Studies on the nature of interaction of iron(III) with alginates

The interactions between the polysaccharide alginate and iron(III) were investigated. The solution properties were studied through pH-metry, viscometry, zeta potential and particle size measurements. In the presence of alginate, iron(III) was stabilized and no precipitation was observed. Studies indicate that iron(III)-alginate system was more stable than iron(III) or alginate alone. The binding constant is of the order of 10(4) M(-1). A case for 'site binding model' for the interaction between alginate and Fe(III) has been made based on the studies using circular dichroism and zeta potential experiments. The number of binding sites per molecule of alginate has been estimated to be 66. This indicates that the alginate can bind more number of Fe(III) ions and thus provide a stable complex which can find wide industrial applications.     

Origin of renal proximal tubular injuries by Fe(III)-nta chelate

Interaction between apo-transferrin and several iron(III) chelates has been investigated in terms of the capillary electrophoresis method. Based on the results, it has been clarified that (i) a binuclear iron(III) unit with an oxo-bridge is necessary for the facile transfer of an iron atom from the iron(III) chelate to apo-transferrin, and (ii) the renal proximal tubular injuries by Fe(III)-nitrilotriacetate (Fe-nta) should be due to the unique binuclear structure of this complex, which gives a peroxide adduct of the binuclear Fe-nta in the presence of glutathione cycle and oxygen.     

Iron (III) induces aggregation of hyperphosphorylated tau and its reduction to iron (II) reverses the aggregation: implications in the formation of neurofibrillary tangles of Alzheimer's disease

Iron as well as aluminum is reported to accumulate in neurons with neurofibrillary tangles (NFTs) of Alzheimer's disease (AD) brain. Previously we demonstrated that aluminum (III) shows phosphate-dependent binding with hyperphosphorylated tau (PHFtau), the major constituent of NFTs, thereby inducing aggregation of PHFtau. Herein we report that iron (III) can also induce aggregation of soluble PHFtau. Importantly, for the aggregation of PHFtau to occur, iron in the oxidized state (III) is essential since iron in the reduced state (II) lacks such ability. Furthermore, iron (III)-induced aggregation is reversed by reducing iron (III) to iron (II). Thus the iron-participating aggregation is mediated not only by tau phosphorylation but also by the transition of iron between reduced (II) and oxidized (III) states. Further incubation of insoluble PHFtau aggregates isolated from AD brain with reducing agents produced liberation of solubilized PHFtau and iron (II), indicating that PHFtau in association with iron (III) constitutes the insoluble pool of PHFtau. These results indicate that iron might play a role in the aggregation of PHFtau leading to the formation of NFTs in AD brain.     

Arene activation by a nonheme iron(III)–hydroperoxo complex: pathways leading to phenol and ketone products

Iron(III)–hydroperoxo complexes are found in various nonheme iron enzymes as catalytic cycle intermediates; however, little is known on their catalytic properties. The recent work of Banse and co-workers on a biomimetic nonheme iron(III)–hydroperoxo complex provided evidence of its involvement in reactivity with arenes. This contrasts the behavior of heme iron(III)–hydroperoxo complexes that are known to be sluggish oxidants. To gain insight into the reaction mechanism of the biomimetic iron(III)–hydroperoxo complex with arenes, we performed a computational (density functional theory) study. The calculations show that iron(III)–hydroperoxo reacts with substrates via low free energies of activation that should be accessible at room temperature. Moreover, a dominant ketone reaction product is observed as primary products rather than the thermodynamically more stable phenols. These product distributions are analyzed and the calculations show that charge interaction between the iron(III)–hydroxo group and the substrate in the intermediate state pushes the transferring proton to the meta-carbon atom of the substrate and guides the selectivity of ketone formation. These studies show that the relative ratio of ketone versus phenol as primary products can be affected by external interactions of the oxidant with the substrate. Moreover, iron(III)–hydroperoxo complexes are shown to selectively give ketone products, whereas iron(IV)–oxo complexes will react with arenes to form phenols instead.     

