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The iron uptake and storage systems of terrestrial/higher plants are now reasonably well understood with two basic strategies being distinguished: strategy I involves the induction of a Fe(III)-chelate reductase (ferrireductase) along with Fe(II) or Fe(III) transporter proteins while strategy II plants have evolved sophisticated systems based on high-affinity, iron specific, binding compounds called phytosiderophores. In contrast, there is little knowledge about the corresponding systems in marine, plant-like lineages. Herein we report a study of the iron uptake and storage mechanisms in the green alga Tetraselmis suecica. Short term radio-iron uptake studies indicate that iron is taken up by Tetraselmis in a time and concentration dependent manner consistent with an active transport process. Based on inhibitor and other studies it appears that a reductive-oxidative pathway such as that found in yeast and the green alga Chlamydomonas reinhardtii is likely. Upon long term exposure to (57)Fe we have been able, using a combination of Mössbauer and X-ray absorption spectroscopies, to identify three metabolites. The first exhibits Mössbauer parameters typical of a [Fe(4)S(4)](2+) cluster and which accounts for approximately 10% of the total intracellular iron pool. The second displays a spectrum typical of a [Fe(II)O(6)] system accounting for approximately 2% of the total pool. The largest component (ca. 85+%) consists of polymeric iron-oxo mineral species with parameters between that of the crystalline ferrihydrite core of animal ferritins and the amorphous hydrated ferric phosphate of bacterial and plant ferritins.

Magnetotactic bacteria synthesize specific organelles, the magnetosomes, which are membrane-enveloped crystals of the magnetic mineral magnetite (Fe 3 O 4 ). The biomineralization of magnetite involves the uptake and intracellular accumulation of large amounts of iron. However, it is not clear how iron uptake and biomineralization are regulated and balanced with the biochemical iron requirement and intracellular homeostasis. In this study, we identified and analyzed a homologue of the f erric u ptake r egulator Fur in Magnetospirillum gryphiswaldense , which was able to complement a fur mutant of Escherichia coli . A fur deletion mutant of M. gryphiswaldense biomineralized fewer and slightly smaller magnetite crystals than did the wild type. Although the total cellular iron accumulation of the mutant was decreased due to reduced magnetite biomineralization, it exhibited an increased level of free intracellular iron, which was bound mostly to a ferritin-like metabolite that was found significantly increased in Mössbauer spectra of the mutant. Compared to that of the wild type, growth of the fur mutant was impaired in the presence of paraquat and under aerobic conditions. Using a Fur titration assay and proteomic analysis, we identified constituents of the Fur regulon. Whereas the expression of most known magnetosome genes was unaffected in the fur mutant, we identified 14 proteins whose expression was altered between the mutant and the wild type, including five proteins whose genes constitute putative iron uptake systems. Our data demonstrate that Fur is a regulator involved in global iron homeostasis, which also affects magnetite biomineralization, probably by balancing the competing demands for biochemical iron supply and magnetite biomineralization.

Very recently, an iron-rich protein, DpsA, was isolated from the extreme halophilic euryarchaeon Halobacterium salinarum JW5 and characterized. The amino acid sequence of DpsA is related to Dps proteins which belong structurally to the ferritin superfamily but differ from ferritins in their function and regulation. Employing Northern and Western blot analysis, the expression of DpsA in H. salinarum was examined throughout all growth phases and under a variety of growth conditions (iron deficiency, iron supplied growth, oxidative stress). DpsA shows increasing expression of dpsA mRNA in iron-rich media and under conditions of oxidative stress (H(2)O(2)), whereas under iron-deficient conditions mRNA-levels decrease. This is in contrast to Dps-type proteins the transcription of which is induced under conditions of iron starvation. Northern blot experiments show that the expression pattern of halobacterial DpsA is the same as that found in the few bacterial non-heme ferritin the expression pattern of which has been analyzed so far. Based on Western-blot analysis post-transcriptional regulation, typical of mammalian ferritins, can be excluded. This protein exhibits features of a non-heme type bacterial ferritin although it shares only little sequence similarity with Ftn from E. coli.

