Ferroxidase Center Research Articles

The crystal structure of Acinetobacter baumannii bacterioferritin reveals a heteropolymer of bacterioferritin and ferritin subunits

Iron storage proteins, e.g., vertebrate ferritin, and the ferritin-like bacterioferritin (Bfr) and bacterial ferritin (Ftn), are spherical, hollow proteins that catalyze the oxidation of Fe2+ at binuclear iron ferroxidase centers (FOC) and store the Fe3+ in their interior, thus protecting cells from unwanted Fe3+/Fe2+ redox cycling and storing iron at concentrations far above the solubility of Fe3+. Vertebrate ferritins are heteropolymers of H and L subunits with only the H subunits having FOC. Bfr and Ftn were thought to coexist in bacteria as homopolymers, but recent evidence indicates these molecules are heteropolymers assembled from Bfr and Ftn subunits. Despite the heteropolymeric nature of vertebrate and bacterial ferritins, structures have been determined only for recombinant proteins constituted by a single subunit type. Herein we report the structure of Acinetobacter baumannii bacterioferritin, the first structural example of a heteropolymeric ferritin or ferritin-like molecule, assembled from completely overlapping Ftn homodimers harboring FOC and Bfr homodimers devoid of FOC but binding heme. The Ftn homodimers function by catalyzing the oxidation of Fe2+ to Fe3+, while the Bfr homodimers bind a cognate ferredoxin (Bfd) which reduces the stored Fe3+ by transferring electrons via the heme, enabling Fe2+ mobilization to the cytosol for incorporation in metabolism.

Pseudomonas aeruginosa gene PA4880 encodes a Dps-like protein with a Dps fold, bacterioferritin-type ferroxidase centers, and endonuclease activity.

We report the biochemical, structural, and functional characterization of the protein coded by gene PA4880 in the P. aeruginosa PAO1 genome. The PA4880 gene had been annotated as coding a probable bacterioferritin. Our structural work shows that the product of gene PA4880 is a protein that adopts the Dps subunit fold, which oligomerizes into a 12-mer quaternary structure. Unlike Dps, however, the ferroxidase di-iron centers and iron coordinating ligands are buried within each subunit, in a manner identical to that observed in the ferroxidase center of P. aeruginosa bacterioferritin. Since these structural characteristics correspond to Dps-like proteins, we term the protein as P. aeruginosa Dps-like, or Pa DpsL. The ferroxidase centers in Pa DpsL catalyze the oxidation of Fe2+ utilizing O2 or H2O2 as oxidant, and the resultant Fe3+ is compartmentalized in the interior cavity. Interestingly, incubating Pa DpsL with plasmid DNA results in efficient nicking of the DNA and at higher concentrations of Pa DpsL the DNA is linearized and eventually degraded. The nickase and endonuclease activities suggest that Pa DpsL, in addition to participating in the defense of P. aeruginosa cells against iron-induced toxicity, may also participate in the innate immune mechanisms consisting of restriction endonucleases and cognate methyl transferases.

Myxococcus xanthus encapsulin cargo protein EncD is a flavin-binding protein with ferric reductase activity

Encapsulins are protein nanocompartments that regulate cellular metabolism in several bacteria and archaea. Myxococcus xanthus encapsulins protect the bacterial cells against oxidative stress by sequestering cytosolic iron. These encapsulins are formed by the shell protein EncA and three cargo proteins: EncB, EncC, and EncD. EncB and EncC form rotationally symmetric decamers with ferroxidase centers (FOCs) that oxidize Fe+2 to Fe+3 for iron storage in mineral form. However, the structure and function of the third cargo protein, EncD, have yet to be determined. Here, we report the x-ray crystal structure of EncD in complex with flavin mononucleotide. EncD forms an α-helical hairpin arranged as an antiparallel dimer, but unlike other flavin-binding proteins, it has no β-sheet, showing that EncD and its homologs represent a unique class of bacterial flavin-binding proteins. The cryo-EM structure of EncA-EncD encapsulins confirms that EncD binds to the interior of the EncA shell via its C-terminal targeting peptide. With only 100 amino acids, the EncD α-helical dimer forms the smallest flavin-binding domain observed to date. Unlike EncB and EncC, EncD lacks a FOC, and our biochemical results show that EncD instead is a NAD(P)H-dependent ferric reductase, indicating that the M. xanthus encapsulins act as an integrated system for iron homeostasis. Overall, this work contributes to our understanding of bacterial metabolism and could lead to the development of technologies for iron biomineralization and the production of iron-containing materials for the treatment of various diseases associated with oxidative stress.

