constants vary by less than 1 order of magnitude for the oxidants studied here and the related oxidant 5,10,15,20-tetrakis(pentafluorophenyl)porphyrinato-iron(IV)-oxo porphyrin radical cation in CH(3)CN solvent. Charge transfer in the transition states for epoxidation reactions of substituted styrenes by oxidants 1 and 2, rho(+) values of -1.9 and -0.9, respectively, mirrors results found previously for related species. Competition kinetic reactions with a catalytic amount of porphyrin iron(III) species and a terminal oxidant give relative rate constants for oxidations of competing substrates that are somewhat smaller than the ratios of absolute rate constants. Water in CH(3)CN solutions has an apparent modest stabilizing effect on oxidant 1 as indicated in slightly reduced rate constants for oxidation reactions. The iron(IV)-oxo porphyrin radical cations are orders of magnitude less reactive than porphyrin-manganese(V)-oxo cations and a corrole-iron(V)-oxo species. The small environment effects found here suggest that high energy demanding hydrocarbon oxidation reactions catalyzed by cytochrome P450 enzymes might require highly reactive iron(V)-oxo transients as oxidants instead of the more stable, isomeric iron(IV)-oxo porphyrin radical cations.