Iron, metalloenzymes and cytotoxic reactions.

There is considerable evidence implicating iron and other redox-active transition metals as progenitors of reactive intermediates of oxygen (ROI), molecules which lead to oxidative stress and contribute to various neurodegenerative processes. An important aspect of such metal-mediated damage to biomolecules is the site-specific nature of such pathological activity. Iron sequestering molecules, such as ferritin, transferrin, lactotransferrin, melanotransferrin, hemosiderin and heme can serve as cytoprotectants against metal-mediated oxidant damage. Metalloenzymes also constitute an important group of iron sequestering molecules. Metalloenzyme-catalyzed reactions in which metal ions at the enzyme active site undergo redox-cycling in association with O2 are site-specific in nature, and may represent a potential source of ROI-mediated damage to biomolecules. Dysregulation of brain iron and alterations in the levels of metalloenzymes involved in reactions with O2 derived molecules can contribute to neuronal damage. Iron may increase the cytotoxicity of neuronal dopamine by increasing its rate of oxidation to quinones and semiquinones, thereby reducing the level of this neurotransmitter. Interestingly, dopamine also may play an important role in the maintenance of transition-metal homeostasis as an iron chelator, since it can form both catecholate and hydroxamate groups, molecules employed by many microorganisms to sequester iron.     

Iron mobilization from asbestos by chelators and ascorbic acid

The ability of chelators and ascorbic acid to mobilize iron from crocidolite, amosite, medium- and short-fiber chrysotile, and tremolite was investigated. Ferrozine, a strong Fe(II) chelator, mobilized Fe(II) from crocidolite (6.6 nmol/mg asbestos/h) and amosite (0.4 nmol/mg/h) in 50 mM NaCl, pH 7.5. Inclusion of ascorbate increased these rates to 11.4 and 4.9 nmol/mg/h, respectively. Ferrozine mobilized Fe(II) from medium-fiber chrysotile (0.6 nmol/mg/h) only in the presence of ascorbate. Citrate and ADP mobilized iron (ferrous and/or ferric) from crocidolite at rates of 4.2 and 0.3 nmol/mg/h, respectively, which increased to 4.8 and 1.0 nmol/mg/h in the presence of ascorbate. Since ascorbate alone mobilized iron from crocidolite (0.5 nmol/mg/h), the increase appeared to result from additional chelation by ascorbate. Citrate also mobilized iron from amosite (1.4 nmol/mg/h) and medium-fiber chrysotile (1.6 nmol/mg/h). Mobilization of iron from asbestos appeared to be a function not only of the chelator, but also of the surface area, crystalline structure, and iron content of the asbestos. These results suggest that iron can be mobilized from asbestos in the cell by low-molecular-weight chelators. If this occurs, it may have deleterious effects since this could result in deregulation of normal iron metabolism by proteins within the cell resulting in iron-catalyzed oxidation of biomolecules.     

Iron binding β-hairpin peptides

Posttranslational modification of tyrosine to 3,4-dihydroxyphenylalanine (dopa) yields a unique functional group in biomolecular systems. Oxidation produces a quinone, which can undergo cross linking while deprotonation is well suited to metal binding. Mussels, tunicates and bacteria chelate iron and other metals with multiple dopa subunits. Solution equilibria between catechols and iron indicate favorable assembly though this interaction has not been studied with highly structured biomolecules, such as peptides, despite their widespread biological applications. Here, a series of β-hairpin peptides are generated. Dopa is involved in an aromatic interaction as the edge position. Despite the presence of the surrounding secondary structure dopa readily undergoes oxidation and cross linking. Formation of bispeptide:iron complexes also occur in the presence of mild to significant aromatic interactions.     

