Mechanism Of Iron Transport Research Articles

Transport genes and chemotaxis in Laribacter hongkongensis: a genome-wide analysis

BackgroundLaribacter hongkongensis is a Gram-negative, sea gull-shaped rod associated with community-acquired gastroenteritis. The bacterium has been found in diverse freshwater environments including fish, frogs and drinking water reservoirs. Using the complete genome sequence data of L. hongkongensis, we performed a comprehensive analysis of putative transport-related genes and genes related to chemotaxis, motility and quorum sensing, which may help the bacterium adapt to the changing environments and combat harmful substances.ResultsA genome-wide analysis using Transport Classification Database TCDB, similarity and keyword searches revealed the presence of a large diversity of transporters (n = 457) and genes related to chemotaxis (n = 52) and flagellar biosynthesis (n = 40) in the L. hongkongensis genome. The transporters included those from all seven major transporter categories, which may allow the uptake of essential nutrients or ions, and extrusion of metabolic end products and hazardous substances. L. hongkongensis is unique among closely related members of Neisseriaceae family in possessing higher number of proteins related to transport of ammonium, urea and dicarboxylate, which may reflect the importance of nitrogen and dicarboxylate metabolism in this assacharolytic bacterium. Structural modeling of two C4-dicarboxylate transporters showed that they possessed similar structures to the determined structures of other DctP-TRAP transporters, with one having an unusual disulfide bond. Diverse mechanisms for iron transport, including hemin transporters for iron acquisition from host proteins, were also identified. In addition to the chemotaxis and flagella-related genes, the L. hongkongensis genome also contained two copies of qseB/qseC homologues of the AI-3 quorum sensing system.ConclusionsThe large number of diverse transporters and genes involved in chemotaxis, motility and quorum sensing suggested that the bacterium may utilize a complex system to adapt to different environments. Structural modeling will provide useful insights on the transporters in L. hongkongensis.

Characterization of Fe(iii) sequestration by an analog of the cytotoxic siderophore brasilibactin A: Implications for the iron transport mechanism in mycobacteria

Mycobacteria such as M. tuberculosis represent a significant health concern throughout much of the developing world. In mycobacteria and other pathogenic bacteria, an important virulence factor is the ability of the bacterium to obtain iron from its host. One means of obtaining iron is through the use of siderophores. Brasilibactin A is a membrane bound siderophore produced by Nocardia brasiliensis with structural similarity to the mycobactin class of siderophore in mycobacteria. A characterization of the protonation constants and Fe(III) affinity of a water soluble Brasilibactin A analog (Bbtan) has been performed. Using protonation constants and competition with EDTA, the stability constant of the 1 : 1 Fe(III)-Bbtan complex was found to be log β(110) = 26.96. The pFe of Bbtan is 22.73, somewhat low for a proposed siderophore molecule. The redox potential of the Fe-Bbtan complex was found to be -300 mV vs. NHE, very high for an iron-siderophore complex. The combination of relatively low complex stability and ease of iron reduction may play a crucial role in the mechanism of mycobactin siderophore-mediated iron uptake in mycobacteria and related organisms.

Brain iron metabolism and its perturbation in neurological diseases

Metal ions are of particular importance in brain function, notably iron. A broad overview of iron metabolism and its homeostasis both at the cellular level (involving regulation at the level of mRNA translation) and the systemic level (involving the peptide 'hormone' hepcidin) is presented. The mechanisms of iron transport both across the blood-brain barrier and within the brain are then examined. The importance of iron in the developing foetus and in early life is underlined. We then review the growing corpus of evidence that many neurodegenerative diseases (NDs) are the consequence of dysregulation of brain iron homeostasis. This results in the production of reactive oxygen species, generating reactive aldehydes, which, together with further oxidative insults, causes oxidative modification of proteins, manifested by carbonyl formation. These misfolded and damaged proteins overwhelm the ubiquitin/proteasome system, accumulating the characteristic inclusion bodies found in many NDs. The involvement of iron in Alzheimer's disease and Parkinson's disease is then examined, with emphasis on recent data linking in particular interactions between iron homeostasis and key disease proteins. We conclude that there is overwhelming evidence for a direct involvement of iron in NDs.

