Ferrous ion formation by ferrioxamine prepared from aged desferrioxamine: A potential prooxidant property

The siderophore desferrioxamine (DEFOM) binds ferric ions in a 1:1 ratio resulting in a ferrioxamine (FOM) complex. When DEFOM is stored or heat degraded, the resulting FOMD undergoes an autoreduction with the transfer of electrons to the bound ferric ions forming ferrous ions, which react with Ferrozine to yield a pink-coloured complex absorbing at 562 nm. Heat-aged DEFOM forms a FOND complex with an absorption maxima changing from 432 nm to 441 nm. When the autoreduced FOMD complex is placed in a phosphate buffer at pH 7.4, ferrous ions autoxidase transferring electrons to molecular oxygen to form superoxide and hydrogen peroxide. Fenton chemistry leading to the formation of hydroxyl radicals can then occur. Studies with a variety of reactive oxygen scavengers support a role for the hydroxyl radical in damage to the detector molecule deoxyribose. However, when EDTA is present, damage to deoxyribose is decreased and the radicals causing deoxyribose degradation no longer appear to be characteristic of the hydroxyl radical.     

THE EFFECT OF CYANIDE AND OF VARIATION IN ALKALINITY ON THE OXIDATION-REDUCTION POTENTIAL OF THE HEMOGLOBIN-METHEMOGLOBIN SYSTEM

The potentiometric method was applied to the study of the influence of cyanide and of hydroxyl ion on methemoglobin. Both of these ions appear to combine with the iron of the methemoglobin molecule and reduce its oxidant activity. From the magnitude of the effect produced by cyanide and by variation in pH on the oxidation-reduction potential of the methemoglobin-hemoglobin system, it is concluded that cyanmethemoglobin and alkaline methemoglobin are undissociated ferric compounds, the first with cyanide and the second with hydroxyl. Electronic formulas, based on the electrical properties of the hemoglobin derivatives, are suggested.     

Stability properties of sperm whale oxymyoglobin

Sperm whale oxymyoglobin was isolated directly from muscle and was examined for its stability properties over the wide range of pH 5–13 in 0.1 m buffer at 25 °C. The remarkable pH dependence for the autoxidation rate was analyzed using the kinetic equation derived in terms of nucleophilic displacement processes of O₂^? from oxymyoglobin by the entering water molecule or hydroxyl ion with the iron resulting in the ferric form. Most of the autoxidation reaction of the oxymyoglobin can be best explained by the proton-catalyzed processes involving the distal histidine as the catalytic residue. The kinetic equation could also be used as an interesting diagnostic probe into differences in the heme reactivity and the heme environment of different types of oxymyoglobin from other sources.     

Bleomycin-dependent damage to the bases in DNA is a minor side reaction.

The antitumor antibiotic bleomycin degrades DNA in the presence of ferric ions and H2O2 or in the presence of ferric ions, oxygen, and ascorbic acid. When DNA degradation is measured as formation of base propenals by the thiobarbituric acid assay, it is not inhibited by superoxide dismutase and scavengers of the hydroxyl radical or by catalase (except that catalase inhibits in the bleomycin/ferric ion/H2O2 system by removing H2O2). Using the technique of gas chromatography/mass spectrometry with selected-ion monitoring, we show that DNA degradation is accompanied by formation of small amounts of modified DNA bases. The products formed are identical with those generated when hydroxyl radicals react with DNA bases. Base modification is significantly inhibited by catalase and partially inhibited by scavengers of the hydroxyl radical and by superoxide dismutase. We suggest that the bleomycin-oxo-iron ion complex that cleaves the DNA to form base propenals can decompose in a minor side reaction to generate hydroxyl radical, which accounts for the base modification in DNA. However, hydroxyl radical makes no detectable contribution to the base propenal formation.     

Hydroxyl radical generation by the tetracycline antibiotics with free radical damage to DNA, lipids and carbohydrate in the presence of iron and copper salts

Tetracycline antibiotics caused the degradation of carbohydrate in the presence of a ferric salt at pH 7.4. This degradation appeared to involve hydroxyl radicals since the damage was substantially reduced by the presence of catalase, superoxide dismutase, scavengers of the hydroxyl radical and metal chelators. Similarly, the tetracycline antibiotics in the presence of a ferric salt greatly stimulated the peroxidation of liposomal membranes. This damage, which did not implicate the hydroxyl radical, was significantly reduced by the addition of chain-breaking antioxidants and metal chelators. Only copper salts in the presence of tetracycline antibiotics, however, caused substantial damage to linear duplex DNA. Studies with inhibitors suggested that damage to DNA did involve hydroxyl radicals.     