Carbamoylphosphate has been shown to be the educt for the synthesis ofthe CN ligands of the NiFe metal centre of hydrogenases from Escherichiacoli. In the absence of carbamoylphosphate, cells accumulate a complex oftwo hydrogenase maturation proteins, namely HypC and HypD for thesynthesis of hydrogenase 3. A procedure for the purification of wild-typeHypD protein or of a biologically active derivative carrying the Strep-tagIIwat the N terminus has been developed. HypD is a monomeric proteinpossessing about 4 mol of iron per mol of protein. Electron paramagneticresonance (EPR) and Mo¿ssbauer spectroscopy demonstrated that the iron ispresent as a diamagnetic [4Fe¿4S]2C cluster. The complex between HypCand HypD can be cross-linked by a number of thiol and primary aminespecificlinkers. When HypD and HypC were overproduced side-by-sidewith HypE, the HypC¿HypD complex contained substoichiometricamounts of HypE whose proportion in the complex could be augmentedwhen HypF was also overproduced. HypE trapped in this complex couldbe carbamoylated by protein HypF and after dehydration transferred thecyano group to the HypC¿HypD part of the complex. Free HypC andHypD were not cyanated by HypE-CN. An active HypC¿HypD complexfrom anaerobic cells was inactivated by incubation with K3[Fe(CN)6] butnot with K4[Fe(CN)6]. The results suggest the existence of a dynamiccomplex between the hydrogenase maturation proteins HypD, HypC,HypE and HypF, which is the site of ligand biosynthesis and attachment tothe iron atom of the NiFe site in hydrogenase 3.

FhuF is a cytoplasmic 2Fe-2S protein of Escherichia coli loosely associated with the cytoplasmic membrane. E. coli fhuF mutants showed reduced growth on plates with ferrioxamine B as the sole iron source, although siderophore uptake was not defective in transport experiments. Removal of iron from coprogen, ferrichrome, and ferrioxamine B was significantly lower in fhuF mutants compared to the corresponding parental strains, which suggested that FhuF is involved in iron removal from these hydroxamate-type siderophores. A redox potential E(1/2) of -310 +/- 25 mV relative to the normal hydrogen electrode was determined for FhuF by EPR redox titration; this redox potential is sufficient to reduce the siderophores coprogen and ferrichrome. Mössbauer spectra revealed that FhuF in its [Fe(2+)-Fe(3+)] state is also capable of direct reduction of ferrioxamine B-bound ferric iron, thus proving its reductase function. This is the first report on a bacterial siderophore-iron reductase which in vivo seems to be specific for a certain group of hydroxamates.

TheYFH1 geneistheyeasthomologueofthehumanFRDAgene,whichencodesthefrataxinprotein.Saccharomyces cerevisiae cellslackingtheYFH1 geneshowedverylowcytochromecontent.InDyfh1strains,thelevelofferrochelatase(Hem15p)wasverylow,asaresultoftranscriptionalrepressionofHEM15.However,thelowamountofHem15pwasnotthecauseofhaemedeficiencyinDyfh1 cells.Ferrochelatase,amitochondrialprotein,abletomediateinsertionofironorzincintotheporphyrinprecursor,madeprimarilythezincprotoporphyrinproduct.ZincprotoporphyrininsteadofhaemeaccumulatedduringgrowthofDyfh1mutantcellsand,furthermore,preferentialformationofzincprotoporphyrinwasobservedinrealtime.Themethodforthesestudiesinvolveddirectpresentationofporphyrintomitochondriaandtoferrochelataseofpermeabilizedcellswithintactarchitecture,therebyspecificallytestingtheirondeliveryportionofthehaemebiosyntheticpathway.ThestudiesshowedthatDyfh1 mutantcellsaredefectiveinironusebyferrochelatase.Mo¨ssbauerspectroscopicanalysisshowedthatironwaspresentasamorphousnano-particlesofferricphosphateinDyfh1 mitochondria,whichcouldexplaintheunavailabilityofironforhaemesynthesis.Ahighfrequencyofsuppressormutationswasobserved,andthephenotypeofsuchmutantswascharacterizedbyrestorationofhaemesynthesisintheabsenceofYfh1p.Suppressorstrainsshowedanormalcytochromecontent,normalrespiration,butremaineddefectiveinFe–Sproteinsandstillaccumulatedironintomitochondriaalthoughtoalesserextent.Yfh1pandHem15pwereshowntointeractin vitro byBiacorestudies.OurresultssuggestthatYfh1mediatesironusebyferrochelatase.INTRODUCTIONThe YFH1 gene is the yeast homologue of the human FRDAgene, which encodes the frataxin protein. Mutations of FRDAassociated with decreased frataxin expression are responsiblefor Friedreich’s ataxia, the most common autosomal-recessiveneurodegenerative disease of Caucasians (1,2). Both genescode for mitochondrial proteins that are involved in ironhomeostasis and cellular respiration (3–8), but their preciseroles are unknown. Cardiac tissues from patients withFriedreich’s ataxia exhibit iron deposition, deficiencies in manyiron–sulphur cluster enzymes and reduced mitochondrial DNA(8,9). In addition, fibroblasts from these patients showhypersensitivity to oxidative stress that can be rescued bytreatment with iron chelators (10). A link to haeme biosynthesishas not been uncovered, and blood, bone marrow and red celldevelopment appear to be normal in patient with frataxindeficits. Frataxin is downregulated during erythroid develop-ment, suggesting that this protein is not involved in the high-volume iron trafficking that accompanies red cell production inthe bone marrow (11). However, activities of haeme enzymes inother tissues of Friedreich’s ataxia patients have not beenassessed, leaving open the possibility that tissue-specific haemedeficiencies may exist.Yeast cells lacking Yfh1p mirror many of the phenotypesobserved in disease tissues from patients with Friedreich’sataxia. These cells have defective respiration (3,5,12–14),unstable mitochondrial DNA and hypersensitivity to oxidative

