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.     

Role of Hydrophobicity in Adhesion of the Dissimilatory Fe(III)-Reducing Bacterium Shewanella alga to Amorphous Fe(III) Oxide

The mechanisms by which the dissimilatory Fe(III)-reducing bacterium Shewanella alga adheres to amorphous Fe(III) oxide were examined through comparative analysis of S. alga BrY and an adhesion-deficient strain of this species, S. alga RAD20. Approximately 100% of S. alga BrY cells typically adhered to amorphous Fe(III) oxide, while less than 50% of S. alga RAD20 cells adhered. Bulk chemical analysis, isoelectric point analysis, and cell surface analysis by time-of-flight secondary-ion mass spectrometry and electron spectroscopy for chemical analysis demonstrated that the surfaces of S. alga BrY cells were predominantly protein but that the surfaces of S. alga RAD20 cells were predominantly exopolysaccharide. Physicochemical analyses and hydrophobic interaction assays demonstrated that S. alga BrY cells were more hydrophobic than S. alga RAD20 cells. This study represents the first quantitative analysis of the adhesion of a dissimilatory Fe(III)-reducing bacterium to amorphous Fe(III) oxide, and the results collectively suggest that hydrophobic interactions are a factor in controlling the adhesion of this bacterium to amorphous Fe(III) oxide. Despite having a reduced ability to adhere, S. alga RAD20 reduced Fe(III) oxide at a rate identical to that of S. alga BrY. This result contrasts with results of previous studies by demonstrating that irreversible cell adhesion is not requisite for microbial reduction of amorphous Fe(III) oxide. These results suggest that the interaction between dissimilatory Fe(III)-reducing bacteria and amorphous Fe(III) oxide is more complex than previously believed.     

The dynamics of (57)Fe nuclei in Fe(III)-DNA condensates.

The dynamics of iron nuclei in the condensates obtained by interaction of Fe(III) with DNA, Fe(III)(DNA monomer)(2), have been investigated by variable temperature (57)Fe M?ssbauer spectroscopy. Studies were effected on gel and freeze-dried samples, obtaining nearly coincident values of the parameters isomer shift and nuclear quadrupole splitting in T ranges 20-260 K. Functions ln(A(T)/A(77.3)) vs. T, here employed to investigate the dynamics of Fe nuclei, showed linear trends in the T ranges 20-150 and 150-260 K, respectively, the latter with larger slopes. Data coincided for gelled and freeze-dried specimens. No variation of delta or Delta E parameters occurred at the two T intervals, which suggests constancy of structure and bonding with the temperature changes. Functions (T) showed trends analogous to the corresponding functions determined for iron proteins, which were attributed to the occurrence of 'conformational substates'.     

Adsorption behaviour of Fe(II) and Fe(III) ions in aqueous solution on chitosan and cross-linked chitosan beads

A batch adsorption system was applied to study the adsorption of Fe(II) and Fe(III) ions from aqueous solution by chitosan and cross-linked chitosan beads. The adsorption capacities and rates of Fe(II) and Fe(III) ions onto chitosan and cross-linked chitosan beads were evaluated. Chitosan beads were cross-linked with glutaraldehyde (GLA), epichlorohydrin (ECH) and ethylene glycol diglycidyl ether (EGDE) in order to enhance the chemical resistance and mechanical strength of chitosan beads. Experiments were carried out as function of pH, agitation period, agitation rate and concentration of Fe(II) and Fe(III) ions. Langmuir and Freundlich adsorption models were applied to describe the isotherms and isotherm constants. Equilibrium data agreed very well with the Langmuir model. The kinetic experimental data correlated well with the second-order kinetic model, indicating that the chemical sorption was the rate-limiting step. Results also showed that chitosan and cross-linked chitosan beads were favourable adsorbers.     

