The role of reduction in iron uptake processes in a unicellular, planktonic cyanobacterium

In many aquatic environments the essential micronutrient iron is predominantly complexed by a heterogeneous pool of strong organic chelators. Research on iron uptake mechanisms of cyanobacteria inhabiting these environments has focused on endogenous siderophore production and internalization. However, as many cyanobacterial species do not produce siderophores, alternative Fe acquisition mechanisms must exist. Here we present a study of the iron uptake pathways in the unicellular, planktonic, non-siderophore producing strain Synechocystis sp. PCC 6803. By applying trace metal clean techniques and a chemically controlled growth medium we obtained reliable and reproducible short-term (radioactive assays) and long-term (growth experiments) iron uptake rates. We found that Synechocystis 6803 is capable of acquiring iron from exogenous ferrisiderophores (Ferrioxamine-B, FeAerobactin) and that unchelated, inorganic Fe is a highly available source of iron. Inhibition of iron uptake by the Fe(II)-specific ligand, ferrozine, indicated that reduction of both inorganic iron and ferrisiderophore complexes occurs before transport through the plasma membrane. Measurements of iron reduction rates and the inhibitory effect of ferrozine on growth supported this conclusion. The reduction-based uptake strategy is well suited for acquiring iron from multiple complexes in dilute aquatic environments and may play an important role in other cyanobacterial strains.     

The uptake of inorganic iron complexes by human melanoma cells

The human melanoma cell line, SK-MEL-28, expresses high levels of melanotransferrin. The uptake of inorganic iron (Fe) complexes compared to transferrin-bound Fe by these cells has been investigated to determine whether melanotransferrin has a role in Fe uptake. The mechanisms of Fe uptake have been characterised using 59Fe complexes of citrate, nitrilotriacetate, desferrioxamine, and 59Fe added to Eagle's minimum essential medium (MEM) and compared with human transferrin (Tf) labelled with 59Fe and iodine-125. Iron uptake from the Fe complexes of citrate, nitrilotriacetate and MEM were similar, and far greater than that from Tf at the same Fe concentration (2.5 microM). Ammonium chloride and a monoclonal antibody to the transferrin receptor (42/6), had no effect on the uptake of Fe from inorganic Fe complexes, suggesting that receptor-mediated endocytosis of Tf was not involved. The monoclonal antibody, 96.5, specific for melanotransferrin did not alter total Fe uptake but slightly increased the proportion of Fe internalised, possibly due to the modulation of the antigen by the antibody. However, from the time required for modulation to occur (approximately 2 h), the small increase in internalisation observed and the fact that no increase in total cell Fe occurred, it is suggested that melanotransferrin has little role in Fe uptake.     

Electron Transport in the Dissimilatory Iron Reducer, GS-15

Mechanisms for electron transport to Fe(III) were investigated in GS-15, a novel anaerobic microorganism which can obtain energy for growth by coupling the complete oxidation of organic acids or aromatic compounds to the reduction of Fe(III) to Fe(II). The results indicate that Fe(III) reduction proceeds through a type b cytochrome and a membrane-bound Fe(III) reductase which is distinct from the nitrate reductase.     

Iron bioavailability to phytoplankton: an empirical approach

Phytoplankton are often limited by iron in aquatic environments. Here we examine Fe bioavailability to phytoplankton by analyzing iron uptake from various Fe substrates by several species of phytoplankton grown under conditions of Fe limitation and comparing the measured uptake rate constants (Fe uptake rate/ substrate concentration). When unchelated iron, Fe′, buffered by an excess of the chelating agent EDTA is used as the Fe substrate, the uptake rate constants of all the eukaryotic phytoplankton species are tightly correlated and proportional to their respective surface areas (S.A.). The same is true when FeDFB is the substrate, but the corresponding uptake constants are one thousand times smaller than for Fe′. The uptake rate constants for the other substrates we examined fall mostly between the values for Fe′ and FeDFB for the same S.A. These two model substrates thus empirically define a bioavailability envelope with Fe′ at the upper and FeDFB at the lower limit of iron bioavailability. This envelope provides a convenient framework to compare the relative bioavailabilities of various Fe substrates to eukaryotic phytoplankton and the Fe uptake abilities of different phytoplankton species. Compared with eukaryotic species, cyanobacteria have similar uptake constants for Fe′ but lower ones for FeDFB. The unique relationship between the uptake rate constants and the S.A. of phytoplankton species suggests that the uptake rate constant of Fe-limited phytoplankton has reached a universal upper limit and provides insight into the underlying uptake mechanism.     