Disruption of Nap14, a plastid‐localized non‐intrinsic ABC protein in Arabidopsis thaliana results in the over‐accumulation of transition metals and in aberrant chloroplast structures

Chloroplasts are the major sink for Fe in shoot tissues because of the requirements of the photosynthetic process and to storage in ferritins. Such requirements are common both to plastids and to their evolutionary progenitors, the cyanobacteria. Here, we examined whether iron transport mechanisms were conserved throughout the evolution of photosynthetic organisms. Comparison of the sequences of putative plastid transporters from Arabidopsis thaliana with those involved in cyanobacterial Fe transport identified two orthologs of the FutC protein, AtNAP11 and AtNAP14. To study their function, we analysed insertional mutants in the genes coding for these proteins. Both nap11/nap11 and nap14/nap14 plants exhibited severe growth defects. Significant changes in transition metal homeostasis were detected only in nap14/nap14. This mutant was found to contain approximately 18 times more Fe in the shoot tissue than in wild-type plants. The increased shoot transition metal content was accompanied by a specific loss of chloroplast structures and by a reduction in transcript levels of Fe homeostasis-related genes. Based on these results, we propose that AtNAP14 plays an important role in plastid transition metal homeostasis. One possibility is that AtNAP14 is part of a chloroplast transporter complex. Alternatively, AtNAP14 function may be in regulating transition metal homeostasis.

Accumulation of Non-Transferrin-Bound Iron by Neurons, Astrocytes, and Microglia

Neurodegenerative conditions such as Alzheimer's disease, Parkinson's disease, and hemorrhagic stroke are associated with increased levels of non-transferrin-bound iron (NTBI) in the brain, which can promote Fenton chemistry. While all types of brain cells can take up NTBI, their efficiency of accumulation and capacity to withstand iron-mediated toxicity has not been directly compared. The present study assessed NTBI accumulation in cultures enriched in neurons, astrocytes, or microglia after exposure to ferric ammonium citrate (FAC). Microglia were found to be the most efficient in accumulating iron, followed by astrocytes, and then neurons. Exposure to 100μM FAC for 24h increased the specific iron content of cultured neurons, astrocytes, and microglial cells by 30-, 80-, and 100-fold, respectively. All cell types accumulated iron against the concentration gradient, resulting in intracellular iron concentrations that were several orders of magnitude higher than the extracellular iron concentrations. Accumulation of these large amounts of iron did not affect the viability of the cell cultures, indicating a high resistance to iron-mediated toxicity. These findings show that neurons, astrocytes and microglia cultured from neonatal mice all have the capacity to accumulate and safely store large quantities of iron, but that glial cells do this more efficiently than neurons. It is concluded that neurodegenerative conditions involving iron-mediated toxicity may be due to a failure of iron transport or storage mechanisms, rather than to the presence of high levels of NTBI.

Nitric oxide contributes to the regulation of iron metabolism in skeletal muscle in vivo and in vitro

Nitric Oxide (NO) plays an important role in iron redistribution during exercise, while its molecular regulatory mechanism is still not clear. Our present studies were to investigate the effects of NO on iron metabolism and to elucidate the regulatory mechanism of iron transport in skeletal muscle both in vivo and in vitro. One group of male Wistar rats (300 +/- 10 g) were subjected to an exercise of 30 min on a treadmill for 5 weeks (exercise group, EG, 6 rats) and the other one was placed on the treadmill without running (control group, CG, 6 rats). The cultured L6 rat skeletal muscle cells were treated with either 0.5 mM SNAP (NO donor) or not for 24 h, and their iron release and intake amount were examined by measuring radiolabelled (55)Fe. The results showed: (1) The NO content (CG, 1.09 +/- 0.18 micromol/g vs. EG, 1.49 +/- 0.17 micromol/g) and non-heme iron in gastrocnemius (CG, 118.35 +/- 11.41 microg/g vs. EG, 216.65 +/- 11.10 microg/g) of EG were significantly increased compared with CG. (2) The expression of DMT1 (IRE) and TfR1 of EG was increased. (3) The iron intake was increased in L6 cells treated with SNAP (P < 0.01). (4) Western blot results showed the protein level of both TfR1 and DMT1 (IRE) in SNAP cells were up-regulated, while the expression of FPN1 was down-regulated (P < 0.05). The data suggested that the induced elevation of NO level by exercise lead to the up-regulation of both TfR1 and DMT1 (IRE), which in turn increasing the iron absorption in skeletal muscle.

