Tannic acid inhibits in vitro iron-dependent free radical formation

The antioxidant activity of tannic acid (TA), a plant polyphenol claimed to possess antimutagenic and anticarcinogenic activities, was studied by monitoring (i) 2-deoxyribose degradation (a technique for OH detection), (ii) ascorbate oxidation, (iii) ascorbate radical formation (determined by EPR analysis) and (iv) oxygen uptake induced by the system, which comprised Fe(III) complexes (EDTA, nitrilotriacetic acid (NTA) or citrate as co-chelators), ascorbate and oxygen. TA removes Fe(III) from the co-chelators (in the case of EDTA, this removal is slower than with NTA or citrate), forming an iron-TA complex less capable of oxidizing ascorbate into ascorbate radical or mediating 2-deoxyribose degradation. The effectiveness of TA against 2-deoxyribose degradation, ascorbate oxidation and ascorbate radical formation was substantially higher in the presence of iron-NTA (or iron-citrate) than with iron-EDTA, which is consistent with the known formation constants of the iron complexes with the co-chelators. Oxygen uptake and 2-deoxyribose degradation induced by Fe(II) autoxidation were also inhibited by TA. These results indicate that TA inhibits OH formation induced by Fe(III)/ascorbate/O(2) mainly by arresting Fe(III)-induced ascorbate oxidation and Fe(II) autoxidation (which generates Fe(II) and H(2)O(2), respectively), thus limiting the production of Fenton reagents and OH formation. We also hypothesize that the Fe(II) complex with TA exhibits an OH trapping activity, which explains the effect of TA on the Fenton reaction.     

Role of Fe(III) in Fe(II)citrate-mediated peroxidation of mitochondrial membrane lipids

In this report we study the effect of Fe(III) on lipid peroxidation induced by Fe(II)citrate in mitochondrial membranes, as assessed by the production of thiobarbituric acid-reactive substances and antimycin A-insensitive oxygen uptake. The presence of Fe(III) stimulates initiation of lipid peroxidation when low citrate:Fe(II) ratios are used ( 4:1). For a citrate:total iron ratio of 1:1 the maximal stimulation of lipid peroxidation by Fe(III) was observed when the Fe(II):Fe(III) ratio was in the range of 1:1 to 1:2. The lag phase that accompanies oxygen uptake was greatly diminished by increasing concentrations of Fe(III) when the citrate:total iron ratio was 1:1, but not when this ratio was higher. It is concluded that the increase of lipid peroxidation by Fe(III) is observed only when low citrate:Fe(II) ratios were used. Similar results were obtained using ATP as a ligand of iron. Monitoring the rate of spontaneous Fe(II) oxidation by measuring oxygen uptake in buffered medium, in the absence of mitochondria, Fe(III)-stimulated oxygen consumption was observed only when a low citrate:Fe(II) ratio was used. This result suggests that Fe(III) may facilitate the initiation and/or propagation of lipid peroxidation by increasing the rate of Fe(II)citrate-generated reactive oxygen species.     

Pyridoxal isonicotinoyl hydrazone inhibits iron-induced ascorbate oxidation and ascorbyl radical formation

Previous work from our laboratory demonstrated that pyridoxal isonicotinoyl hydrazone (PIH) has in vitro antioxidant activity against iron plus ascorbate-induced 2-deoxyribose degradation due to its ability to chelate iron; the resulting Fe(III)-PIH(2) complex is supposedly unable to catalyze oxyradical formation. A putative step in the antioxidant action of PIH is the inhibition of Fe(III)-mediated ascorbate oxidation, which yields the Fenton reagent Fe(II) [Biochim. Biophys. Acta 1523 (2000) 154]. In this work, we demonstrate that PIH inhibits Fe(III)-EDTA-mediated ascorbate oxidation (measured at 265 nm) and the formation of ascorbyl radical (in electron paramagnetic resonance (EPR) studies). The efficiency of PIH against ascorbate oxidation, ascorbyl radical formation and 2-deoxyribose degradation was dose dependent and directly proportional to the period of preincubation of PIH with Fe(III)-EDTA. The efficiency of PIH in inhibiting ascorbate oxidation and ascorbyl radical formation was also inversely proportional to the Fe(III)-EDTA concentration in the media. When EDTA was replaced by the weaker iron ligand nitrilotriacetic acid (NTA), PIH was much more effective in preventing ascorbate oxidation, ascorbyl radical formation and 2-deoxyribose degradation. Moreover, the replacement of EDTA with citrate, a physiological chelator with a low affinity for iron, also resulted in PIH having a higher efficiency in inhibiting iron-mediated ascorbate oxidation and 2-deoxyribose degradation. These results demonstrate that PIH removes iron from EDTA (or from either NTA or citrate), forming an iron-PIH complex that cannot induce ascorbate oxidation effectively, thus inhibiting iron-mediated oxyradical formation. These results are of pharmacological relevance because PIH has been considered for experimental chelating therapy in iron-overload diseases.     