Adhesion of Dissimilatory Fe(III)-Reducing Bacteria to Fe(III) Minerals

The metabolism of dissimilatory iron-reducing bacteria (DIRB) may provide a means of remediating contaminated subsurface soils. The factors controlling the rate and extent of bacterial F(III) mineral reduction are poorly understood. Recent research suggests that molecular-scale interactions between DIRB cells and Fe(III) mineral particles play an important role in this process. One of these interactions, cell adhesion to Fe(III) mineral particles, appears to be a complex process that is, at least in part, mediated by a variety of surface proteins. This study examined the hypothesis that the flagellum serves as an adhesin to different Fe(III) minerals that range in their surface area and degree of crystallinity. Deflagellated cells of the DIRB Shewanella algae BrY showed a reduced ability to adhere to hydrous ferric oxide (HFO) relative to flagellated cells. Flagellated cells were also more hydrophobic than deflagellated cells. This was significant because hydrophobic interactions have been previously shown to dominate S. algae cell adhesion to Fe(III) minerals. Pre-incubating HFO, goethite, or hematite with purified flagella inhibited the adhesion of S. algae BrY cells to these minerals. Transposon mutagenesis was used to generate a flagellum-deficient mutant designated S. algae strain NF. There was a significant difference in the rate and extent of S. algae NF adhesion to HFO, goethite, and hematite relative to that of S. algae BrY. Amiloride, a specific inhibitor of Na + -driven flagellar motors, inhibited S. algae BrY motility but did not affect the adhesion of S. algae BrY to HFO. S.algae NF reduced HFO at the same rate as S. algae BrY. Collectively, the results of this study support the hypothesis that the flagellum of S. algae functions as a specific Fe(III) mineral adhesin. However, these results suggest that flagellum-mediated adhesion is not requisite for Fe(III) mineral reduction.     

Dissimilatory Fe(III) and Mn(IV) reduction.

The oxidation of organic matter coupled to the reduction of Fe(III) or Mn(IV) is one of the most important biogeochemical reactions in aquatic sediments, soils, and groundwater. This process, which may have been the first globally significant mechanism for the oxidation of organic matter to carbon dioxide, plays an important role in the oxidation of natural and contaminant organic compounds in a variety of environments and contributes to other phenomena of widespread significance such as the release of metals and nutrients into water supplies, the magnetization of sediments, and the corrosion of metal. Until recently, much of the Fe(III) and Mn(IV) reduction in sedimentary environments was considered to be the result of nonenzymatic processes. However, microorganisms which can effectively couple the oxidation of organic compounds to the reduction of Fe(III) or Mn(IV) have recently been discovered. With Fe(III) or Mn(IV) as the sole electron acceptor, these organisms can completely oxidize fatty acids, hydrogen, or a variety of monoaromatic compounds. This metabolism provides energy to support growth. Sugars and amino acids can be completely oxidized by the cooperative activity of fermentative microorganisms and hydrogen- and fatty-acid-oxidizing Fe(III) and Mn(IV) reducers. This provides a microbial mechanism for the oxidation of the complex assemblage of sedimentary organic matter in Fe(III)- or Mn(IV)-reducing environments. The available evidence indicates that this enzymatic reduction of Fe(III) or Mn(IV) accounts for most of the oxidation of organic matter coupled to reduction of Fe(III) and Mn(IV) in sedimentary environments. Little is known about the diversity and ecology of the microorganisms responsible for Fe(III) and Mn(IV) reduction, and only preliminary studies have been conducted on the physiology and biochemistry of this process.     

Role of Menaquinones in Fe(III) Reduction by Membrane Fractions of Shewanella putrefaciens

Two Tn5-generated mutants of Shewanella putrefaciens with insertions in menD and menB were isolated and analyzed. Both mutants were deficient in the use of several terminal electron acceptors, including Fe(III). This deficiency was overcome by the addition of menaquinone (vitamin K(2)). Isolated membrane fractions from both mutants were unable to reduce Fe(III) in the absence of added menaquinone when formate was used as the electron donor. These results indicate that menaquinones are essential components for the reduction of Fe(III) by both whole cells and purified membrane fractions when formate or lactate is used as the electron donor.     