Pro-oxidant activity of apocynin radical

Apocynin has been widely used as an NADPH oxidase inhibitor in many experimental models. However, concern regarding the efficacy, selectivity, and oxidative side effects of the inhibitor is increasing. In this study, our aim was to characterize the pro-oxidant properties of apocynin and the structurally-related compounds vanillin and vanillic acid. Glutathione (GSH), cysteine, ovalbumin, and the coenzyme NADPH were chosen as potential target biomolecules that could be affected by transient free radicals from apocynin, vanillin and vanillic acid. Additionally, trolox and rifampicin were used as models of hydroquinone moieties, which are particularly susceptible to oxidation. Transient radicals were generated by horseradish peroxidase/hydrogen peroxide-mediated oxidation. In the presence of apocynin, oxidation of GSH was increased seven-fold, and the product of this reaction was identified as GSSG. Similar results were obtained for oxidation of cysteine and ovalbumin. Oxidation of the coenzyme NADPH increased more than 100-fold in the presence of apocynin. Apocynin also caused rapid oxidation of trolox and rifampicin to their quinone derivatives. In conclusion, the pro-oxidant activity of apocynin is related to its previous oxidation leading to transient free radicals. This characteristic may underlie some of the recent findings regarding beneficial or deleterious effects of the phytochemical.     

Catalysis of iron core formation in Pyrococcus furiosus ferritin

The hollow sphere-shaped 24-meric ferritin can store large amounts of iron as a ferrihydrite-like mineral core. In all subunits of homomeric ferritins and in catalytically active subunits of heteromeric ferritins a diiron binding site is found that is commonly addressed as the ferroxidase center (FC). The FC is involved in the catalytic Fe(II) oxidation by the protein; however, structural differences among different ferritins may be linked to different mechanisms of iron oxidation. Non-heme ferritins are generally believed to operate by the so-called substrate FC model in which the FC cycles by filling with Fe(II), oxidizing the iron, and donating labile Fe(III)–O–Fe(III) units to the cavity. In contrast, the heme-containing bacterial ferritin from Escherichia coli has been proposed to carry a stable FC that indirectly catalyzes Fe(II) oxidation by electron transfer from a core that oxidizes Fe(II). Here, we put forth yet another mechanism for the non-heme archaeal 24-meric ferritin from Pyrococcus furiosus in which a stable iron-containing FC acts as a catalytic center for the oxidation of Fe(II), which is subsequently transferred to a core that is not involved in Fe(II)-oxidation catalysis. The proposal is based on optical spectroscopy and steady-state kinetic measurements of iron oxidation and dioxygen consumption by apoferritin and by ferritin preloaded with different amounts of iron. Oxidation of the first 48 Fe(II) added to apoferritin is spectrally and kinetically different from subsequent iron oxidation and this is interpreted to reflect FC building followed by FC-catalyzed core formation.     

Valence-tautomerism in high-valent iron and manganese porphyrins

Iron and manganese hemes are "high-valent" when the valence state of the metal exceeds III. Redox chemistry of the high valent metal complexes involves redistribution of holes and electrons over the metal ion and the porphyrin and axial ligands, defined as valence tautomerism. Thus, catalytic pathways of heme-containing biomolecules such as peroxidases, catalases and cytochromes P450 involve valence tautomerism, as do pathways of biomimetic oxygen transfer catalysis by manganese porphyrins, robust catalysts with potential commercial value. Determinants of the site of electron abstraction are key to understanding valence tautomerism. In model systems, metal-centered oxidation is supported by hard anionic axial ligands that are also strongly pi-donating, such as oxo, aryl, bix-methoxy and bis-fluoro groups. Manganese(IV) is more stable than iron(IV) and metal-centered one-electron oxidations occur with weaker pi-donating axial ligands such as bisazido, -isocyanato, -hypochlorito and bis chloro groups. Virtually all known high-valent iron porphyrin complexes oxidized by two-electrons above the ferric state are coordinated by the strongly pi-donating oxo or nitrido ligands. In all well-characterized oxo complexes, iron is in the ferryl state and the second oxidizing equivalent resides on the porphyrin. Complexes with iron(V) have not been definitively characterized. One-electron oxidation of oxomanganese(IV) porphyrin complexes gives the oxomanganese(IV) porphyrin pi-cation redicals. In aqueous solution, oxidation of Mn(III) complexes of tetra cationic N-methylpyridiniumylporphyrin isomers by monooxygen donors yields a transient oxomanganese(V) species.     