Iron(III)--phosphoprotein chelates: stoichiometric equilibrium constant for interation of iron(III) and phosphorylserine residues of phosvitin and casein.

Estimates of the strength of iron binding to model phosphoproteins were obtained from equilibrium dialysis experiments. Iron-free phosvitin (chicken and frog) or alpha sl-casein (cow) was dialyzed against the iron(III) chelates of nitrilotriacetate (NTA), )ethylenedinitrilo)tetraacetate (EDTA), or citrate. Protein-bound metal was measured at equilibrium; competition of chelator and phosphoprotein for iron(III) was determined by reference to comprehensive equilibrium equations presented in the Appendix. Analysis of the iron-binding data for phosvitin suggested that clusters of di-O-phosphorylserine residues (SerP.SerP) were the most probable iron-binding sites. A stoichiometric equilibrium constant of 10(18.0) was calculated for the formation of the Fe3+(SerP.SerP) chelate. When comared on the basis of phosphate content, casein bound iron more weakly than phosvitin. However, if the stoichiometric equilibrium constant for the formation of the casein Fe3+(SerP.SerP) chelate (10(17.5) was adjusted to account for the fact that a smaller percentage of casein phosphoserines occurs in di-O-phosphorylserine clusters, the affinity of casein and phosvitin for iron was very similar. A theoretical comparison showed that the "strengths" of the ferric chelates can be ranked: EDTA greater than phosphoprotein di-O-phosphorylserine greater than citrate greater than NTA.     

Effects of Chemical Speciation on the Mineralization of Organic Compounds by Microorganisms

The mineralization of 1.0 to 100 ng each of four complexing compounds—oxalate, citrate, nitrilotriacetate (NTA), and EDTA—per ml was tested in media prepared in accordance with equilibrium calculations by a computer program so that the H, Ca, Mg, Fe, or Al complex (chemical species) was predominant. Sewage microorganisms mineralized calcium citrate more rapidly than iron, aluminum, or hydrogen citrate, and magnesium citrate was degraded slowest. Aluminum, hydrogen, and iron oxalates were mineralized more rapidly than calcium oxalate, and magnesium oxalate was decomposed slowest. Sewage microorganisms mineralized calcium NTA but not aluminum, magnesium, hydrogen, or iron NTA or any of the EDTA complexes. Pseudomonas sp. mineralized calcium and iron citrates but had no activity on hydrogen, aluminum, or magnesium citrate. Pseudomonas pseudoalcaligenes mineralized calcium, iron, hydrogen, and aluminum citrates but had little activity on magnesium citrate. Pseudomonas alcaligenes used calcium, iron, hydrogen, and aluminum oxalates readily, but it used magnesium oxalate at a slower rate. Listeria sp. destroyed calcium NTA but had no effect on hydrogen, iron, or magnesium NTA. Increasing the Ca concentration in the medium enhanced the breakdown of NTA by Listeria sp. The different activities of the bacterial isolates were not a result of the toxicity of the complexes or the lack of availability of a nutrient element. NTA mineralization was not enhanced by the addition of Ca to Beebe Lake water, but it was enhanced when Ca and an NTA-degrading inoculum were added to water from an oligotrophic lake. The data show that chemical speciation influences the mineralization of organic compounds by naturally occurring microbial communities and by individual bacterial populations.     