Maternal and Neonatal Iron Status Impact the Expression of the Heme Transporter, FLVCR, in the Placenta

Mechanisms of placental heme iron transport are largely unknown. To address this, expression of the heme transporter Feline Leukemia Virus, Subgroup C, Receptor (FLVCR) was measured in placental tissue from 84 pregnant teens (13–18 y). Iron (Fe) status indicators, serum ferritin (SF) and serum transferrin receptor (sTfR), were measured at mid‐gestation (25.1±3.5 wks) and in maternal/cord blood at delivery (39.8±1.1 wks). At delivery, 33% (18/54) of teens were anemic (Hb&lt;11 g/dL); hemoglobin (Hb) averaged 11.6±1.5 g/dL (n=54). Maternal SF at delivery was 21.8±13.3 μg/L (n=67); 52% (35/67) had depleted Fe stores (SF&lt;20 μg/L). Neonatal SF averaged 140.8±79.9 μg/L (n=65). At delivery, maternal and neonatal sTfR averaged 5.8±3.7 mg/L (n=66) and 8.0±2.4 mg/L (n=65), respectively. A significant relationship was observed between maternal mid‐gestation SF and neonatal SF (p&lt;0.05, n=52) and between placental FLVCR expression and the maternal delivery: neonatal SF ratio (p&lt;0.01, n=43). Although FLVCR expression was regulated in response to maternal and neonatal Fe status, up‐regulation of this protein was not sufficient to normalize ferritin stores in neonates born to teens with depleted Fe stores. Funded by: NRI Grants 2008‐01857 &amp; 2005‐35200, USDA HATCH (2006‐07‐160), and HHMI.

Mechanisms of Brain Iron Transport: Insight into Neurodegeneration and CNS Disorders

Trace metals such as iron, copper, zinc, manganese, and cobalt are essential cofactors for many cellular enzymes. Extensive research on iron, the most abundant transition metal in biology, has contributed to an increased understanding of the molecular machinery involved in maintaining its homeostasis in mammalian peripheral tissues. However, the cellular and intercellular iron transport mechanisms in the central nervous system (CNS) are still poorly understood. Accumulating evidence suggests that impaired iron metabolism is an initial cause of neurodegeneration, and several common genetic and sporadic neurodegenerative disorders have been proposed to be associated with dysregulated CNS iron homeostasis. This review aims to provide a summary of the molecular mechanisms of brain iron transport. Our discussion is focused on iron transport across endothelial cells of the blood-brain barrier and within the neuro- and glial-vascular units of the brain, with the aim of revealing novel therapeutic targets for neurodegenerative and CNS disorders.

Early martian dynamo generation due to giant impacts

During late-stage planet formation, giant impacts produce localized mantle melt regions within which impactor iron droplets settle to the bottom near a permeability horizon. After accumulation, iron heated by the impact migrates downward to the core through colder, mostly solid mantle. The degree of thermal equilibration and partitioning of viscous heating between impactor iron and silicates depends on the mechanism of iron transport to the core. Simple estimates suggest that, following a giant impact, the temperature difference between iron delivered to the core and the mantle outside the impact heated region can be ∼10 3 K. Hot impactor iron mergers with the core where it may be efficiently mixed or remain stratified due to thermal buoyancy. In either case, collisional energy carried to the core by impactor iron helps establish conditions favorable for early core cooling and dynamo generation. In this study, we consider the end-member scenario in which impactor iron forms a layer at the top of the core. Energy transfer from the impactor iron layer to the mantle is sufficient to power a dynamo for up to ∼30 Myr even in the limit of a very viscous mantle and heat flux limited by conduction. Using two-dimensional finite element calculations of mantle convection, we show that large-scale mantle flow driven by the buoyancy of the impact thermal anomaly focuses plumes in the impact region and increases both dynamo strength and duration. Melting within the mantle thermal boundary layer likely leads to formation of a single superplume in the location of the impact anomaly driven upwelling. We suggest that formation of magnetized southern highland crust may be related to spreading and differentiation of an impact melt region during the impact-induced dynamo episode.