Selective adsorption of apoferritin on immobilized Fe(III): demonstration of Fe(III) binding sites

Immobilized metal ion affinity chromatography has been used to demonstrate and partially characterize Fe(III) binding sites on apoferritin. Binding of Fe(III) to these sites is influenced by pH, but not affected by high ionic strength. These results suggest that both ionic and coordinate covalent interactions are important in the formation of the Fe(III): apoferritin complex. This is, to our knowledge, the first demonstration of direct Fe(III) binding to apoferritin. Other immobilized metal ions, including Zn(II), Ni(II), Cu(II), Cr(III), Co(II), and Tb(III), displayed little or no adsorption of apoferritin. The analytical technique of immobilized metal ion affinity chromatography also shows great promise in the purification of apoferritin, ferritin, and other iron-binding proteins.     

Facilitation of Fe(II) antoxidation by Fe(III) complexing agents

The rate of oxidation of Fe(II) by atmospheric oxygen at pH 7.0 is significantly enhanced by low molecular weight Fe(III)-complexing agents in the order EDTA ≈ nitrilotriacetate > citrate > phosphate > oxalate. This simple effect of Fe(III) binding probably accounts for the “ferroxidase” activity exhibited by transferrin and ferritin.     

Role of Humic-Bound Iron as an Electron Transfer Agent in Dissimilatory Fe(III) Reduction

The dissimilatory Fe(III) reducer Geobacter metallireducens reduced Fe(III) bound in humic substances, but the concentrations of Fe(III) in a wide range of highly purified humic substances were too low to account for a significant portion of the electron-accepting capacities of the humic substances. Furthermore, once reduced, the iron in humic substances could not transfer electrons to Fe(III) oxide. These results suggest that other electron-accepting moieties in humic substances, such as quinones, are the important electron-accepting and shuttling agents under Fe(III)-reducing conditions.     

Mn(III)-containing acid phosphatase: Properties of Fe(III)-substituted enzyme and function of Mn(III) and Fe(III) in plant and mammalian acid phosphatases

The function of Mn(III) in plant acid phosphatase has been investigated by a metal-substitution study, and some properties of the Fe(III)-substituted enzyme were compared with those of the native Mn(III) enzyme and mammalian Fe(III)-containing acid phosphatases. ¹⁹F nuclear magnetic resonance (NMR) and proton relaxation rate measurements showed that inhibitors such as F⁻ and nitrilotriacetic acid interact with paramagnetic Mn(III) active site. The ³¹P-NMR signal of the enzyme-phosphate complex was also broadened by the paramagnetic effect of Mn(III). In the metal-substitution experiments of the Mn(III)-acid phosphatase with Fe(III), Zn(II) and Cu(II), only the iron gave satisfactory substitution. The Fe(III)-substituted plant acid phosphatase exhibited an absorption maximum at 525 nm (ε = 3000), typical high spin ferric ESR signal at g = 4.39, and lower pH optimum (pH 4.8) than the native Mn(III)-enzyme (pH 5.8). The phosphatase activity of the Fe(III)-substituted enzyme was reduced to about 53% of that of the native enzyme. The substrate specificities of both metallophosphatases were remarkably similar, but different from that of the Fe(III)-containing uteroferrin. The present results indicate that Mn(III) and Fe(IIII) in the acid phosphatase play an important role on effective binding of phosphate and acceleration of hydrolysis of phosphomonoesters at pH 4–6.     

Fe(III)-mediated cellular toxicity

Because it can undergo reversible changes in oxidation state, iron is an excellent biocatalyst but also a potentially deleterious metal. Iron-mediated toxicity has been ascribed to Fe(II), which reacts with oxygen to generate free radicals that damage macromolecules and cause cell death. However, we now report that Fe(III) exhibits microbicidal activity towards strains of Salmonella enterica, Escherichia coli and Klebsiella pneumoniae defective in the Fe(III)-responding PmrA/PmrB signal transduction system. Fe(III) bound to a pmrA Salmonella mutant more effectively than to the isogenic wild-type strain and exerted its microbicidal activity even under anaerobic conditions. Moreover, Fe(III) permeabilized the outer membrane of the pmrA mutant, rendering it susceptible to vancomycin, which is normally non-toxic to Gram-negative species. On the other hand, Fe(III) did not affect the viability of a mutant defective in Fur, the major regulator of cytosolic iron homeostasis, which is hypersensitive to Fe(II)-mediated toxicity. A functional pmrA gene was necessary for bacterial survival in soil. Our results indicate that Fe(III) exerts its microbicidal activity by a mechanism that is oxygen independent and different from that mediated by Fe(II).     