Dissimilatory iron reduction in Escherichia coli: identification of CymA of Shewanella oneidensis and NapC of E. coli as ferric reductases

Over geological time scales, microbial reduction of chelated Fe(III) or Fe(III) minerals has profoundly affected today's composition of our bio- and geosphere. However, the electron transfer reactions that are specific and defining for dissimilatory iron(III)-reducing (DIR) bacteria are not well understood. Using a synthetic biology approach involving the reconstruction of the putative electron transport chain of the DIR bacterium Shewanella oneidensis MR-1 in Escherichia coli , we showed that expression of cymA was necessary and sufficient to convert E. coli into a DIR bacterium. In intact cells, the Fe(III)-reducing activity was limited to Fe(III) NTA as electron acceptor. In vitro biochemical analysis indicated that CymA, which is a cytoplasmic membrane-associated tetrahaem c -type cytochrome, carries reductase activity towards Fe(III) NTA, Fe(III) citrate, as well as to AQDS, a humic acid analogue. The in vitro specific activities of Fe(III) citrate reductase and AQDS reductase of E. coli spheroplasts were 10× and 30× higher, respectively, relative to the specific rates observed in intact cells, suggesting that access of chelated and insoluble forms of Fe(III) and AQDS is restricted in whole cells. Interestingly, the E. coli CymA orthologue NapC also carried ferric reductase activity. Our data support the argument that the biochemical mechanism of Fe(III) reduction per se was not the key innovation leading to environmental relevant DIR bacteria. Rather, the evolution of an extension of the electron transfer pathway from the Fe(III) reductase CymA to the cell surface via a system of periplasmic and outer membrane cytochrome proteins enabled access to diffusion-impaired electron acceptors.     

Lipid peroxidation of rabbit small intestinal microvillus membrane vesicles by iron complexes

Fe(II)- and Fe(III)-induced lipid peroxidation of rabbit small intestinal microvillus membrane vesicles was studied. Ferrous ammonium sulphate, ferrous ascorbate at a molar ratio of 10:1, and ferric citrate, at molar ratios of 1:1 and 1:20, did not stimulate lipid peroxidation. Ferrous ascorbate, 1:1, induced low stimulation, while ferrous ascorbate, 1:20 gave higher stimulation of lipid peroxidation. These results show that in our experimental system, ascorbate is a promotor rather than an inhibitor of lipid peroxidation. Ferric nitrilotriacetate (at molar ratios of 1:2 and 1:10), at an iron concentration of 200 microM, was by far the most effective in inducing lipid peroxidation. Superoxide dismutase, mannitol and glutathione had no effect, while catalase, thiourea and vitamin E markedly decreased ferrous ascorbate 1:20-induced lipid peroxidation. Ferric nitrilotriacetate-induced lipid peroxidation was slightly reduced by catalase and mannitol, significantly reduced by superoxide dismutase, and completely inhibited by thiourea. Glutathione caused a 100% increase in the ferric nitrilotriacetate-induced lipid peroxidation. These results suggest that Fe(II) in the presence of trace amounts of Fe(III), or an oxidizing agent and Fe(III) in the presence of Fe(II) or a reducing agent, are potent stimulators of lipid peroxidation of microvillus membrane vesicles. Addition of deferoxamine completely inhibited both ferrous ascorbate, 1:20 and ferric nitrilotriacetate-induced lipid peroxidation, demonstrating the requirement for iron for its stimulation. Iron-induced peroxidation of microvillus membrane may have physiological significance because it could already be demonstrated at 2 microM iron concentration.     

Kinetics and mechanisms of reduction of Cu(II) and Fe(III) complexes by soybean leghemoglobin alpha

The reduction of low-molecular-weight Cu(II) and Fe(III) complexes by soybean leghemoglobin alpha was characterized using both kinetic analysis and 1H-NMR experiments. Whereas Fe(III) (CN)6(3-) was reduced through an outer sphere transfer over the exposed heme edge, all other Cu(II) and Fe(III) complexes investigated were reduced via a site-specific binding of the metal to the protein. Reduction of all metal complexes was enhanced by decreasing pH while only Fe(III)NTA reduction kinetics were altered by changes in ionic strength. Rates of reduction for both Cu(II) and Fe(III) were also affected inversely by the effective binding constant of the metal chelate used. NMR data confirmed that both Cu(II)NTA and Fe(III)NTA were bound to specific sites on the protein. Cu(II) bound preferentially to distal His-61 and Fe(III) exerted its greatest effect on two surface lysine residues with epsilon proton resonances at 3.04 and 3.12 ppm. The Fe(III)NTA complex also had a mild but noticeable line broadening effect on the distal His-61 singlet resonance near 5.3 ppm. Like hemoglobin and myoglobin, leghemoglobin might function not only as an oxygen carrier, but also as a biological reductant for low-molecular-weight Cu(II) and Fe(III) complexes.     