Under iron limitation, the plant pathogen Erwinia chrysanthemi produces the catechol-type siderophore chrysobactin, which acts as a virulence factor. It can also use enterobactin as a xenosiderophore. We began this work by sequencing the 5'-upstream region of the fct-cbsCEBA operon, which encodes the ferric chrysobactin receptor and proteins involved in synthesis of the catechol moiety. We identified a new iron-regulated gene (cbsH) transcribed divergently relative to the fct gene, the translated sequence of which is 45.6% identical to that of Escherichia coli ferric enterobactin esterase. Insertions within this gene interrupt the chrysobactin biosynthetic pathway by exerting a polar effect on a downstream gene with some sequence identity to the E. coli enterobactin synthase gene. These mutations had no effect on the ability of the bacterium to obtain iron from enterobactin, showing that a functional cbsH gene is not required for iron removal from ferric enterobactin in E. chrysanthemi. The cbsH-negative mutants were less able to utilize ferric chrysobactin, and this effect was not caused by a defect in transport per se. In a nonpolar cbsH-negative mutant, chrysobactin accumulated intracellularly. These defects were rescued by the cbsH gene supplied on a plasmid. The amino acid sequence of the CbsH protein revealed characteristics of the S9 prolyl oligopeptidase family. Ferric chrysobactin hydrolysis was detected in cell extracts from a cbsH-positive strain that was inhibited by diisopropyl fluorophosphate. These data are consistent with the fact that chrysobactin is a d-lysyl-l-serine derivative. Mossbauer spectroscopy of whole cells at various states of (57)Fe-labeled chrysobactin uptake showed that this enzyme is not required for iron removal from chrysobactin in vivo. The CbsH protein may therefore be regarded as a peptidase that prevents the bacterial cells from being intracellularly iron-depleted by chrysobactin.

Transport and metabolization of iron bound to the fungal siderophore rhizoferrin was analyzed by transport kinetics, Mossbauer and EPR spectroscopy. Saturation kinetics (vmax=24.4 pmol/(mg min), Km=64.4μM) and energy dependence excluded diffusion and provided evidence for a rhizoferrin transport system in M. smegmatis. Based on the spectroscopic techniques indications for intracellular presence of the ferric rhizoferrin complex were found. This feature could be of practical importance in the search of novel drugs for the treatment of mycobacterial infections. EPR and Mossbauer spectroscopy revealed different ferritin mineral cores depending on the siderophore iron source. This finding was interpreted in terms of different protein shells, i.e. two types of ferritins.