Lipoic acid and acetyl-carnitine reverse iron-induced oxidative stress in human fibroblasts

Iron overload occurs frequently in thalassemia and other disorders that require regular blood transfusions. Excess iron is toxic owing to the generation of free radicals that lead to oxidation of biomolecules and tissue damage. In order to identify compounds that reduce oxidative injury from iron, we evaluated alpha-lipoic acid (LA), a multifunctional antioxidant, in iron-overloaded primary human fibroblasts (IMR-90). Oxidant stress was measured using dichlorodihydrofluorescein diacetate that is converted to the fluorescent dichlorofluorescein (DCF) upon oxidation. Exposure to ferric ammonium citrate (FAC) increased the iron-content of IMR-90 cells and caused a rise in oxidant appearance. The addition of LA improved the cellular redox status and attenuated the iron-mediated rise in oxidants in a dose-dependent manner. The R- and RS-enantiomers of LA demonstrated similar antioxidant activity. N-tert-butyl hydroxylamine (NtBHA) treated cells also exhibited a decrease in DCF fluorescence, but at a much higher concentration compared with LA. The combination acetyl-L-carnitine (ALCAR) and LA exhibited superior antioxidant effect at all dose levels. We conclude that LA is highly effective in reversing oxidative stress arising from iron overload and that its antioxidant efficacy is further enhanced in combination with ALCAR.     

Chemical properties of catechols and their molecular modes of toxic action in cells, from microorganisms to mammals

Catechols can undergo a variety of chemical reactions. In this review, we particularly focus on complex formations and the redox chemistry of catechols, which play an inportant role in the toxicity of catechols. In the presence of heavy metals, such as iron or copper, stable complexes can be formed. In the presence of oxidizing agents, catechols can be oxidized to semiquinone radicals and in a next step to o‐benzoquinones. Heavy metals may catalyse redox reactions in which catechols are involved. Further chemical properties like the acidity constant and the lipophilicity of different catechols are shortly described as well. As a consequence of the chemical properties and the chemical reactions of catechols, many different reactions can occur with biomolecules such as DNA, proteins and membranes, ultimately leading to non‐repairable damage. Reactions with nucleic acids such as adduct formation and strand breaks are discussed among others. Interactions with proteins causing protein and enzyme inactivation are described. The membrane–catechol interactions discussed here are lipid peroxidation and uncoupling. The deleterious effect of the interactions between catechols and the different biomolecules is discussed in the context of the observed toxicities, caused by catechols.     

Efficiency of methemoglobin, hemin and ferric citrate in catalyzing protein tyrosine nitration, protein oxidation and lipid peroxidation in a bovine serum albumin-liposome system: Influence of pH

Protein tyrosine nitration, protein oxidation and lipid peroxidation are nitrative/oxidative modification of protein and lipids. In this paper, a BSA (bovine serum albumin)-lecithin liposome system was used to study the nature of different forms of iron, including methemoglobin, hemin and ferric citrate, in catalyzing H₂O₂-nitrite system to oxidize protein and lipid as well as nitrate protein. It was found that in pH range of 5.0-9.0, in pure BSA solution or pure liposome solution, hemin and methemoglobin catalyzed protein tyrosine nitration and lipid peroxidation were decreased with the increasing of pH, while hemin and methemoglobin catalyzed protein oxidation was significantly and moderately increased, respectively. Lipid completely inhibited hemin catalyzed protein tyrosine nitration but only partially inhibited methemoglobin catalyzed protein tyrosine nitration, and its inhibitory effect on hemin induced protein oxidation was also more pronounced. In addition, BSA showed more efficient in inhibiting hemin and ferric citrate induced lipid peroxidation. At the same condition, ferric citrate was relatively ineffective in all tests. Considering protein tyrosine nitration, protein oxidation and lipid oxidation as overall oxidative damage, these results indicated that methemoglobin is more toxic than hemin and ferric citrate, the degradation procedure of heme containing macromolecules, e.g. hemoglobin to hemin and finally to low molecular weight bounded iron, is step by step detoxification. These results provide fundamental knowledge on oxidative/nitrative of biomolecules in lipid-protein coexistence system.     