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.     

Multiple modes of iron uptake by the filamentous,siderophore‐producing cyanobacterium,Anabaena sp. PCC 7120

Iron is a member of a small group of nutrients that limits aquatic primary production. Mechanisms for utilizing iron have to be efficient and adapted according to the ecological niche. In respect to iron acquisition cyanobacteria, prokaryotic oxygen evolving photosynthetic organisms can be divided into siderophore‐ and non‐siderophore‐producing strains. The results presented in this paper suggest that the situation is far more complex. To understand the bioavailability of different iron substrates and the advantages of various uptake strategies, we examined iron uptake mechanisms in the siderophore‐producing cyanobacterium Anabaena sp. PCC 7120. Comparison of the uptake of iron complexed with exogenous (desferrioxamine B, DFB) or to self‐secreted (schizokinen) siderophores by Anabaena sp. revealed that uptake of the endogenous produced siderophore complexed to iron is more efficient. In addition, Anabaena sp. is able to take up dissolved, ferric iron hydroxide species (Fe′) via a reductive mechanism. Thus, Anabaena sp. exhibits both, siderophore‐ and non‐siderophore‐mediated iron uptake. While assimilation of Fe′ and FeDFB are not induced by iron starvation, FeSchizokinen uptake rates increase with increasing iron starvation. Consequently, we suggest that Fe′ reduction and uptake is advantageous for low‐density cultures, while at higher densities siderophore uptake is preferred.     

Evidence for a specific uptake system for iron phytosiderophores in roots of grasses

Roots of grasses in response to iron deficiency markedly increase the release of chelating substances (`phytosiderophores') which are highly effective in solubilization of sparingly soluble inorganic Fe<sup>III</sup> compounds by formation of Fe<sup>III</sup>phytosiderophores. In barley (Hordeum vulgare L.), the rate of iron uptake from Fe<sup>III</sup>phytosiderophores is 100 to 1000 times faster than the rate from synthetic Fe chelates (e.g. Fe ethylenediaminetetraacetate) or microbial Fe siderophores (e.g. ferrichrome). Reduction of Fe<sup>III</sup> is not involved in the preferential iron uptake from Fe<sup>III</sup>phytosiderophores by barley. This is indicated by experiments with varied pH, addition of bicarbonate or of a strong chelator for Fe<sup>II</sup> (e.g. batho-phenanthrolinedisulfonate). The results indicate the existence of a specific uptake system for Fe<sup>III</sup>phytosiderophores in roots of barley and all other graminaceous species. In contrast to grasses, cucumber plants (Cucumis sativus L.) take up iron from Fe<sup>III</sup>phytosiderophores at rates similar to those from synthetic Fe chelates. Furthermore, under Fe deficiency in cucumber, increased rates of uptake of Fe<sup>III</sup>phytosiderophores are based on the same mechanism as for synthetic Fe chelates, namely enhanced Fe<sup>III</sup> reduction and chelate splitting. Two strategies are evident from the experiments for the acquisition of iron by plants under iron deficiency. Strategy I (in most nongraminaceous species) is characterized by an inducible plasma membrane-bound reductase and enhancement of H<sup>+</sup> release. Strategy II (in grasses) is characterized by enhanced release of phytosiderophores and by a highly specific uptake system for Fe<sup>III</sup>phytosiderophores. Strategy II seems to have several ecological advantages over Strategy I such as solubilization of sparingly soluble inorganic Fe<sup>III</sup> compounds in the rhizosphere, and less inhibition by high pH. The principal differences in the two strategies have to be taken into account in screening methods for resistance to `lime chlorosis'.     