Iron enriched yeast biomass--a promising mineral feed supplement

Yeast biomass enriched with iron could represent a new and safer solution for prevention from anaemia development. Such an iron source is less toxic and has better absorbability in organisms. The purpose of our research was the determination of the most suitable iron source in the cultivation medium for the yeast Saccharomyces cerevisiae, regarding good growth and iron accumulation in cells. Iron(III) citrate, iron(III) chloride, iron(III) nitrate and Fe-EDTA complex were used. The uptake of the chosen iron compound, Fe(III) citrate, by the yeasts Candida intermedia and Kluyveromyces marxianus was also investigated. Different growth behaviour of the three yeast strains in the presence of Fe(III) citrate was observed. The highest amounts of accumulated iron in S. cerevisiae, C. intermedia and K. marxianus biomass were about 13, 20 and 34mgFeg(-1)dry wt., respectively. To optimise the accumulation of iron in K. marxianus and to characterise iron enriched yeast biomass, further experiments are needed.     

Iron-mediated DNA damage: sensitive detection of DNA strand breakage catalyzed by iron.

Oxidative DNA damage is involved in mutagenesis, carcinogenesis, aging, radiation effects, and the action of several anticancer drugs. Accumulated evidence indicates that iron may play an important role in those processes. We studied the in vitro effect of low concentrations of Fe(II) alone or Fe(III) in the presence of reducing agents on supercoiled plasmid DNA. The assay, based on the relaxation and linearization of supercoiled DNA, is simple yet sensitive and quantitative. Iron mediated the production of single and double strand breaks in supercoiled DNA. Iron chelators, free radical scavengers, and enzymes of the oxygen reduction pathways modulated the DNA damage. Fe(III)-nitrilotriacetate (NTA) plus either H2O2, L-ascorbate, or L-cysteine produced single and double strand breaks as a function of reductant concentration. A combination of 0.1 microM Fe(III)-NTA and 100 microM L-ascorbate induced detectable DNA strand breaks after 30 min at 24 degrees C. Whereas superoxide dismutase was inhibitory only in systems containing H2O2 as reductant, catalase inhibited DNA breakage in all the iron-mediated systems studied. The effect of scavengers and enzymes indicates that H2O2 and .OH are involved in the DNA damaging process. These reactions may account for the toxicity and carcinogenicity associated with iron overload.     

Action of iron and iron-complexes onKlebsiella pneumoniae (Klebsiella aerogenes)

Iron in the Fe(III) oxidation state had a negligible effect on the growth ofKlebsiella pneumoniae even at the highest concentration (0.45mm) obtainable without precipitation in a minimal medium containing glucose and inorganic salts together with Tris as the buffer and glycerol 2-phosphate as the phosphorus source. Nevertheless in its presence the toxic action of Cd²⁺, Zn²⁺ and Cu²⁺ was antagonized while that of Co²⁺ and Ni²⁺ was potentiated. Higher iron levels were obtained by supplementing the minimal medium with fructose, glycine, gluconate, tartrate and citrate at a range of concentrations. With fructose and glycine all of the resulting solutions were red-brown and non-toxic. This was also found with the other complexing agents when the ligand:iron ratios were low, but at higher ligand:iron ratios the solutions were green and toxic. Iron-citrate systems were especially toxic but resistance developed and was of the graded type. The results are discussed with particular reference to earlier physico-chemical studies by other workers and it is concluded that the red-brown colour is characteristic of the presence of polymers of high molar mass and that the green colour signifies the formation of low molar mass species.     

Effects of deferrioxamine on iron-catalyzed lipid peroxidation.