The role of labile iron pool in cardiovascular diseases

Although multiple factors are associated with cardiovascular pathology, there is now an impressive body of evidence that free radicals and nonradical oxidants might cause a number of cardiovascular dysfunctions. Both direct damage to cellular components and/or oxidation of extracellular biomolecules, e.g. LDL, might be involved in the aetiology of cardiovascular diseases. The key molecules in this process seem to be iron and copper ions that catalyse formation of the highly reactive hydroxyl radical. Chelation of iron ions has a beneficial effect on the processes associated with the development of atherosclerosis and formation of post-ischemic lesions. These findings are indirectly supported by the increasing body of evidence that stored body iron plays a crucial role in pathogenesis of atherosclerosis and ischemia/reperfusion injury.     

Utilization of Glucose and the Effect of Organic Compounds on the Chemolithotroph Thiobacillus ferrooxidans

The utilization of glucose by the chemolithotroph Thiobacillus ferrooxidans results in a repression of the ability to oxidize iron, the substrate for autotrophic growth. An assay with resting cells was used to measure iron oxidation rates. Concomitant with the decreased iron oxidation rates, the enzyme responsible for carbon dioxide fixation, ribulose diphosphate (RuDP) carboxylase, was also repressed. Maximum iron oxidation rates precede peak RuDP carboxylase levels, consistent with the role of these processes in autotrophic metabolism in nonrepressed cells. The degree of iron oxidation repression depends on the organic substrate supplied, as does the level of RuDP carboxylase. The uptake of glucose parallels an increase in synthesis of glucose-6-phosphate dehydrogenase and the accumulation in cells of poly-beta-hydroxybutyrate. The organism is also capable of growing on glucose and other organic supplements in the absence of its inorganic energy source; growth rates depend on the organic substrate supplied.     

Iron metabolism genes in Antarctic notothenioids: A review

Iron is an essential element for metabolic processes intrinsic to life, but the properties that make iron a necessity also make it potentially deleterious. To avoid harm, iron homeostasis is achieved through iron transport, storage and regulatory proteins. The functions of some of these molecules are well described, for example transferrin and ferritin, whereas the roles of others remain unclear. The past decade has seen the identification of new molecules involved in iron metabolism, such as divalent metal transporter-1, and hepcidin. The present review aims at surveying the studies carried out on some of the most important genes involved in transport and storage of iron in Antarctic Notothenioidei, a dominating fish group endowed of a number of striking adaptive characters, including reduced (or absence of) hematocrit. This unique peculiarity among vertebrates makes this fish group a suitable system to studying the relationship between hemoglobin and iron metabolism and to understanding the adaptive changes occurred in Antarctic fish metabolism during their evolution to avoid the deleterious effects of iron overload in the absence of hemoglobin. The results summarised here indicate that the loss of hemoglobin in the most specialized group of Antarctic notothenioids, belonging to the Channychthyidae family, is accompanied by remodulation of the iron metabolism.     

Ferritin as a source of iron for oxidative damage.