Plasma membrane ferric reductase activity of iron-limited algal cells is inhibited by ferric chelators

Iron-limited cells of the green alga Chlorella kesslerii use a reductive mechanism to acquire Fe(III) from the extracellular environment, in which a plasma membrane ferric reductase reduces Fe(III)-chelates to Fe(II), which is subsequently taken up by the cell. Previous work has demonstrated that synthetic chelators both support ferric reductase activity (when supplied as Fe(III)-chelates) and inhibit ferric reductase. In the present set of experiments we extend these observations to naturally-occurring chelators and their analogues (desferrioxamine B mesylate, schizokinen, two forms of dihydroxybenzoic acid) and also two formulations of the commonly-used herbicide N-(phoshonomethyl)glycine (glyphosate). The ferric forms of the larger siderophores (desferrioxamine B mesylate, schizokinen) and Fe(III)-N-(phoshonomethyl)glycine (as the isopropylamine salt) all supported rapid rates of ferric reductase activity, while the iron-free forms inhibited reductase activity. The smaller siderophores/siderophore precursors, 2,3- and 3,4-dihydroxybenzoic acids, did not support high rates of reductase in the ferric form but did inhibit reductase activity in the iron-free form. Bioassays indicated that Fe(III)-chelates that supported high rates of ferric reductase activity also supported a large stimulation in the growth of iron-limited cells, and that an excess of iron-free chelator decreased the growth rate. With respect to N-(phosphonomethyl)glycine, there were differences between the pure compound (free acid form) and the most common commercial formulation (which also contains isopropylamine) in terms of supporting and inhibiting ferric reductase activity and growth. Overall, these results suggest that photosynthetic organisms that use a reductive strategy for iron acquisition both require, and are potentially simultaneously inhibited by, ferric chelators. Furthermore, these results also may provide an explanation for the frequently contradictory results of N-(phosphonomethyl)glycine application to crops: we suggest that low concentrations of this molecule likely solubilize Fe(III), making it available for plant growth, but that higher (but sub-lethal) concentrations decrease iron acquisition by inhibiting ferric reductase activity.     

Effects of copper and ceruloplasmin on iron transport in the Caco 2 cell intestinal model

Previous studies have implicated copper proteins, including ceruloplasmin, in intestinal iron transport. Polarized Caco2 cells with tight junctions were used to examine the possibilities that (a) ceruloplasmin promotes iron absorption by enhancing release at the basolateral cell surface and (b) copper deficiency reduces intestinal iron transport. Iron uptake and overall transport were followed for 90 min with 1 &mgr;M 59Fe(II) applied to the apical surface of Caco2 cell monolayers. Apotransferrin (38 &mgr;M) was in the basolateral chamber. Induction of iron deficiency with desferrioxamine (100 &mgr;M; 18 h) markedly increased uptake and overall transport of iron. Uptake increased from about 20% to about 65% of dose, and overall 59Fe transport from <1% to 60% of dose. On the basis of actual iron released into the basal chamber (measured with bathophenanthroline), transport increased 8-fold. Desferrioxamine pretreatment reduced cellular Fe by 55%. The addition of freshly isolated, enzymatically active human ceruloplasmin to the basolateral chamber during absorption had no effect on uptake or transport of iron by the cells. Unexpectedly, pretreatment with three different chelators of copper (18 h), which reduced cellular levels about 40%, more than doubled iron uptake and raised overall transport to 20%. This was so, whether or not cells were also made iron deficient with desferrioxamine. Acute addition of 1 &mgr;M Cu(II) to the apical chamber had no significant effect upon iron uptake, retention, or transport in iron deficient or normal cells, in the presence of absence of ascorbate. We conclude that intestinal absorption of Fe(II) is unlikely to depend upon plasma ceruloplasmin, and that cuproproteins involved in this form of iron transport must be binding copper tightly.     

The Use of a Mercury Biosensor to Evaluate the Bioavailability of Mercury-Thiol Complexes and Mechanisms of Mercury Uptake in Bacteria

As mercury (Hg) biosensors are sensitive to only intracellular Hg, they are useful in the investigation of Hg uptake mechanisms and the effects of speciation on Hg bioavailability to microbes. In this study, bacterial biosensors were used to evaluate the roles that several transporters such as the glutathione, cystine/cysteine, and Mer transporters play in the uptake of Hg from Hg-thiol complexes by comparing uptake rates in strains with functioning transport systems to strains where these transporters had been knocked out by deletion of key genes. The Hg uptake into the biosensors was quantified based on the intracellular conversion of inorganic mercury (Hg(II)) to elemental mercury (Hg(0)) by the enzyme MerA. It was found that uptake of Hg from Hg-cysteine (Hg(CYS)₂) and Hg-glutathione (Hg(GSH)₂) complexes occurred at the same rate as that of inorganic complexes of Hg(II) into Escherichia coli strains with and without intact Mer transport systems. However, higher rates of Hg uptake were observed in the strain with a functioning Mer transport system. These results demonstrate that thiol-bound Hg is bioavailable to E. coli and that this bioavailability is higher in Hg-resistant bacteria with a complete Mer system than in non-resistant strains. No difference in the uptake rate of Hg from Hg(GSH)₂ was observed in E. coli strains with or without functioning glutathione transport systems. There was also no difference in uptake rates between a wildtype Bacillus subtilis strain with a functioning cystine/cysteine transport system, and a mutant strain where this transport system had been knocked out. These results cast doubt on the viability of the hypothesis that the entire Hg-thiol complex is taken up into the cell by a thiol transporter. It is more likely that the Hg in the Hg-thiol complex is transferred to a transport protein on the cell membrane and is subsequently internalized.     

