Surface-bound iron: a metal ion buffer in the marine brown alga Ectocarpus siliculosus?

Although the iron uptake and storage mechanisms of terrestrial/higher plants have been well studied, the corresponding systems in marine algae have received far less attention. Studies have shown that while some species of unicellular algae utilize unique mechanisms of iron uptake, many acquire iron through the same general mechanisms as higher plants. In contrast, the iron acquisition strategies of the multicellular macroalgae remain largely unknown. This is especially surprising since many of these organisms represent important ecological and evolutionary niches in the coastal marine environment. It has been well established in both laboratory and environmentally derived samples, that a large amount of iron can be 'non-specifically' adsorbed to the surface of marine algae. While this phenomenon is widely recognized and has prompted the development of experimental protocols to eliminate its contribution to iron uptake studies, its potential biological significance as a concentrated iron source for marine algae is only now being recognized. This study used an interdisciplinary array of techniques to explore the nature of the extensive and powerful iron binding on the surface of both laboratory and environmental samples of the marine brown alga Ectocarpus siliculosus and shows that some of this surface-bound iron is eventually internalized. It is proposed that the surface-binding properties of E. siliculosus allow it to function as a quasibiological metal ion 'buffer', allowing iron uptake under the widely varying external iron concentrations found in coastal marine environments.

Atypical iron storage in marine brown algae: a multidisciplinary study of iron transport and storage in Ectocarpus siliculosus.

Iron is an essential element for all living organisms due to its ubiquitous role in redox and other enzymes, especially in the context of respiration and photosynthesis. The iron uptake and storage systems of terrestrial/higher plants are now reasonably well understood, with two basic strategies for iron uptake being distinguished: strategy I plants use a mechanism involving induction of Fe(III)-chelate reductase (ferrireductase) and Fe(II) transporter proteins, while strategy II plants utilize high-affinity, iron-specific, binding compounds called phytosiderophores. In contrast, little is known about the corresponding systems in marine, plant-like lineages, particularly those of multicellular algae (seaweeds). Herein the first study of the iron uptake and storage mechanisms in the brown alga Ectocarpus siliculosus is reported. Genomic data suggest that Ectocarpus may use a strategy I approach. Short-term radio-iron uptake studies verified that iron is taken up by Ectocarpus in a time- and concentration-dependent manner consistent with an active transport process. Upon long-term exposure to (57)Fe, two metabolites have been identified using a combination of Mössbauer and X-ray absorption spectroscopies. These include an iron-sulphur cluster accounting for ~26% of the total intracellular iron pool and a second component with spectra typical of a polymeric (Fe(3+)O(6)) system with parameters similar to the amorphous phosphorus-rich mineral core of bacterial and plant ferritins. This iron metabolite accounts for ~74% of the cellular iron pool and suggests that Ectocarpus contains a non-ferritin but mineral-based iron storage pool.

Bacterioferritin of Magnetospirillum gryphiswaldense Is a Heterotetraeicosameric Complex Composed of Functionally Distinct Subunits but Is Not Involved in Magnetite Biomineralization.

The biomineralization pathway of magnetite in magnetotactic bacteria is still poorly understood and a matter of intense debates. In particular, the existence, nature, and location of possible mineral precursors of magnetite are not clear. One possible precursor has been suggested to be ferritin-bound ferrihydrite. To clarify its role for magnetite biomineralization, we analyzed and characterized ferritin-like proteins from the magnetotactic alphaproteobacterium Magnetospirillum gryphiswaldense MSR-1, employing genetic, biochemical, and spectroscopic techniques. Transmission Mössbauer spectroscopy of the wild type (WT) and a bacterioferritin (bfr) deletion strain uncovered that the presence of ferrihydrite in cells is coupled to the presence of Bfr. However, bfr and dps deletion mutants, encoding another ferritin-like protein, or even mutants with their codeletion had no impact on magnetite formation in MSR-1. Thus, ferritin-like proteins are not involved in magnetite biomineralization and Bfr-bound ferrihydrite is not a precursor of magnetite biosynthesis. Using transmission electron microscopy and bacterial two-hybrid and electrophoretic methods, we also show that MSR-1 Bfr is an atypical representative of the Bfr subfamily, as it forms tetraeicosameric complexes from two distinct subunits. Furthermore, our analyses revealed that these subunits are functionally divergent, with Bfr1 harboring a ferroxidase activity while only Bfr2 contributes to heme binding. Because of this functional differentiation and the poor formation of homooligomeric Bfr1 complexes, only heterooligomeric Bfr protects cells from oxidative stress in vivo. In summary, our results not only provide novel insights into the biomineralization of magnetite but also reveal the unique properties of so-far-uncharacterized heterooligomeric bacterioferritins.IMPORTANCE Magnetotactic bacteria like Magnetospirillum gryphiswaldense are able to orient along magnetic field lines due to the intracellular formation of magnetite nanoparticles. Biomineralization of magnetite has been suggested to require a yet-unknown ferritin-like ferrihydrite component. Here, we report the identification of a bacterioferritin as the source of ferrihydrite in M. gryphiswaldense and show that, contrary to previous reports, bacterioferritin is not involved in magnetite biomineralization but required for oxidative stress resistance. Additionally, we show that bacterioferritin of M. gryphiswaldense is an unusual member of the bacterioferritin subfamily as it is composed of two functionally distinct subunits. Thus, our findings extend our understanding of the bacterioferritin subfamily and also solve a longstanding question about the magnetite biomineralization pathway.