The kinetics of iron binding by deferrioxamine B mesylate and the ramifications of this process upon iron-catalyzed lipid peroxidation were assessed. The relative rates of Fe(III) binding by deferrioxamine varied for the chelators tested as follows: ADP greater than AMP greater than citrate greater than histidine greater than EDTA. The addition of a fivefold molar excess of deferrioxamine to that of Fe(III) did not result in complete binding (within 10 min) for any of the Fe(III) chelates tested except ADP:Fe(III). The rates of Fe(III) binding by deferrioxamine were greater at lower pH and when the competing chelator concentration was high in relationship to iron. The relatively slow binding of Fe(III) by deferrioxamine also affected lipid peroxidation, an iron-dependent process. The addition of deferrioxamine to an ascorbate- and ADP:Fe(III)-dependent lipid peroxidation system resulted in a time-dependent inhibition or stimulation of malondialdehyde formation (i.e., lipid peroxidation), depending on the ratio of deferrioxamine to iron. Converse to Fe(III), the rates of Fe(II) binding by deferrioxamine from the chelators tested above were rapid and complete (within 1 min), and resulted in the oxidation of Fe(II) to Fe(III). Lipid peroxidation dependent on Fe(II) autoxidation was stimulated by the addition of deferrioxamine. Malondialdehyde formation in this system was inhibited by the addition of catalase, and a similar extent of lipid peroxidation was achieved by substituting hydrogen peroxide for deferrioxamine. Collectively, these results suggest that the kinetics of Fe(III) binding by deferrioxamine is a slow, variable process, whereas Fe(II) binding is considerably faster. The binding of either valence of iron by deferrioxamine may result in variable effects on iron-catalyzed processes, such as lipid peroxidation, either via slow binding of Fe(III) or the rapid binding of Fe(II) with concomitant Fe(II) oxidation.     

Accumulation and distribution of iron, cadmium, lead and nickel in cucumber plants grown in hydroponics containing two different chelated iron supplies

Cucumber plants grown in hydroponics containing 10 μM Cd(II), Ni(II) and Pb(II), and iron supplied as Fe(III) EDTA or Fe(III) citrate in identical concentrations, were investigated by total-reflection X-ray fluorescence spectrometry with special emphasis on the determination of iron accumulation and distribution within the different plant compartments (root, stem, cotyledon and leaves). The extent of Cd, Ni and Pb accumulation and distribution were also determined. Generally, iron and heavy-metal contaminant accumulation was higher when Fe(III) citrate was used. The accumulation of nickel and lead was higher by about 20% and 100%, respectively, if the iron supply was Fe(III) citrate. The accumulation of Cd was similar. In the case of Fe(III) citrate, the total amounts of Fe taken up were similar in the control and heavy-metal-treated plants (27-31 μmol/plant). Further, the amounts of iron transported from the root towards the shoot of the control, lead- and nickel-contaminated plants were independent of the iron(III) form. Although Fe mobility could be characterized as being low, its distribution within the shoot was not significantly affected by the heavy metals investigated.     

Kinetics and mechanism of dissimilative Fe(III) reduction by Pseudomonas sp. 200

The kinetics and mechanism of Fe(III) reduction to Fe(II) were studied in pure batch cultures of Pseudomonas sp. 200. The rate of iron reduction has been mechanistically related to aqueous phase iron speciation. In the absence of microbial activity the iron reduction rate was negligible. Initial rates of microbial iron reduction were accelerated more than 20-fold by the addition of equimolar quantities of nitrilotriacetic acid (NTA) to media initially containing 1.86 x 10(-3)M total Fe(III). Numerical techniques were utilized to quantify relationships between the observed rate of Fe(II) production and the calculated (equilibrium) aqueous phase speciation. These results indicate that soluble ferric iron species are not equivalent in terms of their susceptibility to bacterial (dissimilative) iron reduction. The concentration of Fe(NTA)(OH)(2) (2-) correlated strongly with observed iron reduction rates. Ferrous iron species appeared to inhibit the reduction process.     

A comparison of two procedures used for complexing Fe(III) with human apotransferrin. II. Uptake of Fe(III) by K-562 cells from 55Fe . transferrins and Fe . [3H]transferrins

Samples of human apotransferrin (apo . HTr) were saturated with Fe(III) by two different techniques, a method employing excess trisodium citrate to chelate Fe(III) and a nonchelating approach which involves the ferroxidase activity of ceruloplasmin to convert Fe(II)----Fe(III). The samples were radiolabelled with either 55Fe or 3H. Using an initial molar Fe/apo . HTr ratio of 2.0-2.1, preparations of human transferrin with bound Fe (Fe . HTr) using the citrate method invariably contained 2.2-2.4 atoms Fe/molecule, whereas Fe . HTr (ceruloplasmin method) contained 2.0 atoms/molecule as shown by spectrophotometric and radioactivity measurements. Uptake of Fe from these Fe . HTr preparations by K-562 cells grown in a serum-free medium was marginally, but consistently, more rapid from 55Fe . HTr (citrate) than from 55Fe . HTr (ceruloplasmin). Taking account of the different Fe contents of the Fe . HTr preparations, the rate measured over a 2-h period amounted to approximately 12,700 and 16,100 Fe atoms/(cell . min) for Fe . HTr (ceruloplasmin) and Fe . HTr (citrate), respectively. However, cell binding by the two Fe . [3H]HTr preparations did not differ significantly over the 8-h incubation period. Furthermore, from the 3H distribution, the quantities of Fe . HTr bound reversibly at the cell surface and contained within the cell were similar for the two Fe . HTr preparations. The results indicate that apo . HTr may bind Fe in different ways depending on the method of Fe presentation and that the Fe . HTr product can donate Fe to K-562 cells at a rate which may reflect the method used for Fe-complex formation.     