The Ferroxidase Centre of Escherichia coli Bacterioferritin Plays a Key Role in the Reductive Mobilisation of the Mineral Iron Core

AbstractFerritins are multimeric cage‐forming proteins that play a crucial role in cellular iron homeostasis. All H‐chain‐type ferritins harbour a diiron site, the ferroxidase centre, at the centre of a 4 α‐helical bundle, but bacterioferritins are unique in also binding 12 hemes per 24 meric assembly. The ferroxidase centre is known to be required for the rapid oxidation of Fe2+ during deposition of an immobilised ferric mineral core within the protein's hollow interior. In contrast, the heme of bacterioferritin is required for the efficient reduction of the mineral core during iron release, but has little effect on the rate of either oxidation or mineralisation of iron. Thus, the current view is that these two cofactors function in iron uptake and release, respectively, with no functional overlap. However, rapid electron transfer between the heme and ferroxidase centre of bacterioferritin from Escherichia coli was recently demonstrated, suggesting that the two cofactors may be functionally connected. Here we report absorbance and (magnetic) circular dichroism spectroscopies, together with in vitro assays of iron‐release kinetics, which demonstrate that the ferroxidase centre plays an important role in the reductive mobilisation of the bacterioferritin mineral core, which is dependent on the heme‐ferroxidase centre electron transfer pathway.

The Ferroxidase Centre of Escherichia coli Bacterioferritin Plays a Key Role in the Reductive Mobilisation of the Mineral Iron Core.

Ferritins are multimeric cage-forming proteins that play a crucial role in cellular iron homeostasis. All H-chain-type ferritins harbour a diiron site, the ferroxidase centre, at the centre of a 4 α-helical bundle, but bacterioferritins are unique in also binding 12 hemes per 24 meric assembly. The ferroxidase centre is known to be required for the rapid oxidation of Fe2+ during deposition of an immobilised ferric mineral core within the protein's hollow interior. In contrast, the heme of bacterioferritin is required for the efficient reduction of the mineral core during iron release, but has little effect on the rate of either oxidation or mineralisation of iron. Thus, the current view is that these two cofactors function in iron uptake and release, respectively, with no functional overlap. However, rapid electron transfer between the heme and ferroxidase centre of bacterioferritin from Escherichia coli was recently demonstrated, suggesting that the two cofactors may be functionally connected. Here we report absorbance and (magnetic) circular dichroism spectroscopies, together with in vitro assays of iron-release kinetics, which demonstrate that the ferroxidase centre plays an important role in the reductive mobilisation of the bacterioferritin mineral core, which is dependent on the heme-ferroxidase centre electron transfer pathway.

Structural Insights into the Reaction between Hydrogen Peroxide and Di-iron Complexes at the Ferroxidase Center of Ferritin.

The Fe(II) oxidation mechanism in the ferroxidase center of heavy chain ferritin has been studied extensively. However, the actual production of H2O2 was found to be substantially lower than expected at low flux of Fe(II) to ferritin subunits. Here, we demonstrated that H2O2 could interact with the di-iron nuclear center, leading to the production of hydroxyl radicals and oxygen. Two reaction intermediates were captured in the ferroxidase center by using the time-lapse crystallographic techniques in a shellfish ferritin. The crystal structures revealed the binding of H2O2 as a μ -1,2-peroxo-diferric species and the binding of O2 to the diferric structure. This investigation sheds light on the reaction between the di-iron nuclear center and H2O2 and provides insights for the exploitation of metalloenzymes.