The influence of chelating agents upon the dissimilatory reduction of Fe(III) by Shewanella putrefaciens

The ability of S. putrefaciens to reduce Fe(III) complexed by a variety of ligands has been investigated. All of the ligands tested caused the cation to be more susceptible to reduction by harvested whole cells than when uncomplexed, although some complexes were more readily reduced than others. Monitoring rates of reduction by a ferrozine assay for Fe(II) formation proved inadequate using Fe(III) ligands giving Fe(II) complexes of low kinetic lability (e.g. EDTA). A more suitable assay for Fe(III) reduction in the presence of such ligands proved to be the observation of associated cytochrome oxidation and re-reduction. Where possible, an assay for Fe(III) reduction based upon the disappearance of Fe(III) complex was also employed. Reduction of all Fe(III) complexes tested was totally inhibited by the presence of O₂, partially inhibited by HQNO and slower in the absence of a physiological electron donor. Upon cell fractionation, Fe(III) reductase activity was detected exclusively in the membranes. Using different physiological electron donors in assays on membranes, relative reduction rates of Fe(III) complexes complemented the data from whole cells. The differences in susceptibility to reduction of the various complexes are discussed, as is evidence for the respiratory nature of the reduction.     

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.     

Iron transport in Streptomyces pilosus mediated by ferrichrome siderophores, rhodotorulic acid, and enantio-rhodotorulic acid

Streptomyces pilosus is one of several microbes which produce ferrioxamine siderophores. In the accompanying paper (G. Müller and K. Raymond, J. Bacteriol. 160:304-312), the mechanism of iron uptake mediated by the endogenous ferrioxamines B, D1, D2, and E was examined. Here we report iron transport behavior in S. pilosus as mediated by the exogenous siderophores ferrichrome, ferrichrysin, rhodotorulic acid (RA), and synthetic enantio-RA. In each case iron acquisition depended on metabolic energy and had uptake rates comparable to that of [55Fe]ferrioxamine B. However, the synthetic ferric enantio-RA (which has the same preferred chirality at the metal center as ferrichrome) was twice as effective in supplying iron as was the natural ferric RA complex, suggesting that stereospecific recognition at the metal center is involved in the transport process. Iron uptake mediated by ferrichrome and ferric enantio-RA was strongly inhibited by kinetically inert chromic complexes of desferrioxamine B. These inhibition experiments indicate that iron from these exogenous siderophores is transported by the same uptake system as ferrioxamine B. Since the ligands have no structural similarity to ferrioxamine B except for the presence of three hydoxamate groups, we conclude that only the hydroxamate iron center and its direct surroundings are important for recognition and uptake. This hypothesis is supported by the fact that ferrichrome A and ferrirubin, which are both substituted at the hydroxamate carbonyl groups, were not (or were poorly) effective in supplying iron to S. pilosus.     

Induction of iron(III) and copper(II) reduction in pea (Pisum sativum L.) roots by Fe and Cu status: Does the root-cell plasmalemma Fe(III)-chelate reductase perform a general role in regulating cation uptake?