Heme Transfer to the Bacterial Cell Envelope Occurs via a Secreted Hemophore in the Gram-positive Pathogen Bacillus anthracis

To initiate and sustain an infection in mammals, bacterial pathogens must acquire host iron. However, the host's compartmentalization of large amounts of iron in heme, which is bound primarily by hemoglobin in red blood cells, acts as a barrier to bacterial iron assimilation. Bacillus anthracis, the causative agent of the disease anthrax, secretes two NEAT (near iron transporter) proteins, IsdX1 and IsdX2, which scavenge heme from host hemoglobin and promote growth under low iron conditions. The mechanism of heme transfer from these hemophores to the bacterial cell is not known. We present evidence that the heme-bound form of IsdX1 rapidly and directionally transfers heme to IsdC, a NEAT protein covalently attached to the cell wall, as well as to IsdX2. In both cases, the transfer of heme is mediated by a physical association between the donor and recipient. Unlike Staphylococcus aureus, whose NEAT proteins acquire heme from hemoglobin directly at the bacterial surface, B. anthracis secretes IsdX1 to capture heme in the extracellular milieu and relies on NEAT-NEAT interactions to deliver the bound heme to the envelope via IsdC. Understanding the mechanism of NEAT-mediated iron transport into pathogenic Gram-positive bacteria may provide an avenue for the development of therapeutics to combat infection.

Age-dependent expression of hephaestin in the brain of ceruloplasmin-deficient mice

Aceruloplasminemia is an autosomal recessive disorder caused by mutations in the ceruloplasmin (CP) gene. It is characterized by iron accumulation in the brain and in visceral organs. However, little is known about the mechanism of iron transport in these regions. Adult CP null (CP −/−) mice show increased iron deposition in several regions of brain, such as the cerebellum and brainstem. In this study, we investigated the expression of the ceruloplasmin homolog hephaestin (Heph) in the brain of CP −/− mice as a function of age. In the cerebral cortex and caudate putamen of 80-week-old CP −/− mice, the expression of Heph increased significantly whilst iron levels remain normal [Patel BN, Dunn RJ, Jeong SY, Zhu Q, Julien JP, David S. Ceruloplasmin regulates iron levels in the CNS and prevents free radical injury. J Neurosci 2002;22(15):6578–6], indicating that Heph might compensate for the loss of CP. In contrast, the substantia nigra and cerebellum of 80-week-old CP −/− mice accumulate iron but do not express high levels or significant decrease of Heph, suggesting that Heph does not replace CP in these regions. These data suggest that Heph may compensate for the loss of CP in a region-specific manner.

OsYSL18 is a rice iron(III)–deoxymugineic acid transporter specifically expressed in reproductive organs and phloem of lamina joints