Adhesion of the dissimilatory Fe(III)-reducing bacterium Shewanella alga BrY to crystalline Fe(III) oxides

Shewanella alga BrY adhesion to hydrous ferric oxide, goethite, and hematite was examined. Adhesion to each oxide followed the Langmuir adsorption model. No correlation between adhesion and Fe(III) oxide surface area or crystallinity was observed. Zeta potential measurements suggested that electrostatic interactions do not influence S. alga BrY adhesion to these minerals. Cell adhesion does not appear to explain the recalcitrance of crystalline Fe(III) oxides to bacterial reduction. Received: 12 May 2000 / Accepted: 19 June 2000     

Mechanisms for Accessing Insoluble Fe(III) Oxide during Dissimilatory Fe(III) Reduction by Geothrix fermentans

Mechanisms for Fe(III) oxide reduction were investigated in Geothrix fermentans, a dissimilatory Fe(III)-reducing microorganism found within the Fe(III) reduction zone of subsurface environments. Culture filtrates of G. fermentans stimulated the reduction of poorly crystalline Fe(III) oxide by washed cell suspensions, suggesting that G. fermentans released one or more extracellular compounds that promoted Fe(III) oxide reduction. In order to determine if G. fermentans released electron-shuttling compounds, poorly crystalline Fe(III) oxide was incorporated into microporous alginate beads, which prevented contact between G. fermentans and the Fe(III) oxide. G. fermentans reduced the Fe(III) within the beads, suggesting that one of the compounds that G. fermentans releases is an electron-shuttling compound that can transfer electrons from the cell to Fe(III) oxide that is not in contact with the organism. Analysis of culture filtrates by thin-layer chromatography suggested that the electron shuttle has characteristics similar to those of a water-soluble quinone. Analysis of filtrates by ion chromatography demonstrated that there was as much as 250 μM dissolved Fe(III) in cultures of G. fermentans growing with Fe(III) oxide as the electron acceptor, suggesting that G. fermentans released one or more compounds capable of chelating and solubilizing Fe(III). Solubilizing Fe(III) is another strategy for alleviating the need for contact between cells and Fe(III) oxide for Fe(III) reduction. This is the first demonstration of a microorganism that, in defined medium without added electron shuttles or chelators, can reduce Fe(III) derived from Fe(III) oxide without directly contacting the Fe(III) oxide. These results are in marked contrast to those with Geobacter metallireducens, which does not produce electron shuttles or Fe(III) chelators. These results demonstrate that phylogenetically distinct Fe(III)-reducing microorganisms may use significantly different strategies for Fe(III) reduction. Thus, it is important to know which Fe(III)-reducing microorganisms predominate in a given environment in order to understand the mechanisms for Fe(III) reduction in the environment of interest.     

Mechanisms for accessing insoluble Fe(III) oxide during dissimilatory Fe(III) reduction by Geothrix fermentans