Differential role of ferritins in iron metabolism and virulence of the plant-pathogenic bacterium Erwinia chrysanthemi 3937.

During infection, the phytopathogenic enterobacterium Erwinia chrysanthemi has to cope with iron-limiting conditions and the production of reactive oxygen species by plant cells. Previous studies have shown that a tight control of the bacterial intracellular iron content is necessary for full virulence. The E. chrysanthemi genome possesses two loci that could be devoted to iron storage: the bfr gene, encoding a heme-containing bacterioferritin, and the ftnA gene, coding for a paradigmatic ferritin. To assess the role of these proteins in the physiology of this pathogen, we constructed ferritin-deficient mutants by reverse genetics. Unlike the bfr mutant, the ftnA mutant had increased sensitivity to iron deficiency and to redox stress conditions. Interestingly, the bfr ftnA mutant displayed an intermediate phenotype for sensitivity to these stresses. Whole-cell analysis by Mössbauer spectroscopy showed that the main iron storage protein is FtnA and that there is an increase in the ferrous iron/ferric iron ratio in the ftnA and bfr ftnA mutants. We found that ftnA gene expression is positively controlled by iron and the transcriptional repressor Fur via the small antisense RNA RyhB. bfr gene expression is induced at the stationary phase of growth. The sigmaS transcriptional factor is necessary for this control. Pathogenicity tests showed that FtnA and the Bfr contribute differentially to the virulence of E. chrysanthemi depending on the host, indicating the importance of a perfect control of iron homeostasis in this bacterial species during infection.

The DpsA-homologue of the archaeon Halobacterium salinarum is a ferritin.

An iron-rich protein, DpsA(Hsal), was isolated from the archaeon Halobacterium salinarum sharing a sequence identity of 35% with the starvation-induced DNA-binding protein, DpsA, of Synechecoccus sp. PCC7942. It consists of 20-kDa subunits forming a dodecameric structure. The protein exhibits a ferric iron loading of up to 100 Fe ions per mole of holoprotein. CD spectra and secondary structure calculations are consistent with an alpha-helical contribution of 60%. The UV/VIS spectrum provides no evidence for the presence of heme groups. This protein exhibits features of a non-heme type bacterial ferritin (Ftn) although it shares only little sequence homology with Ftn. Molecular modelling disclosed a high structural similarity to E. coli Dps.

The complex between hydrogenase-maturation proteins HypC and HypD is an intermediate in the supply of cyanide to the active site iron of [NiFe]-hydrogenases.