Iron uptake and translocation in plants are important processes for both plant and human nutrition, whereas relatively little is known about the molecular mechanisms of iron transport within the plant body. Several reports have shown that yellow stripe 1 (YS1) and YS1-like (YSL) transporters mediate metal-phytosiderophore uptake and/or metal-nicotianamine translocation. Among the 18 YSL genes in rice (OsYSLs), OsYSL18 is predicted to encode a polypeptide of 679 amino acids containing 13 putative transmembrane domains. An OsYSL18-green fluorescent protein (GFP) fusion was localized to the plasma membrane when transiently expressed in onion epidermal cells. Electrophysiological measurements using Xenopus laevis oocytes showed that OsYSL18 transports iron(III)–deoxymugineic acid, but not iron(II)–nicotianamine, zinc(II)–deoxymugineic acid, or zinc(II)–nicotianamine. Reverse transcriptase PCR analysis revealed more OsYSL18 transcripts in flowers than in shoots or roots. OsYSL18 promoter-β-glucuronidase (GUS) analysis revealed that OsYSL18 was expressed in reproductive organs including the pollen tube. In vegetative organs, OsYSL18 was specifically expressed in lamina joints, the inner cortex of crown roots, and phloem parenchyma and companion cells at the basal part of every leaf sheath. These results suggest that OsYSL18 is an iron-phytosiderophore transporter involved in the translocation of iron in reproductive organs and phloem in joints.

Novel insights into iron metabolism by integrating deletome and transcriptome analysis in an iron deficiency model of the yeast Saccharomyces cerevisiae

BackgroundIron-deficiency anemia is the most prevalent form of anemia world-wide. The yeast Saccharomyces cerevisiae has been used as a model of cellular iron deficiency, in part because many of its cellular pathways are conserved. To better understand how cells respond to changes in iron availability, we profiled the yeast genome with a parallel analysis of homozygous deletion mutants to identify essential components and cellular processes required for optimal growth under iron-limited conditions. To complement this analysis, we compared those genes identified as important for fitness to those that were differentially-expressed in the same conditions. The resulting analysis provides a global perspective on the cellular processes involved in iron metabolism.ResultsUsing functional profiling, we identified several genes known to be involved in high affinity iron uptake, in addition to novel genes that may play a role in iron metabolism. Our results provide support for the primary involvement in iron homeostasis of vacuolar and endosomal compartments, as well as vesicular transport to and from these compartments. We also observed an unexpected importance of the peroxisome for growth in iron-limited media. Although these components were essential for growth in low-iron conditions, most of them were not differentially-expressed. Genes with altered expression in iron deficiency were mainly associated with iron uptake and transport mechanisms, with little overlap with those that were functionally required. To better understand this relationship, we used expression-profiling of selected mutants that exhibited slow growth in iron-deficient conditions, and as a result, obtained additional insight into the roles of CTI6, DAP1, MRS4 and YHR045W in iron metabolism.ConclusionComparison between functional and gene expression data in iron deficiency highlighted the complementary utility of these two approaches to identify important functional components. This should be taken into consideration when designing and analyzing data from these type of studies. We used this and other published data to develop a molecular interaction network of iron metabolism in yeast.

Small molecule inhibitors of divalent metal transporter-1

Divalent metal transporter-1 (DMT1) is a divalent cation transporter that plays a key role in iron metabolism by mediating ferrous iron uptake across the small intestine. We have previously identified several small molecule inhibitors of iron uptake (4). Using a cell line that stably overexpresses DMT1, we screened the ability of these inhibitors to specifically block this transporter's activity. One compound, NSC306711, inhibited DMT1-mediated iron uptake in a reversible and competitive manner. This inhibitor is a polysulfonated dye containing two copper centers. Although one of these two sites could be chelated by Triethylenetetramine copper chelation did not perturb NSC306711 inhibition of DMT1 activity. Several other polysulfonated dyes with structural features similar to NSC306711 were identified as potential DMT1 transport inhibitors. This study characterizes important pharmacological tools that can be used to probe DMT1's mechanism of iron transport and its role in iron metabolism.

Iron transport in Francisella in the absence of a recognizable TonB protein still requires energy generated by the proton motive force

The mechanism of iron transport in Francisella is still a puzzle since none of the sequenced Francisella strains appears to encode a TonB protein, the energy transducer of the proton motive force necessary to act on the bacterial outer membrane siderophore receptor to allow the internalization of iron. In this work we demonstrate using kinetic experiments of radioactive Fe(3+) utilization, that iron uptake in Francisella novicida, although with no recognizable TonB protein, is indeed dependent on energy generated by the proton motive force. Moreover, mutants of a predicted outer membrane receptor still transport iron and are sensitive to the iron dependent antimicrobial compound streptonigrin. Our studies suggest that alternative pathways to internalize iron might exist in Francisella.