Mechanisms for Fe(III) oxide reduction were investigated in Geothrix fermentans, a dissimilatory Fe(III)-reducing microorganism found within the Fe(III) reduction zone of subsurface environments. Culture filtrates of G. fermentans stimulated the reduction of poorly crystalline Fe(III) oxide by washed cell suspensions, suggesting that G. fermentans released one or more extracellular compounds that promoted Fe(III) oxide reduction. In order to determine if G. fermentans released electron-shuttling compounds, poorly crystalline Fe(III) oxide was incorporated into microporous alginate beads, which prevented contact between G. fermentans and the Fe(III) oxide. G. fermentans reduced the Fe(III) within the beads, suggesting that one of the compounds that G. fermentans releases is an electron-shuttling compound that can transfer electrons from the cell to Fe(III) oxide that is not in contact with the organism. Analysis of culture filtrates by thin-layer chromatography suggested that the electron shuttle has characteristics similar to those of a water-soluble quinone. Analysis of filtrates by ion chromatography demonstrated that there was as much as 250 microM dissolved Fe(III) in cultures of G. fermentans growing with Fe(III) oxide as the electron acceptor, suggesting that G. fermentans released one or more compounds capable of chelating and solubilizing Fe(III). Solubilizing Fe(III) is another strategy for alleviating the need for contact between cells and Fe(III) oxide for Fe(III) reduction. This is the first demonstration of a microorganism that, in defined medium without added electron shuttles or chelators, can reduce Fe(III) derived from Fe(III) oxide without directly contacting the Fe(III) oxide. These results are in marked contrast to those with Geobacter metallireducens, which does not produce electron shuttles or Fe(III) chelators. These results demonstrate that phylogenetically distinct Fe(III)-reducing microorganisms may use significantly different strategies for Fe(III) reduction. Thus, it is important to know which Fe(III)-reducing microorganisms predominate in a given environment in order to understand the mechanisms for Fe(III) reduction in the environment of interest.     

The influence of chelating agents upon the dissimilatory reduction of Fe(III) byShewanella putrefaciens. Part 2. Oxo-and hydroxo-bridged polynuclear Fe(III) complexes

The susceptibility to dissimilatory reduction of polynuclear oxo- and hydroxo-bridged Fe(III) complexes byShewanella putrefaciens intact cells and membranes has been investigated. These complexes were ligated by the potential tetradentates heidi (H₃heidi =N-(2-hydroxyethyl)iminodiacetic acid) or nta (H₃nta = nitrilotriacetic acid), or the potential tridentate ida (H₂ida = iminodiacetic acid). A number of defined small complexes with varied nuclearity and solubility properties were employed, as well as undefined species prepared by mixing different molar ratios of ida or heidi:Fe(III) in solution. The rates of Fe(III) reduction determined by an assay for Fe(II) formation with ferrozine were validated by monitoringc-type cytochrome oxidation and re-reduction associated with electron transport. For the undefined Fe(III) polymeric species, reduction rates in whole cells and membranes were considerably faster in the presence of heidi compared to ida. This is believed to result from generally smaller and more reactive clusters forming with heidi as a consequence of the alkoxo function of this ligand being able to bridge between Fe(III) nuclei, with access to an Fe(III) reductase located at the cytoplasmic membrane being of some importance. The increases in reduction rates of the undefined ida species with Fe(III) using membranes relative to whole cells reinforce such a view. Using soluble synthetic Fe(III) clusters, slow reduction was noted for an oxo-bridged dimer coordinatively saturated with ida and featuring unligated carboxylates. This suggests that sterically hindering the cation can influence enzyme action. A heidi dimer and a heidi multimer (17 or 19 Fe(III) nuclei), which are both of poor solubility, were found to be reduced by whole cells, but dissimilation rates increased markedly using membranes. These data suggest that Fe(III) reductase activity may be located at both the outer membrane and the cytoplasmic membrane ofS. putrefaciens. Slower reduction of the heidi multimer relative to the heidi dimer reflects the presence of a central hydroxo(oxo)-bridged core containing nine Fe(III) nuclei within the former cluster. This unit is a poor substrate for dissimilation, owing to the fact that the Fe(III) is not ligated by aminocarboxylate. The faster reduction noted for the heidi dimer in membranes than for a soluble ida monomer suggests that the presence of ligating water molecules may relieve steric hindrance to enzyme attack. Furthermore, reduction of an insoluble oxo-bridged nta dimer featuring ligating water molecules in intact cells was faster than that of a soluble monomer coordinatively saturated by nta and possessing an unligated carboxylate. This suggests that steric factors may override solubility considerations with respect to the susceptibility to reduction of certain Fe(III) complexes by the bacterium.Previous paper in this series: Dobbin PS, Powell AK, McEwan AG, Richardson DJ. 1995 The influence of chelating agents upon the dissimilatory reduction of Fe(III) byShewanella putefraciens.BioMetals 8, 163–173.