We previously used fhuF as a sensitive reporter gene of the iron status of Escherichia coli. In this report, the fhuF gene was identified as open reading frame f262b at 99.2 min on the genome sequence map of E. coli K-12. The FhuF protein was labeled with a His-tag and then purified to electrophoretic homogeneity. Based on sulfur determinations and Mössbauer and EPR spectroscopy, FhuF was identified as a [2Fe-2S] protein. The g values (gx = 1.886, gy = 1.961, gz = 1.994) and some of the Mössbauer parameters of FhuF obtained [oxidized protein as isolated: delta EQ,4.2K = 0.474 mm s-1; Fe3+ (reduced protein): delta EQ = 0.978 mm s-1] are not typical of common [2Fe-2S] proteins and indicate that FhuF has unusual structural properties. The primary sequence of FhuF does not show any sequence similarities to known [2Fe-2S] proteins. By site-directed mutagenesis, each of the six cysteines of FhuF was replaced by serine. EPR of the six reduced mutant proteins revealed that the terminal cysteine residues 244, 245, 256, and 259 form the [2Fe-2S]Cys4 cluster. Mutants having the Cys-to-Ser replacement at positions 244, 245, 256, or 259 did not complement a fhuF mutant. The motif Cys-Cys-Xaa10-Cys-Xaa2-Cys in FhuF differs considerably from the motif Cys-Xaa2-Cys-Xaa9-15-Cys-Xaa2-Cys found in other [2Fe-2S] proteins. The unusual Cys-Cys terminal group of the cluster may explain the atypical EPR and Mössbauer spectroscopic properties of the FhuF protein; possibly the tetrahedral symmetry at the ferric ion site is distorted. The phenotype of fhuF mutants and the structural features of the FhuF protein suggest that FhuF is involved in the reduction of ferric iron in cytoplasmic ferrioxamine B.

Iron uptake of the 57Fe-siderophores ferricrocin, rhizoferrin and citrate in M. smegmatis was analyzed by in situ Mossbauer spectroscopy. Siderophore dependent uptake and metabolic utilization patterns of 57Fe were found. Rhizoferrin is accumulated in the organism and, therefore, represents a suitable candidate for the synthesis of novel siderophore-antibiotics conjugates.

The interactions between ferric ions and the anticancer antibiotic adriamycin have been investigated by 57 Fe Mossbauer and electronic spectroscopies. The Mossbauer parameters are markedly dependent on the preparation procedure, the equilibration time, the metal-to-ligand ratio, and the concentration of the drug. At millimolar drug concentration, the 4.2 K Mossbauer spectra exhibit a broad Fe(III) magnetic sextet attributed to polynuclear aggregates of high magnetic anisotropy, and a quadrupole split Fe(III) doublet attributed to a species of lower magnetic anisotropy, which exhibits superparamagnetic behavior. The two species are in equilibrium, as indicated by the time evolution of both Mossbauer and electronic spectra. At 3.0 10 −5 M drug concentration, when adriamycin is mainly monomeric, the 4.2 K Mossbauer spectra exhibit a quadrupole split doublet, connected with a superparamagnetic system, as for the concentrated preparations, and a broad magnetic sextet whose relative area increases with aging. The species responsible for this sextet should be some polymerizable hydrolysis product of the Fe(III) ions present in the dilute solution. In addition, the presence of a transient Fe(II) quadrupole doublet, both in this preparation and when the [Fe] / [ADR] ratio is 1:5 at millimolar drug concentration, is explained with the known intramolecular one-electron redox reaction which ferric-adriamycin undergoes under anaerobic conditions. The present results confirm that adriamycin is markedly more reactive than daunomycin due to its hydroxymethyl side-chain, and suggest that the stacking of the drug molecules plays a role in the observed cooperative phenomena.

The interactions of the iron complexes of the anthracycline antitumour drugs daunomycin (DN) and adriamycin (ADM) with the mononucleotide AMP, herring sperm DNA, plasmic pBR322 and immortalized 3T3 fibroblasts were studied. By means of Mössbauer spectroscopy it was demonstrated that DNA is a powerful ferric iron chelator as compared with AMP, which is not able to compete with DN or acetohydroxamic acid for ferric iron. The difference between AMP and DNA is postulated to be based on the chelate effect. The Mössbauer spectra of the ternary Fe-anthracycline-DNA systems differ from Fe-anthracycline binary complexes, indicating rearrangement reactions. Dialysis experiments clearly disclose the formation of a ternary Fe-ADM-pBR322 complex, the topology of which differs substantially from intercalating ADM. The effect of Fe-ADM complexes (3:1) on the growth of immortalized mouse embryonal fibroblasts (NIH-3T3) was studied in comparison with ADM alone. No significant difference on the inhibition of cell growth was noticed, suggesting comparable cytotoxicity for the compounds. In contrast to literature data, no evidence was found for DNA cleavage by ferric ADM at molar ratios as high as 1/100 (ADM/base pair), even if the ternary systems were prepared in the light and in the presence of reducing or oxidizing agents. Based on our observations it seems that the cytotoxicity of both ADM and Fe-ADM oligomer is not based primarily on intercalation or direct interaction with DNA.