Formation of spherical iron(III) oxyhydroxide nanoparticles sterically stabilized by chitosan in aqueous solutions

The interactions between the cationic polymer chitosan (Chit) and iron(III) were investigated. The solution properties were studied by pH-metry, viscometry and dynamic light scattering. Solid state iron(III)-Chit samples were also prepared and characterized by IR spectroscopy and electron microscopy. In aqueous solutions, the precipitation pH of the iron(III) oxyhydroxide (FeOOH) is significantly shifted towards the higher pH values in the presence of Chit indicating that some interaction takes place between the iron(III) and the polymer. However, the additivity of the pH-metric titration curves, the lack of variation both in the viscometric and IR spectra of Chit in the presence and absence of iron(III), indicate the lack of direct complexation between the Chit and ferric ions. Isolated FeOOH nanospheres of 5-10 nm diameter were observed on the transmission electron microscopic pictures of samples obtained from solutions containing iron(III) and Chit, while from DLS measurements hydrodynamic units with a few hundred nm in diameter were identified. Our data support that Chit acts as steric stabilizer and inhibits the macroscopic aggregation of the subcolloidal FeOOH particles. Thus the iron(III)-Chit interactions offer a simple and economic way to fabricate nanometric size FeOOH spheres, morphologically similar to the core of iron(III)-storage protein, ferritin.     

Localization and Solubilization of the Iron(III) Reductase of Geobacter sulfurreducens

The iron(III) reductase activity of Geobacter sulfurreducens was determined with the electron donor NADH and the artificial electron donor horse heart cytochrome c. The highest reduction rates were obtained with Fe(III) complexed by nitrilotriacetic acid as an electron acceptor. Fractionation experiments indicated that no iron(III) reductase activity was present in the cytoplasm, that approximately one-third was found in the periplasmic fraction, and that two-thirds were associated with the membrane fraction. Sucrose gradient separation of the outer and cytoplasmic membranes showed that about 80% of the iron(III) reductase was present in the outer membrane. The iron(III) reductase could be solubilized from the membrane fraction with 0.5 M KCl showing that the iron(III) reductase was weakly bound to the membranes. In addition, solubilization of the iron(III) reductase from whole cells with 0.5 M KCl, without disruption of cells, indicated that the iron(III) reductase is a peripheral protein on the outside of the outer membrane. Redox difference spectra of KCl extracts showed the presence of c-type cytochromes which could be oxidized by ferrihydrite. Only one activity band was observed in native polyacrylamide gels stained for the iron(III) reductase activity. Excision of the active band from a preparative gel followed by extraction of the proteins and sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed the presence of high-molecular-mass, cytochrome-containing proteins in this iron(III) reductase activity band. From these experimental data it can be hypothesized that the iron(III) reductase of G. sulfurreducens is a peripheral outer membrane protein that might contain a c-type cytochrome.     

Hydroxylated phytosiderophore species possess an enhanced chelate stability and affinity for iron(III)

Graminaceous plant species acquire soil iron by the release of phytosiderophores and subsequent uptake of iron(III)-phytosiderophore complexes. As plant species differ in their ability for phytosiderophore hydroxylation prior to release, an electrophoretic method was set up to determine whether hydroxylation affects the net charge of iron(III)-phytosiderophore complexes, and thus chelate stability. At pH 7.0, non-hydroxylated (deoxymugineic acid) and hydroxylated (mugineic acid; epi-hydroxymugineic acid) phytosiderophores form single negatively charged iron(III) complexes, in contrast to iron(III)-nicotianamine. As the degree of phytosiderophore hydroxylation increases, the corresponding iron(III) complex was found to be less readily protonated. Measured pKa values of the amino groups and calculated free iron(III) concentrations in presence of a 10-fold chelator excess were also found to decrease with increasing degree of hydroxylation, confirming that phytosiderophore hydroxylation protects against acid-induced protonation of the iron(III)-phytosiderophore complex. These effects are almost certainly associated with intramolecular hydrogen bonding between the hydroxyl and amino functions. We conclude that introduction of hydroxyl groups into the phytosiderophore skeleton increases iron(III)-chelate stability in acid environments such as those found in the rhizosphere or the root apoplasm and may contribute to an enhanced iron acquisition.     