Role of globin moiety in the autoxidation reaction of oxymyoglobin: effect of 8 M urea.

It is in the ferrous form that myoglobin or hemoglobin can bind molecular oxygen reversibly and carry out its function. To understand the possible role of the globin moiety in stabilizing the FeO2 bond in these proteins, we examined the autoxidation rate of bovine heart oxymyoglobin (MbO2) to its ferric met-form (metMb) in the presence of 8 M urea at 25 degrees C and found that the rate was markedly enhanced above the normal autoxidation in buffer alone over the whole range of pH 5-13. Taking into account the concomitant process of unfolding of the protein in 8 M urea, we then formulated a kinetic procedure to estimate the autoxidation rate of the unfolded form of MbO2 that might appear transiently in the possible pathway of denaturation. As a result, the fully denatured MbO2 was disclosed to be extremely susceptible to autoxidation with an almost constant rate over a wide range of pH 5-11. At pH 8.5, for instance, its rate was nearly 1000 times higher than the corresponding value of native MbO2. These findings lead us to conclude that the unfolding of the globin moiety allows much easier attack of the solvent water molecule or hydroxyl ion on the FeO2 center and causes a very rapid formation of the ferric met-species by the nucleophilic displacement mechanism. In the molecular evolution from simple ferrous complexes to myoglobin and hemoglobin molecules, therefore, the protein matrix can be depicted as a breakwater of the FeO2 bonding against protic, aqueous solvents.     

Hydrixyl radical generation by the tetracycline antibiotics with free radical damage to DNA, lipids and carbohydrate in the presence of iron and copper salts

Tetracycline antibiotics caused the degradation of carbohydrate in the presence of a ferric salt at pH 7.4. This degradation appeared to involve hydroxyl radicals since the damage was substantially reduced by the presence of catalase, superoxide dismutase, scavengers of the hydroxyl radical and metal chelators. Similarly, the tetracycline antibiotics in the presence of a ferric salt greatly stimulated the peroxidation of liposomal membranes. This damage, which did not implicate the hydroxyl radical, was significantly reduced by the addition of chain-breaking antioxidants and metal chelators. Only copper salts in the presence of tetracycline antibiotics, however, caused substantial damage to linear duplex DNA. Studies with inhibitors suggested that damage to DNA did involve hydroxyl radicals.     

Damage to the bases in DNA induced by hydrogen peroxide and ferric ion chelates

Incubation of a number of ferric ion chelates with H2O2 at pH 7.4 generated a reactive species able to produce chemical modifications of the bases in DNA that are very similar to those produced in DNA by the hypoxanthine/xanthine oxidase system (Aruoma, O.I., Halliwell, B., and Dizdaroglu, M. (1989) J. Biol. Chem. 264, 13024-13028). Products were identified and quantitated by the use of gas chromatography-mass spectrometry with selected-ion monitoring. Compared with other complexes used, ferric ion-nitrilotriacetic acid produced by far the largest amount of the base products. Typical hydroxyl radical scavengers and superoxide dismutase provided significant decreases in the yields of the products. On this basis, it is proposed that ferric ion complexes react with H2O2 to produce hydroxyl radical; this was also shown using the deoxyribose assay. Inhibition of product formation by superoxide dismutase suggests the involvement of superoxide radical in this reaction. It is likely that hydroxyl radical generated by reaction of the ferric ion-nitrilotriacetic acid complex with H2O2 contributes to the carcinogenicity and nephrotoxicity associated with this chelating agent.     

Determination of rate constants for the reaction of hydroxyl radicals with some purines and pyrimidines using sunlight

Sunlight mediated hydroxyl radical production from aqueous ferric perchlorate at low pH has been investigated using deoxyribose-thiobarbituric acid assay. The rate of production of hydroxyl radical was found to be dependent on the time of irradiation. Hydroxyl radical scavengers can compete with deoxyribose for hydroxyl radicals produced in the system leading to a decreased yield of thiobarbituric acid chromogen. The second-order rate constants of the added scavengers can be determined using a simple competition kinetic method. The rate constants for the reaction of hydroxyl radical with a number of purine and pyrimidine derivatives were determined using this method. The rate constants obtained (1-7 x 10(9) dm(3) mol(-1) s(-1)) were found to be in good agreement with those reported using pulse radiolysis technique. The rate constants of dimethyluracil, xanthosine, amino and methyl substituted pyrimidines, cytidine monophosphate and uridine monophosphate were also determined by this method. It is proposed that sunlight mediated production of hydroxyl radical coupled with deoxyribose-thiobarbituric acid assay is a simple and efficient method for the determination of rate constants for the reaction of hydroxyl radical with a wide range of biomolecules.     