Pseudomonas aeruginosa Dps (PA0962) Functions in H2O2 Mediated Oxidative Stress Defense and Exhibits In Vitro DNA Cleaving Activity.

We report the structural, biochemical, and functional characterization of the product of gene PA0962 from Pseudomonas aeruginosa PAO1. The protein, termed Pa Dps, adopts the Dps subunit fold and oligomerizes into a nearly spherical 12-mer quaternary structure at pH 6.0 or in the presence of divalent cations at neutral pH and above. The 12-Mer Pa Dps contains two di-iron centers at the interface of each subunit dimer, coordinated by conserved His, Glu, and Asp residues. In vitro, the di-iron centers catalyze the oxidation of Fe2+ utilizing H2O2 (not O2) as an oxidant, suggesting Pa Dps functions to aid P. aeruginosa to survive H2O2-mediated oxidative stress. In agreement, a P. aeruginosa Δdps mutant is significantly more susceptible to H2O2 than the parent strain. The Pa Dps structure harbors a novel network of Tyr residues at the interface of each subunit dimer between the two di-iron centers, which captures radicals generated during Fe2+ oxidation at the ferroxidase centers and forms di-tyrosine linkages, thus effectively trapping the radicals within the Dps shell. Surprisingly, incubating Pa Dps and DNA revealed unprecedented DNA cleaving activity that is independent of H2O2 or O2 but requires divalent cations and 12-mer Pa Dps.

Potential of ferritin 2 as an antigen for the development of a universal vaccine for avian mites, poultry red mites, tropical fowl mites, and northern fowl mites.

Poultry red mites (PRMs, Dermanyssus gallinae), blood-sucking ectoparasites, are a threat to the poultry industry because of reduced production caused by infestation. In addition, tropical fowl mites (TFMs, Ornithonyssus bursa) and northern fowl mites (NFMs, Ornithonyssus sylviarum) are hematophagous, distributed in various regions, genetically and morphologically close to PRMs, and cause similar problems to the poultry industry. Vaccine approaches have been studied for PRM control, and several molecules have been identified in PRMs as candidates for effective vaccine antigens. The development of an anti-PRM vaccine as a universal vaccine with broad efficacy against avian mites could improve the productivity of poultry farms worldwide. Molecules that are highly conserved among avian mites and have critical functions in the physiology and growth of mites could be ideal antigen candidates for the development of universal vaccines. Ferritin 2 (FER2), an iron-binding protein, is critical for the reproduction and survival of PRMs and has been reported as a useful vaccine antigen for the control of PRMs and a candidate for the universal vaccine antigen in some tick species. Herein, we identified and characterized FER2 in TFMs and NFM. Compared with the sequence of PRM, the ferroxidase centers of the heavy chain subunits were conserved in FER2 of TFMs and NFMs. Phylogenetic analysis revealed that FER2 belongs to clusters of secretory ferritins of mites and other arthropods. Recombinant FER2 (rFER2) proteins from PRMs, TFMs, and NFMs exhibited iron-binding abilities. Immunization with each rFER2 induced strong antibody responses in chickens, and each immune plasma cross-reacted with rFER2 from different mites. Moreover, mortality rates of PRMs fed with immune plasma against rFER2 from TFMs or NFMs, in addition to PRMs, were higher than those of control plasma. rFER2 from each avian mite exhibited anti-PRM effects. This data suggests that it has the potential to be used as an antigen candidate for a universal vaccine against avian mites. Further studies are needed to access the usefulness of FER2 as a universal vaccine for the control of avian mites.