Iron induces protection and necrosis in cultured cardiomyocytes: Role of reactive oxygen species and nitric oxide

We investigate here the role of reactive oxygen species and nitric oxide in iron-induced cardiomyocyte hypertrophy or cell death. Cultured rat cardiomyocytes incubated with 20 μM iron (added as FeCl₃–Na nitrilotriacetate, Fe–NTA) displayed hypertrophy features that included increased protein synthesis and cell size, plus realignment of F-actin filaments along with sarcomeres and activation of the atrial natriuretic factor gene promoter. Incubation with higher Fe–NTA concentrations (100 μM) produced cardiomyocyte death by necrosis. Incubation for 24 h with Fe–NTA (20–40 μM) or the nitric oxide donor Δ-nonoate increased iNOS mRNA but decreased iNOS protein levels; under these conditions, iron stimulated the activity and the dimerization of iNOS. Fe–NTA (20 μM) promoted short- and long-term generation of reactive oxygen species, whereas preincubation with l-arginine suppressed this response. Preincubation with 20 μM Fe–NTA also attenuated the necrotic cell death triggered by 100 μM Fe–NTA, suggesting that these preincubation conditions have cardioprotective effects. Inhibition of iNOS activity with 1400 W enhanced iron-induced ROS generation and prevented both iron-dependent cardiomyocyte hypertrophy and cardioprotection. In conclusion, we propose that Fe–NTA (20 μM) stimulates iNOS activity and that the enhanced NO production, by promoting hypertrophy and enhancing survival mechanisms through ROS reduction, is beneficial to cardiomyocytes. At higher concentrations, however, iron triggers cardiomyocyte death by necrosis.     

Mobilization of iron from crocidolite asbestos by certain chelators results in enhanced crocidolite-dependent oxygen consumption.

The reactivity of iron on crocidolite asbestos with dioxygen was determined and compared with iron mobilized from crocidolite. Ferrozine, a strong Fe(II) chelator, was used to demonstrate that iron on crocidolite was redox active. More Fe(II) was mobilized from crocidolite (1 mg/ml) by ferrozine anaerobically (11.2 nmol/mg crocidolite/h) than aerobically (6.6 nmol/mg/h) in 50 mM NaCl, pH 7.5, suggesting that Fe(II) on crocidolite reacts with O2 upon aqueous suspension. However, suspension of crocidolite in 50 mM NaCl, pH 7.5, did not result in a measurable amount of O2 consumption. The addition of reducing agents (1 mM) increased the amount of Fe(II) on crocidolite, and addition of ascorbate resulted in 0.4 nmol O2 consumed/mg crocidolite/min. Therefore, iron on crocidolite had limited redox activity in the presence of ascorbate. However, mobilization of iron from crocidolite increased its redox activity. Citrate, nitrilotriacetate (NTA), or EDTA (1 mM) mobilized 79, 32, or 58 microM iron, respectively, in preincubations up to 76 h, and increased O2 consumption upon addition of ascorbate to 2.8, 7.6, or 22.0 nmol O2 consumed/mg/min, respectively. This activity depended only upon the presence of a component(s) mobilized from crocidolite by the chelators. Pretreatment of crocidolite with the iron chelator desferrioxamine B (10 mM) inhibited O2 consumption. The results of the present study suggest that iron on or in crocidolite is responsible for the redox activity of crocidolite, but that mobilization of iron by chelators such as citrate, NTA, or EDTA greatly enhances its redox activity. Thus, iron mobilization from crocidolite in vivo by low-molecular-weight chelators may lead to the increased production of reactive oxygen species which may damage biomolecules, such as DNA.     