Carbamoylphosphate has been shown to be the educt for the synthesis of the CN ligands of the NiFe metal centre of hydrogenases from Escherichia coli. In the absence of carbamoylphosphate, cells accumulate a complex of two hydrogenase maturation proteins, namely HypC and HypD for the synthesis of hydrogenase 3. A procedure for the purification of wild-type HypD protein or of a biologically active derivative carrying the Strep-tagII((R)) at the N terminus has been developed. HypD is a monomeric protein possessing about 4 mol of iron per mol of protein. Electron paramagnetic resonance (EPR) and Mossbauer spectroscopy demonstrated that the iron is present as a diamagnetic [4Fe-4S](2+) cluster. The complex between HypC and HypD can be cross-linked by a number of thiol and primary amine-specific linkers. When HypD and HypC were overproduced side-by-side with HypE, the HypC-HypD complex contained substoichiometric amounts of HypE whose proportion in the complex could be augmented when HypF was also overproduced. HypE trapped in this complex could be carbamoylated by protein HypF and after dehydration transferred the cyano group to the HypC-HypD part of the complex. Free HypC and HypD were not cyanated by HypE-CN. An active HypC-HypD complex from anaerobic cells was inactivated by incubation with K(3)[Fe(CN)(6)] but not with K(4)[Fe(CN)(6)]. The results suggest the existence of a dynamic complex between the hydrogenase maturation proteins HypD, HypC, HypE and HypF, which is the site of ligand biosynthesis and attachment to the iron atom of the NiFe site in hydrogenase 3.

Synthesis and Characterization of a Series of Edge-Sharing Octahedral-Tetrahedral-Octahedral Linear Trinuclear Complexes [M(3)(L1O)(4)](2+), Where M = Mn(2+), Co(2+), Ni(2+), Cu(2+), and Zn(2+) and L1OH Is the "Heteroscorpionate" Ligand (2-Hydroxyphenyl)bis(pyrazolyl)methane.

The mixed functionality pyrazole/phenol ligand (2-hydroxyphenyl)bis(pyrazolyl)methane, L1OH, has been used to prepare a series of linear trimetallic systems with the general structural motif [M(3)(L1O)(4)](2+), where M = Mn(2+), Co(2+), Ni(2+), Cu(2+), and Zn(2+). Each of these complexes has been structurally characterized by X-ray crystallography giving the following structural parameters: [Zn(3)(L1O)(4)][BF(4)](2).H(2)O, C(52)H(44)N(16)B(2)F(8)O(5)Zn(3), monoclinic, a = 18.572(4) Å, b = 22.400(5) Å, c = 15.921(3), beta = 112.439(8) degrees, space group C2/c, Z = 4; [Cu(3)(L1O)(4)] [BF(4)](2).2MeCN, C(56)H(44)N(18)B(2)Cu(3)F(8)O(4), monoclinic, a = 40.574(2) Å, b = 16.701(1) Å, c = 19.841(2) Å, beta = 111.388(5) degrees, space group C2/c, Z = 8; [Ni(3)(L1O)(4)][ClO(4)](2).MeCN.0.5H(2)O, C(54)H(44)N(17)Cl(2)Ni(3)O(12.5), monoclinic, a = 12.324(4) Å, b = 26.537(2) Å, c = 18.829(3) Å, beta = 102.78(1) degrees, space group C2/c, Z = 4; [Co(3)(L1O)(4)][BF(4)](2).MeCN, C(54)H(44)N(17)B(2)Co(3)F(8)O(4), monoclinic, a = 12.395(2) Å, b = 26.483(3) Å, c = 18.703(4) Å, beta = 103.22(2) degrees, space group C2/c, Z = 4; [Mn(3)(L1O)(4)(MeCN)][ClO(4)](2).1.4MeCN, C(56.68)H(44)N(18.34)Cl(2)Mn(3)O(12), orthorhombic, a = 15.471(2) Å, b = 17.364(2) Å, c = 24.216(2) Å, space group Pbcn, Z = 4. For Zn(2+), Cu(2+), Ni(2+), and Co(2+) the central metal atom of the linear trimetallic [M(3)(L1O)(4)](2+) unit is four coordinate and has a pseudotetrahedral geometry with a dihedral angle, omega, between the two M(central)O(2)M(terminal) planes of 79.9 degrees (Zn), 61.2 degrees (Co), 60.4 degrees (Ni), and 46.8 degrees (Cu). The central Mn(2+) atom of [Mn(3)(L1O)(4)(MeCN)][ClO(4)](2).1.4MeCN is five-coordinate, with a trigonal bipyramidal stereochemistry, the result of an equatorially coordinated MeCN solvent molecule. Variable-temperature magnetic data indicate that the Ni, Cu, and Mn complexes display modest antiferromagnetic coupling between the metal centers, while the Co derivative is strongly ferromagnetically coupled.

Iron transport and storage in the coccolithophore: Emiliania huxleyi.