We investigated the effects of Fe and Cu status of pea (Pisum sativum L.) seedlings on the regulation of the putative root plasma-membrane Fe(III)-chelate reductase that is involved in Fe(III)-chelate reduction and Fe²⁺ absorption in dicotyledons and nongraminaceous monocotyledons. Additionally, we investigated the ability of this reductase system to reduce Cu(II)-chelates as well as Fe(III)-chelates. Pea seedlings were grown in full nutrient solutions under control, -Fe, and -Cu conditions for up to 18 d. Iron(III) and Cu(II) reductase activity was visualized by placing roots in an agarose gel containing either Fe(III)-EDTA and the Fe(II) chelate, Na₂bathophenanthrolinedisulfonic acid (BPDS), for Fe(III) reduction, or CuSO₄, Na₃citrate, and Na₂-2,9-dimethyl-4,7-diphenyl-1, 10-phenanthrolinedisulfonic acid (BCDS) for Cu(II) reduction. Rates of root Fe(III) and Cu(II) reduction were determined via spectrophotometric assay of the Fe(II)-BPDS or the Cu(I)-BCDS chromophore. Reductase activity was induced or stimulated by either Fe deficiency or Cu depletion of the seedlings. Roots from both Fe-deficient and Cu-depleted plants were able to reduce exogenous Cu(II)-chelate as well as Fe(III)-chelate. When this reductase was induced by Fe deficiency, the accumulation of a number of mineral cations (i.e., Cu, Mn, Fe, Mg, and K) in leaves of pea seedlings was significantly increased. We suggest that, in addition to playing a critical role in Fe absorption, this plasma-membrane reductase system also plays a more general role in the regulation of cation absorption by root cells, possibly via the reduction of critical sulfhydryl groups in transport proteins involved in divalent-cation transport (divalent-cation channels?) across the root-cell plasmalemma.     

Direct Measurement of 59Fe-Labeled Fe2+ Influx in Roots of Pea Using a Chelator Buffer System to Control Free Fe2+ in Solution

Fe2+ transport in plants has been difficult to quantify because of the inability to control Fe2+ activity in aerated solutions and non-specific binding of Fe to cell walls. In this study, a Fe(II)-3-(2-pyridyl)-5,6-diphenyl-1,2,4-triazine-4[prime]4"-disulfonic acid buffer system was used to control free Fe2+ in uptake solutions. Additionally, desorption methodologies were developed to adequately remove nonspecifically bound Fe from the root apoplasm. This enabled us to quantify unidirectional Fe2+ influx via radiotracer (59Fe) uptake in roots of pea (Pisum sativum cv Sparkle) and its single gene mutant brz, an Fe hyperaccumulator. Fe influx into roots was dramatically inhibited by low temperature, indicating that the measured Fe accumulation in these roots was due to true influx across the plasma membrane rather than nonspecific binding to the root apoplasm. Both Fe2+ influx and Fe translocation to the shoots were stimulated by Fe deficiency in Sparkle. Additionally, brz, a mutant that constitutively exhibits high ferric reductase activity, exhibited higher Fe2+ influx rates than +Fe-grown Sparkle. These results suggest that either Fe deficiency triggers the induction of the Fe2+ transporter or that the enhanced ferric reductase activity somehow stimulates the activity of the existing Fe2+ transport protein.     

Ascorbate Efflux as a New Strategy for Iron Reduction and Transport in Plants

Iron (Fe) is essential for virtually all living organisms. The identification of the chemical forms of iron (the speciation) circulating in and between cells is crucial to further understand the mechanisms of iron delivery to its final targets. Here we analyzed how iron is transported to the seeds by the chemical identification of iron complexes that are delivered to embryos, followed by the biochemical characterization of the transport of these complexes by the embryo, using the pea (Pisum sativum) as a model species. We have found that iron circulates as ferric complexes with citrate and malate (Fe(III)₃Cit₂Mal₂, Fe(III)₃Cit₃Mal₁, Fe(III)Cit₂). Because dicotyledonous plants only transport ferrous iron, we checked whether embryos were capable of reducing iron of these complexes. Indeed, embryos did express a constitutively high ferric reduction activity. Surprisingly, iron(III) reduction is not catalyzed by the expected membrane-bound ferric reductase. Instead, embryos efflux high amounts of ascorbate that chemically reduce iron(III) from citrate-malate complexes. In vitro transport experiments on isolated embryos using radiolabeled ⁵⁵Fe demonstrated that this ascorbate-mediated reduction is an obligatory step for the uptake of iron(II). Moreover, the ascorbate efflux activity was also measured in Arabidopsis embryos, suggesting that this new iron transport system may be generic to dicotyledonous plants. Finally, in embryos of the ascorbate-deficient mutants vtc2-4, vtc5-1, and vtc5-2, the reducing activity and the iron concentration were reduced significantly. Taken together, our results identified a new iron transport mechanism in plants that could play a major role to control iron loading in seeds.     