An iron hydroxide moiety in the 1.35 Å resolution structure of hydrogen peroxide derived myoglobin compound II at pH 5.2

The biological conversions of O(2) and peroxides to water as well as certain incorporations of oxygen atoms into small organic molecules can be catalyzed by metal ions in different clusters or cofactors. The catalytic cycle of these reactions passes through similar metal-based complexes in which one oxygen- or peroxide-derived oxygen atom is coordinated to an oxidized form of the catalytic metal center. In haem-based peroxidases or oxygenases the ferryl (Fe(IV)O) form is important in compound I and compound II, which are two and one oxidation equivalents higher than the ferric (Fe(III)) form, respectively. In this study we report the 1.35 A structure of a compound II model protein, obtained by reacting hydrogen peroxide with ferric myoglobin at pH 5.2. The molecular geometry is virtually unchanged compared to the ferric form, indicating that these reactive intermediates do not undergo large structural changes. It is further suggested that at low pH the dominating compound II resonance form is a hydroxyl radical ferric iron rather than an oxo-ferryl form, based on the short hydrogen bonding to the distal histidine (2.70 A) and the Fe...O distance. The 1.92 A Fe...O distance is in agreement with an EXAFS study of compound II in horseradish peroxidase.     

Axial coordination of ferric Aplysia myoglobin

Resonance Raman spectra of ferric Aplysia myoglobin in the ligand-free and the azide-bound forms have been studied over a wide pH range to determine the coordination states of the heme iron atom. In the hydroxide form at high pH (approximately 9) the iron is six-coordinate and is in a high/low spin equilibrium. As the pH is lowered below the acid/alkaline transition (pKa = 7.5), the heme becomes five-coordinate. When the pH is lowered even further no other changes in the resonance Raman spectrum are detected; thus, the heme remains five-coordinate down to pH 4, the lowest value studied. For ferric azide-bound Aplysia myoglobin, the iron is six-coordinate in a high/low spin equilibrium at all pH values (4.8-9). These data indicate (i) that the unusual reactivity toward azide previously observed at neutral pH is indeed related to the absence of a coordinated water molecule, and (ii) that causes other than the heme coordination are responsible for the spectral differences and the ligand-binding kinetics differences observed below pH 6.     

The hydroxylation of tryptophan.

Products of the chemical hydroxylation of tryptophan by Fenton and Udenfriend reactions are similar to those obtained by ionizing radiation. When tryptophan is exposed to either of these systems, a mixture of four hydroxytryptophans, oxindole-3-alanine, and N-formylkynurenine is formed. This observation indicates that the hydroxyl radical attacks the aromatic nucleus as well as the 2 and 3 positions of the pyrrole ring. During gamma-radiolysis of nitrous oxide-saturated tryptophan solution and in the absence of oxygen or ferric edta, the hydroxyl radical adduct (or hydroxycyclohexadienyl radical) of tryptophan undergoes dimerization and polymerization, which results in a yellow product with maximal absorbance at 425 nm. In the presence of ferric edta, or in a Fenton system, the hydroxyl radical adduct disproportionates, and hydroxylated derivatives are formed. The yields of the hydroxytryptophans are proportional to the concentration of ferric edta to a limiting yield of 54% of the theoretical yield, which is taken to be one hydroxylated product per two hydroxyl radicals. Under these conditions, 4-, 5-, 6-, and 7-hydroxy-derivatives of tryptophan are found in the proportion 4:2:2:3, respectively. The presence of dioxygen during gamma-radiolysis increases the yield of N-formylkynurenine, but does not affect the total yield of hydroxytryptophans. Similarly, tryptophan subjected to the Udenfriend reaction yields 4-, 5-, 6-, and 7-hydroxytryptophan and N-formylkynurenine in approximately equal amounts.     

Copper salt-dependent hydroxyl radical formation. Damage to proteins acting as antioxidants

Cupric ions (Cu2+) and ferric ions (Fe3+) added to hydrogen peroxide generate hydroxyl radicals (OH) capable of degrading deoxyribose with the formation of thiobarbituric acid-reactive products. This damage can be inhibited by catalase, OH radical scavengers and specific metal ion chelators. All proteins tested nonspecifically inhibited copper-dependent damage but have little effect on the iron-dependent reaction. Copper ions appear to bind to the proteins which prevents formation of OH radicals in free solution. However, OH radicals are still generated at a site-specific location on the protein molecule. Protein damage is detected as fluorescent changes in amino acid residues.     