The Iron-regulated Transporter, MbNRAMP1, Isolated from Malus baccata is Involved in Fe, Mn and Cd Trafficking

Iron deficiency is one of the most common nutritional disorders in plants, especially in fruit trees grown in calcareous soil. Malus baccata is widely used as an apple rootstock in north China and is highly resistant to low temperatures. There are few studies on iron absorption by this species at the molecular level. It is very important to understand the mechanism of iron uptake and transport in such woody plants. As a helpful tool, the aim of the present study was the cloning and functional analysis of NRAMP (natural resistance-associated macrophage protein) genes from the apple tree in relation to trafficking of micronutrients (Fe, Mn and Cd). Reverse transcription-PCR (RT-PCR) combined with RACE (rapid amplification of cDNA ends) was adopted to isolate the full-length NRAMP1 cDNA. Southern blotting was used to test gene copy information, and northern blot was used to detect the gene's expression level. Complementation experiments using the yeast mutant strains DEY1453 and SLY8 were employed to confirm the iron- and manganese-transporting ability of NRAMP1 from apple, and inductively coupled plasma (ICP) spectrometry was used to measure Cd accumulation in yeast. NRAMP1-green fluorescent protein (GFP) fusion protein was used to determine the cellular localization in yeast. A 2090 bp cDNA was isolated and named MbNRAMP1. It encodes a predicted polypeptide of 551 amino acids. MbNRAMP1 exists in the M. baccata genome as a single copy and was expressed mainly in roots. MbNRAMP1 rescued the phenotype of yeast mutant strains DEY1453 and SLY8, and also increased Cd2+ sensitivity and accumulation. MbNRAMP1 expression in yeast was largely influenced by iron status, and the expression pattern of MbNRAMP1-GFP varied with the environmental iron nutrition status. MbNRAMP1 encodes a functional metal transporter capable of mediating the distribution of ions as well as transport of the micronutrients, Fe and Mn, and the toxic metal, Cd.

Use of Nramp2-transfected Chinese hamster ovary cells and reticulocytes from mk/mk mice to study iron transport mechanisms

We investigated mechanisms involved in iron (Fe) transport by DMT1 (endosomal Fe(II) exporter, encoded by the Nramp2 gene) using wild-type Chinese hamster ovary (CHO) cells and Nramp2-transfected CHO cells, as well as reticulocytes from normal and mk/mk mice that have a defect in DMT1. CHO cells and reticulocytes were incubated with 59Fe bound to various ligands. The radioiron was present in its Fe(II) or Fe(III) forms or bound to transferrin (Tf), and the internalized 59Fe measured under varying experimental conditions. Additionally, 125I-Tf interaction with reticulocytes was investigated and 59Fe incorporation into their heme was determined. Hyperexpression of DMT1 in CHO cells greatly increases their capacity to acquire ferrous iron. Although CHO-Nramp2 cells showed an increase in Fe(III) uptake as compared to CHO cells, they transported Fe(III) with much lower efficacy than Fe(II). In addition to their defect in Fe uptake, mk/mk reticulocytes also showed a decrease in Tf receptor levels. Given that CHO cells acquire iron from Fe(II)-ascorbate with much higher rates than from Fe(III)-Tf, Tf-receptor levels represent the rate-limiting step in their iron uptake. As Fe(III) transport by CHO-Nramp2 cells can be inhibited by the impermeable oxidant K3Fe(CN)6, a membrane ferric reductase is probably needed for reduction of Fe(III) to Fe(II), which is then transported by DMT1. DMT1 is not a limiting factor in Fe acquisition by normal reticulocytes and their heme synthesis.