The generation of deleterious activated oxygen species capable of damaging DNA, lipids, and proteins requires a catalyst such as iron. Once released, ferritin iron is capable of catalyzing these reactions. Thus, agents that promote iron release may lead to increased oxidative damage. The superoxide anion formed enzymatically, radiolytically, via metal-catalyzed oxidations, or by redox cycling xenobiotics reductively mobilizes ferritin iron and promotes oxidative damage. In addition, a growing list of compounds capable of undergoing single electron oxidation/reduction reactions exemplified by paraquat, adriamycin, and alloxan have been reported to release iron from ferritin. Because the rapid removal of iron from ferritin requires reduction of the iron core, it is not surprising that the reduction potential of a compound is a primary factor that determines whether a compound will mobilize ferritin iron. The reduction potential does not, however, predict the rate of iron release. Therefore, ferritin-dependent oxidative damage may be involved in the pathogenesis of diseases where increased superoxide formation occurs and the toxicity of chemicals that increase superoxide production or have an adequate reduction potential to mobilize ferritin iron.     

Induction of Oxidant Stress by Iron Available in Advanced Forms of Plasmodium Falciparum

Oxidative stress has been incriminated as a deleterious factor in the development of malaria parasites. Various chemical reductones which can undergo cyclic oxidation and reduction, such as ascorbate have been shown to cause oxidative stress to red blood cells. This, naturally-occurring and redox-active compound, can induce the formation of active oxygen derived species, such as superoxide radicals (.O^?₂), hydrogen peroxide (H₂O₂) and hydroxyl radical (OH.), The formation of the hydroxyl radical, the ultimate deleterious species, is mediated by the redox-active and available transition metals iron and copper in the Haber-Weiss reaction.

During the development of the parasite, hemoglobin is progressively digested and a concurrent release of high levels of iron-containing breakdown products takes place within the red blood cell. Indications for the progressive increase in redox-active iron during the growth of P. falciparum have been recently found in our lab: a) adventitious ascorbatc proved highly detrimental to the parasite when added to the mature forms. In contrast, if the parasitized erythrocytes were in the early phase following invasion, and only low levels of iron-containing structures had been liberated. then the observed effect was a small promotion of parasite development. b) erythrocytes containing mature parasites were more potent than erythrocytes containing ring forms as a source for redox-active iron in the acerbate-driven metal-mediated degradation of DNA. The addition of extracts from parasitized erythrocytes and ascorbate to DNA causcd a dose and time dependent DNA degradation. Non-infected erythrocytes had no effect. These findings could also propose that the parasite-dependent accumulation of redox-active forms of iron within the erythrocytes serve as a biological clock triggering the rupture of the red blood cell membrane at the right moment, when the parasite reaches its maturity.     

Iron uptake and metabolism in the new millennium

Iron is an essential element for metabolic processes intrinsic to life, and yet the properties that make iron a necessity also make it potentially deleterious. To avoid harm, iron homeostasis is achieved through iron transport, storage and regulatory proteins. The functions of some of these molecules are well described, for example transferrin and transferrin receptor-1, whereas the roles of others, such as the transferrin homolog melanotransferrin, remain unclear. The past decade has seen the identification of new molecules involved in iron metabolism, such as divalent metal transporter-1, ferroportin-1, hepcidin, hemojuvelin and heme carrier protein-1. Here, we focus on these intriguing new molecules and the insights gained from them into cellular iron uptake and the regulation of iron metabolism.     

Inhibition of growth, iron, and sulfur oxidation in Thiobacillus ferrooxidans by simple organic compounds.

Iron and sulfur oxidation by Thiobacillus ferrooxidans as well as growth on ferrous iron were inhibited by a variety of low molecular weight organic compounds. The influences of chemical structure of the organic inhibitors, pH, temperature, physical treatment of cells, and added inhibitory or stimulatory inorganic ions and iron oxidation suggest that a major factor contributing to the inhibitory effects on iron oxidation is the relative electronegativity of the organic molecule. The data also suggest that inhibitory organic compounds may (i) directly affect the iron-oxidizing enzyme system, (ii) react abiologically with ferrous iron outside the cell, (iii) interfere with the roles of phosphate and sulfate in iron oxidation, and (iv) nonselectively disrupt the cell envelope or membrane.     