Iron(III) binding in DNA solutions: complex formation and catalytic activity in the oxidation of hydrazine derivatives.

A strong interaction between iron(III) and calf thymus DNA at pH 7.4 was demonstrated in the present study by separation of the complex by column chromatography and by the slow kinetics of iron(III) removal from DNA by disodium-1,2-dihydroxybenzene-3,5-disulfonate (Tiron). An equilibrium constant of 2.1 x 10(14) was calculated by measurements of bound iron(III) by flame atomic absorption spectroscopy and assuming a one iron to two nucleotide stoichiometry. Graphic analysis of the interaction however, indicated that DNA has two binding sites for iron(III) characterized by a stoichiometry of one iron to 12 nucleotides and one iron to 2 nucleotides, and association constants of 4.8 x 10(12) and 2.3 x 10(11), respectively. The DNA-iron(III) complex isolated by column chromatography was shown to catalyze the oxidation of both 2-phenylethylhydrazine and methylhydrazine by spin-trapping experiments with alpha-(4-pyridyl 1-oxide)-N-tert-butylnitrone (POBN). By contrast, oxidation of 1,2-dimethylhydrazine was not catalyzed. Catalysis of 2-phenylethylhydrazine oxidation was confirmed by oxygen consumption studies. The results suggest that iron chelated to DNA may be significant in DNA damage induced by oxidizable chemicals.     

Potentiometric and ³¹P NMR studies on inositol phosphates and their interaction with iron(III) ions

Potentiometric, conductometric and 31P NMR titrations have been applied to study interactions between myo-inositol hexakisphosphate (phytic acid), (±)-myo-inositol 1,2,3,5-tetrakisphosphate and (±)-myo-inositol 1,2,3-trisphosphate with iron(III) ions. Potentiometric and conductometric titrations of myo-inositol phosphates show that addition of iron increases acidity and consumption of hydroxide titrant. By increasing the Fe(III)/InsP(6) ratio (from 0.5 to 4) 3 mol of protons are released per 2 mol of iron(III). At first, phytates coordinate iron octahedrally between P2 and P1,3. The second coordination site represents P5 and neighbouring P4,6 phosphate groups. Complexation is accompanied with the deprotonation of P1,3 and P4,6 phosphate oxygens. At higher concentration of iron(III) intermolecular P-O-Fe-O-P bonds trigger formation of a polymeric network and precipitation of the amorphous Fe(III)-InsP(6) aggregates. (31)P NMR titration data complement the above results and display the largest chemical shift changes at pD values between 5 and 10 in agreement with strong interactions between iron and myo-inositol phosphates. The differences in T(1) relaxation times of phosphorous atoms have shown that phosphate groups at positions 1, 2 and 3 are complexated with iron(III). The interactions between iron(III) ions and inositol phosphates depend significantly on the metal to ligand ratio and an attempt to coordinate more than two irons per InsP(6) molecule results in an unstable heterogeneous system.     

Studies of the reactions of iron(II) ascorbate mixtures with molecular oxygen in solution

Mössbauer and electronic absorbance spectroscopic studies on the reactions of iron(II): ascorbic acid with molecular oxygen in aqueous and methanolic solutions are reported. Both spectroscopic techniques show that in the starting mixtures there are no iron(II): ascorbate complexes. On mixing the iron(II)/ascorbate solution with solutions containing molecular oxygen at pH 6–7 high spin iron(III) is observed in the Mössbauer spectrum. Coloured intermediates, the lifetimes of which are solvent dependent, are seen by stopped-flow spectrophotometry. We assign these coloured intermediates as iron(III) ascorbate complexes. The stoichiometry of the initial reaction between iron(II) and oxygen is shown to be 2Fe(II):O₂ by stopped-flow methods. A scheme for the overall reactions is discussed.     