Bovine heart microsomes contain an Mr = 66,000 non-heme iron protein which stimulates NADPH oxidation

Bovine heart microsomes have been found to contain a non-heme iron protein which serves as an electron acceptor for NADPH-cytochrome P-450 reductase and therefore stimulates NADPH oxidation. This protein, tentatively referred to as Microsomal Iron Protein (MIP), has been extracted with Triton N-101 and purified by ion exchange chromatography on CM- and DEAE-celluloses and gel filtration on Sepharose 6B. MIP is an Mr = 66,000 monomer with 17 atoms of Fe(III)/molecule. Incubation with dithionite removes iron from MIP and abolishes the stimulation of NADPH oxidation, but subsequent incubation with nitrilotriacetic-Fe(III) reincorporates iron and restores the stimulation of NADPH oxidation. Oxygen is the ultimate electron acceptor. In the presence of oxygen, the enzymatic reduction of MIP Fe(III) is followed by the reoxidation of Fe(II) at the expense of oxygen, generating superoxide anion and regenerating MIP Fe(III) for the continuous oxidation of NADPH. In the absence of oxygen, electron transfer from the reductase to MIP Fe(III) causes the release of Fe(II), which limits the ability of MIP to serve as an electron acceptor and stimulate NADPH oxidation. The--NH2-terminal of MIP has been sequenced, and no homology has been found with the sequence of other iron storage or transport proteins such as ferritin or transferrin.     

Reductive dissolution of Fe(III) oxides by Pseudomonas sp. 200

The kinetics and mechanism of reductive dissolution of Fe(III) oxides were examined in pure, batch cultures of Pseudomonassp. 200. Primary factors controlling hematite dissolution kinetics were mineral surface area (or concentration of high-energy surface sites), ligand concentration, and cell number. In the presence of nitrilotriacetic acid (NTA), saturation kinetics were apparent in the relationship governing reductive dissolution of hematite. A kinetic expression was developed in which overall iron-reduction rate is functionally related to the concentrations of both NTA and Fe(III).Addition of NTA resulted in a 20-fold increase in the microbial rate of mineral (reductive) dissolution. Mechanisms in which NTA served as a bridging ligand, shuttling respiratory electrons from the membrane-bound microbial electron transport chain to the metal center of the iron oxide, or accelerated the departure of Fe(II) centers (bound to ligand) from the oxide surface following reduction have been postulated. Experimental results indicated that cell-mineral contact was essential for reductive dissolution of goethite.     

Redox Cycling of Iron Supports Growth and Magnetite Synthesis by Aquaspirillum magnetotacticum

Under anaerobic conditions and in the absence of alternative electron acceptors, growth of the magnetic bacterium Aquaspirillum magnetotacticum MSI was iron concentration dependent. Weak chelation of the iron (with quinate, oxalate, or 2,3-dihydroxybenzoate) enhanced growth, whereas strong chelation (with EDTA, citrate, or nitrilotriacetic acid) retarded the growth of strain MSI relative to that of controls lacking chelators. Growth was proportional to the percentage of unchelated iron in medium containing EDTA in various molar ratios to iron. Addition of the respiratory inhibitors antimycin A (5 μM), NaCN (10 mM), and NaN₃ (10 mM) inhibited growth with Fe(III) or NO₃^- as the terminal electron acceptor. Growth with O₂ and NO₃^- was inhibited by 2-heptyl-4-hydroxyquinolone-N-oxide (HOQNO) but not with 2 mM Fe(III). Under strongly reducing conditions, strain MS1 survived but grew poorly and became irreversibly nonmagnetic. Growth and iron reduction in anaerobic cultures were stimulated by the provision of small amounts of O₂ or H₂O₂. Slow infusion of air to cultures which had reduced virtually all of the Fe(III) in the medium (2 mM) supported a high rate of iron reoxidation (relative to killed controls) and growth in proportion to the amount of iron reoxidized. Oxygen consumption by iron-reducing cultures was predominantly biological, since NaCN and HOQNO both inhibited consumption. Inhibition of oxygen consumption (and iron reoxidation) by the addition of ferrozine and the inhibition of iron oxidation (and oxygen consumption) by the addition of HOQNO suggest that iron oxidation by strain MS1 is an aerobic respiratory process, perhaps tied to energy conservation. Iron oxidation was also necessary for magnetite synthesis, since in microaerobic denitrifying cultures, sequestration of reduced iron by ferrozine present in 10-fold molar excess to the available iron resulted in loss of magnetism and a severe drop in the average magnetosome number of the cells.