We have analyzed ferrioxamine-E-mediated iron uptake and metabolization in Erwinia herbicola K4 (Pantoea agglomerans) by means of in vivo Mössbauer spectroscopy and radioactive labeling techniques. A comparison of cell spectra with the spectrum of ferrioxamine clearly demonstrates that ferrioxamine E is not accumulated in the cell, indicating a fast metal transfer. Only two major components of iron metabolism can be detected, a ferric and a ferrous species. At 30 min after uptake, 86% of the internalized metal corresponded to a ferrous ion compound and 14% to a ferric iron species. Metal transfer apparently involves a reductive process. With progressing growth, the oxidized species of the two major proteins becomes dominant. The two iron metabolites closely resemble species previously isolated from Escherichia coli. These components of iron metabolism differ from bacterio-ferritin, cytochromes and most iron-sulfur proteins. All other iron-containing cellular components are at least one order of magnitude lower in concentration. We suggest that the ferrous and ferric iron species correspond to two different oxidation states of a low-molecular mass protein.

Siderophores (from the Greek: iron carrier) are low molecular mass (500–1500 Da) iron chelators that are synthesized in bacteria and fungi under conditions of iron deficiency. Siderophores exhibit extraordinarily high complex formation constants for ferric iron with β-values ranging from 1020 to approximately 1050. Fe2+-siderophores are some 20 orders of magnitude less stable than their Fe3+ counterparts. The d5 electronic configuration of Fe3+ rules out any crystal field stabilization energy and makes ferric iron complexes relatively labile with respect to isomerization and ligand exchange. Siderophores display a selectivity for iron that is reflected in the corresponding complex stability constants that are higher with Fe3+ than with Al3+, or with bivalent cations like Ca2+, Cu2+ or Zn2+. Microorganisms excrete desferrisiderophores in order to scavenge iron from the environment. The competition for iron by siderophores, the mechanisms of siderophore uptake through microbial membranes, and the intracellular pathways of siderophore-iron utilization strongly depend on thermodynamic, kinetic, and structural features of the ferric iron siderophore complexes. The structural features of siderophores are diverse. The ligating groups contain oxygen atoms of hydroxamate, catecholate, α-hydroxy carboxylic and salicylic acids, or oxazoline and thiazoline nitrogen. Reduction potentials of ferric siderophore complexes vary between −700 and −150 mV. In particular at the low potential end below −450 mV, special biological strategies of reductive iron removal are required because these potentials are too negative for typical cellular reductases. The coordination and redox chemistry of siderophores is also reflected in the mechanisms of siderophore-mediated iron uptake in microorganisms. A classic example is the intracellular removal of iron from enterobactin. The permeation of cell walls or bacterial membranes by siderophores is in most microbes a highly specific process requiring an array of up to eight proteins. The advent of modern molecular biology delivered a cornucopia of methods enabling high-yield production of specific gene products relevant to siderophore synthesis and transport, analyses of structure-function relationships (employing site directed mutagenesis), and detailed insights into the regulation of the corresponding processes. One siderophore, ferrioxamine B, serves as a detoxifier in iron overload diseases and in the treatment of β-thalassemia. Siderophores and siderophore analogs also play a role in MRI and are employed as basic models for actinide chelators in order to remove these metals from the environment or from a contaminated body. Keywords: siderophore; chromic siderophores; complex formation constants; redox potentials; microbial iron transport; siderophore receptors; infection; medical therapy; actinides

The effects of iron limitation on the electron transport chain of the extremely halophilic Euryarchaeon Halobacterium salinarum were analyzed. When iron was growth-limiting, the respiratory rates as well as the inhibition pattern of the membranes were significantly different from membranes of iron replete cells. Changes in the availability of iron cause the formation of different respiratory pathways including different entry sites for electrons, different terminal oxidases of the respiratory chain, and drastic changes of the cytochrome composition and of the relative amounts of cytochromes. Under iron-limiting conditions, mainly low-potential cytochromes were measured. EPR spectroscopic studies revealed that the amount of proteins containing iron-sulfur clusters is reduced in membranes under iron-limiting growth conditions. Taken together, our results strongly suggest for the first time an important role of iron supply for the bioenergetics of an Archaeon.

Based on in vivo Mössbauer spectroscopy it has previously been demonstrated that the intracellular iron pool of Escherichia coli, grown in iron deficient media supplemented with siderophores as the sole iron source, is dominated by a single Fe2+ and a single Fe3+ species. We have isolated the ferrous ion species and have purified it employing native column PAGE, chromatography and ultrafiltration. The purified compound displays an Mapp of 2.2 kDa and an extremely low isoelectric point (pI) of 1.05. It is shown that this ferrous ion binding compound is neither a protein nor a nucleotide, rather it is composed mainly of phosphorylated sugar derivatives. This compound binds approximately 40% of the cytoplasmic iron. Therefore it is proposed that this oligomeric ferrous carbohydrate phosphate represents the long sought after mobile, low molecular mass iron pool.