Hydroxyl radical formation and iron-binding proteins. Stimulation by the purple acid phosphatases

The effect of the purple acid phosphatases with binuclear iron centers (uteroferrin and bovine spleen phosphatase) on hydroxyl radical formation by iron-catalyzed Haber-Weiss-Fenton chemistry has been compared to that of lactoferrin and transferrin. Using 5,5-dimethyl-1-pyrroline-1-oxide to detect superoxide and hydroxyl radicals and the xanthine-xanthine oxidase system to generate superoxide and hydrogen peroxide, we have observed by ESR spectroscopy that both phosphatases were able to promote hydroxyl radical formation. Lactoferrin and transferrin were found incapable of giving rise to these reactive species. This can be explained by the fact that lactoferrin and transferrin carry two Fe(III) atoms per molecule, neither of which are readily reduced by biological reductants. In contrast, the phosphatases possess a binuclear iron center in which one of the iron atoms is stabilized in the ferric state, but the other freely undergoes one-electron redox reactions. The redox-active iron may act as a catalyst of the Haber-Weiss-Fenton sequence, thus enabling the reactions generating hydroxyl radical to proceed. The iron complex of diethylenetriamine penta-acetic acid, also redox active, was investigated and found as well to promote Haber-Weiss-Fenton chemistry.     

An EXAFS study of the interaction of substrate with the ferric active site of protocatechuate 3,4-dioxygenase

X-ray crystallographic studies of the intradiol cleaving protocatechuate 3,4-dioxygenase from Pseudomonas aeruginosa have shown that the enzyme has a trigonal bipyramidal ferric active site with two histidines, two tyrosines, and a solvent molecule as ligands [Ohlendorf, D.H., Lipscomb, J.D., & Weber, P.C. (1988) Nature 336, 403-405]. Fe K-edge EXAFS studies of the spectroscopically similar protocatechuate 3,4-dioxygenase from Brevibacterium fuscum are consistent with a pentacoordinate geometry of the iron active site with 3 O/N ligands at 1.90 A and 2 O/N ligands at 2.08 A. The 2.08-A bonds are assigned to the two histidines, while the 1.90-A bonds are associated with the two tyrosines and the coordinated solvent. The short Fe-O distance for the solvent suggests that it coordinates as hydroxide rather than water. When the inhibitor terephthalate is bound to the enzyme, the XANES data indicate that the ferric site becomes 6-coordinate and the EXAFS data show a beat pattern which can only be simulated with an additional Fe-O/N interaction at 2.46 A. Together, the data suggest that the oxygens of the carboxylate group in terephthalate displace the hydroxide and chelate to the ferric site but in an asymmetric fashion. In contrast, protocatechuate 3,4-dioxygenase remains 5-coordinate upon the addition of the slow substrate homoprotocatechuic acid (HPCA). Previous EPR data have indicated that HPCA forms an iron chelate via the two hydroxyl functions.(ABSTRACT TRUNCATED AT 250 WORDS)     

Evaluation of electron-shuttling compounds in microbial ferric iron reduction

Iron-reducing bacteria can transfer electrons to ferric iron oxides which are barely soluble at neutral pH, and electron-shuttling compounds or chelators are discussed to be involved in this process. Experiments using semipermeable membranes for separation of ferric iron-reducing bacteria from ferric iron oxides do not provide conclusive results in this respect. Here, we used ferrihydrite embedded in 1% agar to check for electron-shuttling compounds in pure and in enrichment cultures. Geobacter sulfurreducens reduced spatially distant ferrihydrite only in the presence of anthraquinone-2,6-disulfonate, a small molecule known to shuttle electrons between the bacterial cell and ferrihydrite. However, indications for the production and excretion of electron-shuttling compounds or chelators were found in ferrihydrite-containing agar dilution cultures that were inoculated with ferric iron-reducing enrichment cultures.     

Generation of hydroxyl radical from linoleic acid hydroperoxide in the presence of epinephrine and iron.

When linoleic acid hydroperoxide was reacted with ferrous iron, the electron spin resonance signals characteristic of the spin adduct of 5,5-dimethyl-1-pyrroline-N-oxide and the hydroxyl radical were detected. Although the signals were not detected with the hydroperoxide and ferric iron, they were actually found if epinephrine was added, indicating that the hydroxyl radical could be generated from the hydroperoxide upon reduction of the latter with ferrous iron formed by epinephrine. The possible generation of the hydroxyl radical from lipid peroxides in vivo was discussed from the viewpoint of pathogenesis of lipid peroxide-related diseases.     