Synthesis and structural characterization of soluble neuromelanin analogs provides important clues to its biosynthesis

Elucidating the structure and biosynthesis of neuromelanin (NM) would be an important step towards understanding its putative role in the pathogenesis of Parkinson’s disease. A useful complement to studies aimed at unraveling the origin and properties of this essentially insoluble natural substance is the preparation of synthetic derivatives that resemble NM. With this aim in mind, water-soluble conjugates between dopamine-derived melanin and bovine serum albumin (BSA) were synthesized. Melanin–BSA adducts were prepared with both eumelanic oligomers obtained through the oxidative polymerization of dopamine and pheomelanic oligomers obtained under the same conditions from dopamine and cysteine. Iron ions were added during the synthesis to understand the interaction between the pigment and this metal ion, as the NM in neurons in several human brain regions contains significant amounts of iron. The structures of the conjugates were analyzed by ¹H NMR spectroscopy and controlled proteolysis/MS experiments. The binding of iron(III) ions was evaluated by ICP analysis and EPR spectroscopy. The EPR signal from bound iron(III) indicated high-spin octahedral sites and, as also seen for NM, the signal is coupled to a signal from a radical associated with the melanic components of the conjugates. However, the intensity of the EPR signal from iron suggested a reduced fraction of the total iron, indicating that most of the iron is strongly coupled in clusters within the matrix. The amount of paramagnetic, mononuclear iron(III) was greater in the pheomelanin–BSA conjugates, suggesting that iron clustering is reduced in the sulfur-containing pigment. Thus, the melanin–BSA conjugates appear to be good models for the natural pigment.     

Neither human hephaestin nor ceruloplasmin forms a stable complex with transferrin

Iron homeostasis is essential for maintaining the physiological requirement for iron while preventing iron overload. Cell toxicity is caused by the generation of hydroxyl-free radicals that result from redox reactions involving Fe(II). Multicopper ferroxidases regulate the oxidation of Fe(II) to Fe(III), circumventing the generation of these harmful by-products. Ceruloplasmin (Cp) is the major multicopper ferroxidase in blood; however, hephaestin (Hp), a membrane-bound Cp homolog, was recently discovered and has been implicated in the export of iron from duodenal enterocytes into blood. In the intracellular milieu, it is likely that iron exists as reduced Fe(II), yet transferrin (Tf), the plasma iron transporter, is only capable of binding oxidized Fe(III). Due to the insoluble and reactive nature of free Fe(III), the oxidation of Fe(II) upon exiting the duodenal enterocyte may require an interaction between a ferroxidase and the iron transporter. As such, it has been suggested that as a means of preventing the release of unbound Fe(III), a direct protein-protein interaction may occur between Tf and Hp during intestinal iron export. In the present study, the putative interaction between Tf and both Cp and a soluble form of recombinant human Hp was investigated. Utilizing native polyacrylamide gel electrophoresis, covalent cross-linking and surface plasmon resonance (SPR), a stable interaction between the two proteins was not detected. We conclude that a stable complex between these ferroxidases and Tf does not occur under the experimental conditions used. We suggest alternative models for loading Tf with Fe(III) during intestinal iron export.     

Studies on the reactions of ferric iron with ascorbic acid. A study of solution chemistry using Mössbauer spectroscopy and stopped-flow techniques

We report studies on the interaction of iron(III) and ascorbic acid as a function of pH in pure water, pure methanol and mixtures of these solvents.Mössbauer data indicates the iron(III) is reduced in water at low pH to iron(II). Rapid mixing studies and pH jump investigations using stopped flow spectrophotometry have been used to follow the reactions and show evidence for blue intermediates in the reduction pathway of iron at low pH values. A scheme is proposed to account for the complex reaction between iron and ascorbate in aqueous solvent. Binding constants between iron(III) and ascorbate are given.     