Iron is an essential element for all living organisms due to its ubiquitous role in redox and other enzymes, especially in the context of respiration and photosynthesis. The iron uptake and storage systems of terrestrial/higher plants are now reasonably well understood with two basic strategies for iron uptake being distinguished: strategy I plants use a mechanism involving soil acidification and induction of Fe(III)-chelate reductase (ferrireductase) and Fe(II) 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 coccolithophore Emiliania huxleyi. Short term radio-iron uptake studies indicate that iron is taken up by Emiliania in a time and concentration dependent manner consistent with an active transport process. Based on inhibitor studies it appears that iron is taken up directly as Fe(iii). However if a reductive step is involved the Fe(II) must not be accessible to the external environment. Upon long term exposure to (57)Fe we have been able, using a combination of Mössbauer and XAS spectroscopies, to identify a single metabolite which displays spectral features similar to the phosphorus-rich mineral core of bacterial and plant ferritins.

A multidisciplinary study of iron transport and storage in the marine green alga Tetraselmis suecica.

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.

The Fe(III) and Ga(III) coordination chemistry of 3-(1-hydroxymethylidene) and 3-(1-hydroxydecylidene)-5-(2-hydroxyethyl)pyrrolidine-2,4-dione: novel tetramic acid degradation products of homoserine lactone bacterial quorum sensing molecules.

Bacteria use small diffusible molecules to exchange information in a process called quorum sensing (QS). An important class of quorum sensing molecules used by Gram-negative bacteria is the family of N-acylhomoserine lactones (HSL). It was recently discovered that a degradation product of the QS molecule 3-oxo-C(12)-homoserine lactone, the tetramic acid 3-(1-hydroxydecylidene)-5-(2-hydroxyethyl)pyrrolidine-2,4-dione, is a potent antibacterial agent, thus implying roles for QS outside of simply communication. Because these tetramic acids also appear to bind iron with appreciable affinity it was suggested that metal binding might contribute to their biological activity. Here, using a variety of spectroscopic tools, we describe the coordination chemistry of both the methylidene and decylidene tetramic acid derivatives with Fe(III) and Ga(III) and discuss the potential biological significance of such metal binding.

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.

Expression and regulation pattern of ferritin-like DpsA in the archaeon Halobacterium salinarum.

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 (H2O2), 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.

Expression and regulation pattern of ferritin-like DpsA in the archaeon Halobacterium Salinarum.

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.

Effects of iron limitation on the respiratory chain and the membrane cytochrome pattern of the Euryarchaeon Halobacterium salinarum.

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.

Chrysobactin-dependent iron acquisition in Erwinia chrysanthemi. Functional study of a homolog of the Escherichia coli ferric enterobactin esterase.

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. Mössbauer 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.

FhuF, part of a siderophore-reductase system.

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.

Iron use for haeme synthesis is under control of the yeast frataxin homologue (Yfh1).

The YFH1 gene is the yeast homologue of the human FRDA gene, which encodes the frataxin protein. Saccharomyces cerevisiae cells lacking the YFH1 gene showed very low cytochrome content. In Deltayfh1 strains, the level of ferrochelatase (Hem15p) was very low, as a result of transcriptional repression of HEM15. However, the low amount of Hem15p was not the cause of haeme deficiency in Deltayfh1 cells. Ferrochelatase, a mitochondrial protein, able to mediate insertion of iron or zinc into the porphyrin precursor, made primarily the zinc protoporphyrin product. Zinc protoporphyrin instead of haeme accumulated during growth of Deltayfh1 mutant cells and, furthermore, preferential formation of zinc protoporphyrin was observed in real time. The method for these studies involved direct presentation of porphyrin to mitochondria and to ferrochelatase of permeabilized cells with intact architecture, thereby specifically testing the iron delivery portion of the haeme biosynthetic pathway. The studies showed that Deltayfh1 mutant cells are defective in iron use by ferrochelatase. Mössbauer spectroscopic analysis showed that iron was present as amorphous nano-particles of ferric phosphate in Deltayfh1 mitochondria, which could explain the unavailability of iron for haeme synthesis. A high frequency of suppressor mutations was observed, and the phenotype of such mutants was characterized by restoration of haeme synthesis in the absence of Yfh1p. Suppressor strains showed a normal cytochrome content, normal respiration, but remained defective in Fe-S proteins and still accumulated iron into mitochondria although to a lesser extent. Yfh1p and Hem15p were shown to interact in vitro by Biacore studies. Our results suggest that Yfh1 mediates iron use by ferrochelatase.