Comparison of the ability of ferric complexes to catalyze microsomal chemiluminescence, lipid peroxidation, and hydroxyl radical generation

The interaction of microsomes with iron and NADPH to generate active oxygen radicals was determined by assaying for low level chemiluminescence. The ability of several ferric complexes to catalyze light emission was compared to their effect on microsomal lipid peroxidation or hydroxyl radical generation. In the absence of added iron, microsomal light emission was very low; chemiluminescence could be enhanced by several cycles of freeze-thawing of the microsomes. The addition of ferric ammonium sulfate, ferric-citrate, or ferric-ADP produced an increase in chemiluminescence, whereas ferric-EDTA or -diethylenetriaminepentaacetic acid (detapac) were inhibitory. The same response to these ferric complexes was found when assaying for malondialdehyde as an index of microsomal lipid peroxidation. In contrast, hydroxyl radical generation, assessed as oxidation of chemical scavengers, was significantly enhanced in the presence of ferric-EDTA and -detapac and only weakly elevated by the other ferric complexes. Ferric-desferrioxamine was essentially inert in catalyzing any of these reactions. Chemiluminescence and lipid peroxidation were not affected by superoxide dismutase, catalase, or competitive hydroxyl radical scavengers whereas hydroxyl radical production was decreased by the latter two but not by superoxide dismutase. Chemiluminescence was decreased by the antioxidants propylgallate or glutathione and by inhibiting NADPH-cytochrome P-450 reductase with copper, but was not inhibited by metyrapone or carbon monoxide. The similar pattern exhibited by ferric complexes on microsomal light emission and lipid peroxidation, and the same response of both processes to radical scavenging agents, suggests a close association between chemiluminescence and lipid peroxidation, whereas both processes can be readily dissociated from free hydroxyl radical generation by microsomes.     

Effect of pH and oxalic acid on the reduction of Fe3+ by a biomimetic chelator and on Fe3+ desorption/adsorption onto wood: Implications for brown-rot decay

Fenton reaction is thought to play an important role in wood degradation by brown-rot fungi. In this context, the effect of oxalic acid and pH on iron reduction by a biomimetic fungal chelator and on the adsorption/desorption of iron to/from wood was investigated. The results presented in this work indicate that at pH 2.0 and 4.5 and in the presence of oxalic acid, the phenolate chelator 2,3-dihydroxybenzoic acid (2,3-DHBA) is capable of reducing ferric iron only when the iron is complexed with oxalate to form Fe³⁺-mono-oxalate (Fe(C₂O₄)⁺). Within the pH range tested in this work, this complex formation occurs when the oxalate:Fe³⁺ molar ratio is less than 20 (pH 2.0) or less than 10 (pH 4.5). When aqueous ferric iron was passed through a column packed with milled red spruce (Picea rubens) wood equilibrated at pH 2.0 and 4.5, it was observed that ferric iron binds to wood at pH 4.5 but not at pH 2.0, and the bound iron could then be released by application of oxalic acid at pH 4.5. The release of bound iron was dependent on the amount of oxalic acid applied in the column. When the amount of oxalate was at least 20-fold greater than the amount of iron bound to the wood, all bound iron was released. When Fe–oxalate complexes were applied to the milled wood column equilibrated in the pH range of 2–4.5, iron from Fe–oxalate complexes was bound to the wood only when the pH was 3.6 or higher and the oxalate:Fe³⁺ molar ratio was less than 10. When 2,3-DHBA was evaluated for its ability to release iron bound to the milled wood, it was found that 2,3-DHBA possessed a greater affinity for ferric iron than the wood as 2,3-DHBA was capable of releasing the ferric iron bound to the wood in the pH range 3.6–5.5. These results further the understanding of the mechanisms employed by brown-rot fungi in wood biodegradation processes.     

A catalyst function for MPTP in superoxide formation

We demonstrate that 1-methyl-4-phenyl-1,2-dihydropyridine (MPDP) can be generated, in an alternate pathway, from the catalyst action of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) upon the iron redox equilibrium reaction. Superoxide and ferric iron are instantaneously produced after addition of MPTP to a solution of ferrous iron. This reaction is oxygen and pH dependent. Superoxide, through a iron dependent Haber-Weiss reaction with peroxide, can generate the cytotoxic hydroxyl radical. A small portion of the superoxide reacts with MPTP to produce the reactive species X. which, in the presence of Fe+3 can also generate MPDP.