The interaction of the antitumor agent daunomycin (DN) with ferric iron has been analysed by Mössbauer spectroscopy, EPR, extended X-ray absorption fine structure (EXAFS), and magnetic susceptibility measurements. In contrast to literature data, at millimolar iron and anthracycline concentrations no solitary Fe(DN)3 complexes are formed in appreciable amounts. The Mössbauer spectroscopic analysis revealed severe dependencies on temperature, on the preparation procedure, the time allowed for equilibration, and on the metal/ligand ratio. The Mössbauer spectra exhibit two components: a broad magnetic sextet and a quadrupole doublet at an Fe/DN molar ratio of 1:3 and exclusively a doublet at a molar ratio of 1:20, indicating an equilibrium of these two spectral components. The EPR spectra are dominated by a signal at g(eff) = 2. Double integration of the EPR signals enabled the determination of their spin density and a correlation between EPR and Mössbauer spectra. The Mössbauer sextet species is EPR invisible and corresponds to magnetically ordered polynuclear aggregates with high magnetic anisotropy. EXAFS and susceptibility measurements provide additional evidence for the formation of polynuclear aggregates of ferric daunomycin. The quadrupole doublet species in the Mössbauer spectra correlates with the g = 2 signal in EPR. This species is also related to a magnetically ordered system, exhibiting, however, superparamagnetic behavior due to less magnetic anisotropy. Since daunomycin forms dimers in aqueous solution at millimolar concentrations, we conclude that the cooperative phenomena observed in EPR and Mössbauer spectra are a consequence of its stacking effects.

Ferric ion uptake via the fhuE receptor of E. coli is stereospecific; it prefers the delta-absolute configuration of the metal complexes. The importance of the metal center and its adjacent functionalities for the stereospecific recognition of ferrichromes via TonA is discussed.

The mobilization of iron from intracellular ferritin by ascorbic acid has been analysed in situ by electron paramagnetic resonance (EPR) spectroscopy. EPR enables a distinction between ferritins and other Fe3+-binding cellular components. The ordered iron core of ferritin gives rise to a resonance signal which can be observed only at temperatures above 50 K. In the present study we clearly demonstrate that ascorbic acid is capable of mobilizing iron from ferritin in the cellular system by reduction of the ferric ion core in neuroblastoma SK-N-SH cells. This mechanism may open new ways in the therapy of this hardly curable tumor in stage IV, especially in combination with some cytostatic drugs.

To date it has barely been recognized that the nature of about 75% of the Escherichia coli iron pool is unknown. Here we report the isolation of two iron species representing major components of iron metabolism in various growth states of E. coli. In vivo Mössbauer spectroscopy was applied to obtain information on the intracellular distribution pattern of iron in E. coli K12 W3110. Only two types of iron could be detected in the cell spectra: hexacoordinated Fe2+ and Fe3+ high-spin complexes. Other iron-requiring compounds are at least one order of magnitude less abundant in E. coli. The Mössbauer parameters of these complexes fit neither cytochromes nor iron-sulfur proteins nor ferric holo-bacterioferritin. They are sensitive to metabolic changes and inhibitors. The ratio of Fe/subunit, Fe2+/Fe3+ interconversion, chromatographic and electrophoretic data exclude bacterioferritin as the main iron metabolite in E. coli. Bacterioferritin can be observed only at very high ferric ion concentrations in the medium. The 55Fe fluorograms of both cytoplasmic and membrane fractions exhibit two exclusive bands with apparent molecular masses of 17 and 15 kDa, respectively. The two bands comprised 70% of the applied radioactivity. In gel filtration the main iron peak elutes at 155 kDa yielding two bands with apparent molecular masses of 17 and 15 kDa on SDS/PAGE. We therefore conclude that the iron species form a protein with an apparent molecular mass of 155 kDa containing 17-kDa and 15-kDa subunits. The iron content of the protein is 44 micrograms Fe/mg protein which corresponds to approximately 13 iron ions/subunit. No iron protein exhibiting the observed features has been described so far. Additional Mössbauer experiments suggest that these novel iron proteins are not restricted to E. coli but that similar components are detectable in several bacterial and fungal systems, thus pointing to a general occurrence.