Iron chelation with salicylaldehyde isonicotinoyl hydrazone protects against catecholamine autoxidation and cardiotoxicity

Elevated catecholamine levels are known to induce damage of the cardiac tissue. This catecholamine cardiotoxicity may stem from their ability to undergo oxidative conversion to aminochromes and concomitant production of reactive oxygen species (ROS), which damage cardiomyocytes via the iron-catalyzed Fenton-type reaction. This suggests the possibility of cardioprotection by iron chelation. Our in vitro experiments have demonstrated a spontaneous decrease in the concentration of the catecholamines epinephrine and isoprenaline during their 24-h preincubation in buffered solution as well as their gradual conversion to oxidation products. These changes were significantly augmented by addition of iron ions and reduced by the iron-chelating agent salicylaldehyde isonicotinoyl hydrazone (SIH). Oxidized catecholamines were shown to form complexes with iron that had significant redox activity, which could be suppressed by SIH. Experiments using the H9c2 cardiomyoblast cell line revealed higher cytotoxicity of oxidized catecholamines than of the parent compounds, apparently through the induction of caspase-independent cell death, whereas co-incubation of cells with SIH was able to significantly preserve cell viability. A significant increase in intracellular ROS formation was observed after the incubation of cells with catecholamine oxidation products; this could be significantly reduced by SIH. In contrast, parent catecholamines did not increase, but rather decreased, cellular ROS production. Hence, our results demonstrate an important role for redox-active iron in catecholamine autoxidation and subsequent toxicity. The iron chelator SIH has shown considerable potential to protect cardiac cells by both inhibition of deleterious catecholamine oxidation to reactive intermediates and prevention of ROS-mediated cardiotoxicity.     

Molecular and cellular bases of iron metabolism in humans

Iron is a microelement with the most completely studied biological functions. Its wide dissemination in nature and involvement in key metabolic pathways determine the great importance of this metal for uniand multicellular organisms. The biological role of iron is characterized by its indispensability in cell respiration and various biochemical processes providing normal functioning of cells and organs of the human body. Iron also plays an important role in the generation of free radicals, which under different conditions can be useful or damaging to biomolecules and cells. In the literature, there are many reviews devoted to iron metabolism and its regulation in proand eukaryotes. Significant progress has been achieved recently in understanding molecular bases of iron metabolism. The purpose of this review is to systematize available data on mechanisms of iron assimilation, distribution, and elimination from the human body, as well as on its biological importance and on the major iron-containing proteins. The review summarizes recent ideas about iron metabolism. Special attention is paid to mechanisms of iron absorption in the small intestine and to interrelationships of cellular and extracellular pools of this metal in the human body.     

Chloramine-T in radiolabeling techniques. III. Radioiodination of biomolecules containing thioether groups

A previously reported method for iodination of the tyrosine moiety of oxidation-sensitive biomolecules was found to cause unacceptable damage to biomolecules containing thiols and thioether groups. This was due to the oxidation of the sulfur-containing residues by molecular iodine (I(2)). To selectively iodinate the tyrosine moiety with minimum oxidation to the sulfur functionality, studies of the kinetics of the reactions between I-(3) and various amino acids and small peptides at various pH values in phosphate buffer were undertaken. Within the pH range studied (5.5-8.2), the results showed that the iodination reaction is strongly catalyzed by hydroxide ions, whereas the oxidation of the sulfur group was insensitive to pH. The results also showed that both reactions are strongly catalyzed by HPO-(4) ion. In a complex molecule, such as methionine-enkephalin, oxidation of the methionine residue (undesirable reaction) proceeds in parallel with iodination of the tyrosine residue (desirable reaction). If such a molecule was iodinated in 0.01 M phosphate buffer at pH values above 7.5, the iodination reaction would proceed much more rapidly than the oxidation reaction, resulting in a high yield of iodinated substrate with little oxidative damage.