Genetic evidence that induction of root Fe(III) chelate reductase activity is necessary for iron uptake under iron deficiency†

Reduction of Fe(III) to Fe(II) by Fe(III) chelate reductase is thought to be an obligatory step in iron uptake as well as the primary factor in making iron available for absorption by all plants except grasses. Fe(III) chelate reductase has also been suggested to play a more general role in the regulation of cation absorption. In order to experimentally address the importance of Fe(III) chelate reductase activity in the mineral nutrition of plants, three Arabidopsis thaliana mutants (frd1-1, frd1-2 and frd1-3), that do not show induction of Fe(III) chelate reductase activity under iron-deficient growth conditions, have been isolated and characterized. These mutants are still capable of acidifying the rhizosphere under iron-deficiency and accumulate more Zn and Mn in their shoots relative to wild-type plants regardless of iron status. frd1 mutants do not translocate radiolabeled iron to the shoots when roots are presented with a tightly chelated form of Fe(III). These results: (1) confirm that iron must be reduced before it can be transported, (2) show that Fe(III) reduction can be uncoupled from proton release, the other major iron-deficiency response, and (3) demonstrate that Fe(III) chelate reductase activity per se is not necessarily responsible for accumulation of cations previously observed in pea and tomato mutants with constitutively high levels of Fe(III) chelate reductase activity.     

Redox Cycling in Iron Uptake,Efflux, and Trafficking

Aerobic organisms are faced with a dilemma. Environmental iron is found primarily in the relatively inert Fe(III) form, whereas the more metabolically active ferrous form is a strong pro-oxidant. This conundrum is solved by the redox cycling of iron between Fe(III) and Fe(II) at every step in the iron metabolic pathway. As a transition metal ion, iron can be “metabolized” only by this redox cycling, which is catalyzed in aerobes by the coupled activities of ferric iron reductases (ferrireductases) and ferrous iron oxidases (ferroxidases).     

Interaction between iron(II) and hydroxamic acids: oxidation of iron(II) to iron(III) by desferrioxamine B under anaerobic conditions

Interaction between iron(II) and acetohydroxamic acid (Aha), alpha-alaninehydroxamic acid (alpha-Alaha), beta-alaninehydroxamic acid (beta-Alaha), hexanedioic acid bis(3-hydroxycarbamoyl-methyl)amide (Dha) or desferrioxamine B (DFB) under anaerobic conditions was studied by pH-metric and UV-Visible spectrophotometric methods. The stability constants of complexes formed with Aha, alpha-Alaha, beta-Alaha and Dha were calculated and turned out to be much lower than those of the corresponding iron(II) complexes. Stability constants of the iron(II)-hydroxamate complexes are compared with those of other divalent 3d-block metal ions and the Irving-Williams series of stabilities was found to be observed. Above pH 4, in the reactions between iron(II) and desferrioxamine B, the oxidation of the metal ion to iron(III) by the ligand was found. The overall reaction that resulted in the formation of the tris-hydroxamato complex [Fe(HDFB)]+ and monoamide derivative of DFB at pH 6 is: 2Fe2+ + 3H4DFB+ = 2[Fe(HDFB)]+ + H3DFB-monoamide+ + H2O + 4H+. Based on these results, the conclusion is that desferrioxamine B can uptake iron in iron(III) form under anaerobic conditions.     

Reversible inhibition of caspase-3 activity by iron(III): potential role in physiological control of apoptosis

Desferoxamine is known to induce apoptosis in cancer cells, but the mechanisms are still not fully understood. We have shown that iron(III) is a potent caspase-3 inhibitor, and the inhibition is reversible by the iron chelating agent desferoxamine. Also, protein disulfide isomerase (PDI) is capable of activating caspase-3 inhibited by iron(III), likely by formation of iron-sulfur complex through its active site thiols. Data presented here suggests that iron(III) could be a potential inhibitor of apoptosis in vivo, by caspase-3-dependent inhibition with a possibility of recovery through PDI overexpression.     