Insight from TonB Hybrid Proteins into the Mechanism of Iron Transport through the Outer Membrane

We created hybrid proteins to study the functions of TonB. We first fused the portion of Escherichia coli tonB that encodes the C-terminal 69 amino acids (amino acids 170 to 239) of TonB downstream from E. coli malE (MalE-TonB69C). Production of MalE-TonB69C in tonB(+) bacteria inhibited siderophore transport. After overexpression and purification of the fusion protein on an amylose column, we proteolytically released the TonB C terminus and characterized it. Fluorescence spectra positioned its sole tryptophan (W213) in a weakly polar site in the protein interior, shielded from quenchers. Affinity chromatography showed the binding of the TonB C-domain to other proteins: immobilized TonB-dependent (FepA and colicin B) and TonB-independent (FepADelta3-17, OmpA, and lysozyme) proteins adsorbed MalE-TonB69C, revealing a general affinity of the C terminus for other proteins. Additional constructions fused full-length TonB upstream or downstream of green fluorescent protein (GFP). TonB-GFP constructs had partial functionality but no fluorescence; GFP-TonB fusion proteins were functional and fluorescent. The activity of the latter constructs, which localized GFP in the cytoplasm and TonB in the cell envelope, indicate that the TonB N terminus remains in the inner membrane during its biological function. Finally, sequence analyses revealed homology in the TonB C terminus to E. coli YcfS, a proline-rich protein that contains the lysin (LysM) peptidoglycan-binding motif. LysM structural mimicry occurs in two positions of the dimeric TonB C-domain, and experiments confirmed that it physically binds to the murein sacculus. Together, these findings infer that the TonB N terminus remains associated with the inner membrane, while the downstream region bridges the cell envelope from the affinity of the C terminus for peptidoglycan. This architecture suggests a membrane surveillance model of action, in which TonB finds occupied receptor proteins by surveying the underside of peptidoglycan-associated outer membrane proteins.

The Structure of the Iron-binding Protein, FutA1, from Synechocystis 6803

Cyanobacteria account for a significant percentage of aquatic primary productivity even in areas where the concentrations of essential micronutrients are extremely low. To better understand the mechanism of iron selectivity and transport, the structure of the solute binding domain of an ATP binding cassette iron transporter, FutA1, was determined in the presence and absence of iron. The iron ion is bound within the "C-clamp" structure via four tyrosine and one histidine residues. There are extensive interactions between these ligating residues and the rest of the protein such that the conformations of the side chains remain relatively unchanged as the iron is released by the opening of the metal binding cleft. This is in stark contrast to the zinc-binding protein, ZnuA, where the domains of the metal-binding protein remain relatively fixed, whereas the ligating residues rotate out of the binding pocket upon metal release. The rotation of the domains in FutA1 is facilitated by two flexible beta-strands running along the back of the protein that act like a hinge during domain motion. This motion may require relatively little energy since total contact area between the domains is the same whether the protein is in the open or closed conformation. Consistent with the pH dependence of iron binding, the main trigger for iron release is likely the histidine in the iron-binding site. Finally, neither FutA1 nor FutA2 binds iron as a siderophore complex or in the presence of anions, and both preferentially bind ferrous over ferric ions.

Iron-uptake in the Euryarchaeon Halobacterium salinarum

Iron-uptake is well studied in a plethora of pro- and eukaryotic organisms with the exception of Archaea, which thrive mainly in extreme environments. In this study, the mechanism of iron transport in the extremely halophilic Euryarchaeon Halobacterium salinarum strain JW 5 was analyzed. Under low-iron growth conditions no siderophores were detectable in culture supernatants. However, various xenosiderophores support growth of H. salinarum. In [55Fe]-[14C] double-label experiments, H. salinarum displays uptake of iron but not of the chelator citrate. Uptake of iron was inhibited by cyanide and at higher concentrations by Ga. Furthermore, a K(M) for iron uptake in cells of 2.36 microM and a Vmax of approximately 67 pmol Fe/min/mg protein was determined. [55Fe]-uptake kinetics were measured in the absence and presence of Ga. Uptake of iron was inhibited merely at very high Ga concentrations. The results indicate an energy dependent iron uptake process in H. salinarum and suggest reduction of the metal at the membrane level.