Iron oxide solubilization by organic matter and its effect on iron availability

The solubility of Fe in soils is largely controlled by Fe oxides; ferrihydrite, amorphous ferric hydroxide, and soil-Fe are generally believed to exert the major control. Fe(III) hydrolysis species constitute the major Fe species in solution. Other inorganic Fe complexes are present, but their concentrations are much less than the hydrolysis species. Organic complexes of Fe including those of organic acids like citrate, oxalate, and malate contribute slightly to increased Fe solubility in acid soils, but not in alkaline soils.The most important influence that organic matter has on the solubilization of Fe is through reduction. Respiration of organic matter creates reduction microsites in soil where Fe²⁺ concentrations increase above those of the Fe(III) hydrolysis species. Fluctuating redox conditions in these microsites are conducive to the formation of a mixed valency ferrosic hydroxide. This metastable precipitate maintains an elevated level of soluble inorganic Fe for prolonged periods and increases Fe availability to plants. The release of reducing agents and acids next to roots, as well as the production of siderophores by microorganisms within the rhizosphere, contribute to the solubilization and increased availability of Fe to plants.     

Mechanistic analysis of iron accumulation by endothelial cells of the BBB

The mechanism(s) by which iron in blood is transported across the blood-brain barrier (BBB) remains controversial. Here we have examined the first step of this trans-cellular pathway, namely the mechanism(s) of iron uptake into human brain microvascular endothelial cells (hBMVEC). We show that hBMVEC actively reduce non-transferrin bound Fe(III) (NTBI) and transferrin-bound Fe(III) (TBI); this activity is associated with one or more ferrireductases. Efficient, exo-cytoplasmic ferri-reduction from TBI is dependent upon transferrin receptor (TfR), also. Blocking holo-Tf binding with an anti-TfR antibody significantly decreases the reduction of iron from transferrin by hBMVEC, suggesting that holo-Tf needs to bind to TfR in order for efficient reduction to occur. Ferri-reduction from TBI significantly decreases when hBMVEC are pre-treated with Pt(II), an inhibitor of cell surface reductase activity. Uptake of (59)Fe from (59)Fe-Tf by endothelial cells is inhibited by 50?% when ferrozine is added to solution; in contrast, no inhibition occurs when cells are alkalinized with NH(4)Cl. This indicates that the iron reduced from holo-transferrin at the plasma membrane accounts for at least 50?% of the iron uptake observed. hBMVEC-dependent reduction and uptake of NTBI utilizes a Pt(II)-insensitive reductase. Reductase-independent uptake of Fe(II) by hBMVEC is inhibited up to 50?% by Zn(II) and/or Mn(II) by a saturable process suggesting that redundant Fe(II) transporters exist in the hBMVEC plasma membrane. These results are the first to demonstrate multiple mechanism(s) of TBI and NTBI reduction and uptake by endothelial cells (EC) of the BBB.     

Complexation of desferricoprogen with trivalent Fe, Al, Ga, In and divalent Fe, Ni, Cu, Zn metal ions: effects of the linking chain structure on the metal binding ability of hydroxamate based siderophores

Complexes of the natural siderophore, desferricoprogen (DFC), with several trivalent and divalent metal ions in aqueous solution were studied by pH-potentiometry, UV-Vis spectrophotometry and cyclic voltammetry. DFC was found to be an effective metal binding ligand, which, in addition to Fe(III), forms complexes of high stability with Ga(III), Al(III), In(III), Cu(II), Ni(II) and Zn(II). Fe(II), however, is oxidized by DFC under anaerobic conditions and Fe(III) complexes are formed. By comparing the results with those of desferrioxamine B (DFB), it can be concluded that the conjugated beta-double bond slightly increases the stability of the hydroxamate chelates, consequently increases the stability of mono-chelated complexes of DFC. Any steric effect by the connecting chains arises only in the bis- and tris-chelated complexes. With metal ions possessing a relatively big ionic radius (Cu(II), Ni(II), Zn(II), In(III)) DFC, containing a bit longer chains than DFB, forms slightly more stable complexes. With smaller metal ions the trend is the opposite. Also a notable difference is that stable trinuclear complex, [Cu(3)L(2)], is formed with DFC but not with DFB. Possible bio-relevance of the Fe(II)/Fe(III) results is also discussed in the paper.