Selective Increase of Iron in Substantia Nigra Zona Compacta of Parkinsonian Brains

Histochemical and biochemical determinations of total iron, iron (II), and iron (III) contents in brain regions from Parkinson's and Alzheimer's diseases have demonstrated a selective increase of total iron content in parkinsonian substantia nigra zona compacta but not in the zona reticulata. The increase of iron content is mainly in iron (III). The ratio of iron (II):iron (III) in zona compacta changes from almost 2:1 to 1:2. This change is thought to be relevant and may contribute to the selective elevation of basal lipid peroxidation in substantia nigra reported previously. Iron may be available in a free state and thus can participate in autooxidation of dopamine with the resultant generation of H2O2 and oxygen free radicals.     

Ability of ferric nitrilotriacetate complex with three pH-dependent conformations to induce lipid peroxidation

This study examined the generation of reactive oxygen species (ROS) and the induction of lipid peroxidation by carcinogenic iron(III)-NTA complex (1:1), which has three conformations with two pKa values (pKa1 approximately 4, pKa2 approximately 8). These conformations are type (a) in acidic conditions of pH 1-6, type (n) in neutral conditions of pH 3-9, and type (b) in basic conditions of pH 7-10. The iron(III)-NTA complex was reduced to iron(II) complex under cool-white fluorescent light without the presence of any reducer. The reduction rates of three species of iron(III)-NTA were in the order type (a) > type (n) > type (b). Iron(III)-NTA-dependent lipid peroxidation was induced in the presence and absence of preformed lipid peroxides (L-OOH) through processes associated with and without photoreduction of iron(III). The order of the abilities of the three species of iron(III)-NTA to initiate the three mechanisms of lipid peroxidation was: (1) type (a) > type (n) > type (b) in lipid peroxidation that is induced L-OOH- and H2O2-dependently and mediated by the photoreduction of iron(III); (2) type (b) > type (n) > type (a) in lipid peroxidation that is induced L-OOH- and H2O2-dependently but not mediated by the photoreduction of iron(III); (3) type (n) > type (b) > type (a) in lipid peroxidation that is induced peroxide-independently and mediated by the photoactivation but not by the photoreduction of iron(III). The rate of lipid peroxidation induced L-OOH-dependently is faster than that induced H2O2-dependently in the mechanism (1), but the rate of lipid peroxidation induced H2O2-dependently is faster than that induced L-OOH-dependently in the mechanism (2). In the lag process of mechanism (3), L-OOH and/or some free radical species, not 1O2, were generated by photoactivation of iron(III)-NTA. These multiple pro-oxidant properties that depend on the species of iron(III)-NTA were postulated to be a principal cause of its carcinogenicity.     

Importance of iron location in iron-induced hydroxyl radical production by brain slices

Iron imbalance has been implicated in oxidative injury associated with many brain diseases. The present study investigated the importance of iron location in hydroxyl radical (.OH) generation and the link between .OH production evaluated by the salicylate method and lipid peroxidation monitored by thiobarbituric acid-reactive substances assay. Brain slices were exposed to increasing doses (2, 10 and 50 microM) of Fe(III) that was complexed either to a lipophilic (8-hydroxyquinoline, HQ) or to a hydrophilic (ammoniacal citrate) ligand. Both iron complexes resulted in an increased salicylate hydroxylation and lipid peroxidation, these effects being significantly more potent in presence of Fe(III)-HQ. Salicylate hydroxylation was linearly correlated to the intensity of TBARS formation but the slope of the curve was found to be higher with Fe(III)-HQ. The present results demonstrate that 1) cell-associated reactive iron is more prone than extracellular iron to induce .OH generation, 2) the level of lipid peroxidation depending on the site of .OH production, cannot be used as an index of the level of total .OH formation, 3) the salicylate method is a convenient method to detect .OH formed intracellularly, at least in vitro.