Structural and Functional Insights into the Roles of Potential Metal-Binding Sites in Apostichopus japonicus Ferritin.

Ferritin is widely acknowledged as a conservative iron storage protein found in almost all living kingdoms. Apostichopus japonicus (Selenka) is among the oldest echinoderm fauna and has unique regenerative potential, but the catalytic mechanism of iron oxidation in A. japonicus ferritin (AjFER) remains elusive. We previously identified several potential metal-binding sites at the ferroxidase center, the three- and four-fold channels in AjFER. Herein, we prepared AjFER, AjFER-E25A/E60A/E105A, AjFER-D129A/E132A, and AjFER-E168A mutants, investigated their structures, and functionally characterized these ferritins with respect to Fe2+ uptake using X-ray techniques together with biochemical analytical methods. A crystallographic model of the AjFER-D129A/E132A mutant, which was solved to a resolution of 1.98 Å, suggested that the substitutions had a significant influence on the quaternary structure of the three-fold channel compared to that of AjFER. The structures of these ferritins in solution were determined based on the molecular envelopes of AjFER and its variants by small-angle X-ray scattering, and the structures were almost consistent with the characteristics of well-folded and globular-shaped proteins. Comparative biochemical analyses indicated that site-directed mutagenesis of metal-binding sites in AjFER presented relatively low rates of iron oxidation and thermostability, as well as weak iron-binding affinity, suggesting that these potential metal-binding sites play critical roles in the catalytic activity of ferritin. These findings provide profound insight into the structure-function relationships related to marine invertebrate ferritins.

Molecular insights into two ferritin subunits from red-lip mullet (Liza haematocheila): Detectable antibacterial activity with its expressional response against immune stimulants

Iron (Fe) is considered as an essential micronutrient due to its diverse functions in living systems. However, regulation of free iron levels is essential because free Fe ions, in excess, induce biological toxicity through different routes, including production of reactive oxygen species. Ferritin proteins play a vital role in controlling free Fe ion homeostasis by sequestering excess iron in the body. Ferritins comprise an H subunit with a ferroxidase center and an L subunit with a Fe nucleation site. However, lower vertebrates such as fish harbor an additional subunit termed ferritin M, which shows the characteristic features of both H and L. In this study, two ferritin subunits (H and M) with ferroxidase centers were identified and characterized from red-lip mullet (Liza haematocheila). The open reading frames of red-lip mullet ferritin H (LhFerH) and ferritin M−like (LhFerM) subunits comprise 534 and 531 bps, which encode for putative polypeptides of 177 and 176 amino acids, respectively. LhFerH and LhFerM were found to retain well-conserved residues, including seven ferroxidase di-iron centers, characteristic domains, and signatures of their known homologs. We cloned the open reading frames of the two ferritin subunits to overexpress the corresponding proteins in Escherichia coli and subsequently demonstrated their iron sequestration activity along with antibacterial activity against E. coli using respective purified recombinant proteins in vitro. A basal expression analysis of two LhFer genes in selected tissues using qPCR assays showed pronounced expression in blood cells with respect to both genes. A relatively high expression level of LhFerH was also detected in muscle tissues. The expression level of LhFer in the head kidney was significantly up-regulated following lipopolysaccharides (LPS) and Lactococcus garvieae injection. The resulting gene expression pattern upon immune stimulation suggests that ferritin may contribute to the defense against harmful pathogen infection. Collectively, our results indicate that both LhFerH and LhFerM potentially participate in the homeostasis of free Fe ions and in the host immune defense of red-lip mullet.

Distinct structural characteristics define a new subfamily of Mycoplasma ferritin

Ferritins can generally be divided into four subfamilies based on their structural characteristics, namely, the classic ferritins (Ftns), bacterioferritins (Bfrs), DNA-binding proteins from starved cells (Dps’), and encapsulated ferritins (EncFtns). However, the ferritin from Mycoplasma penetrans (Mpef) possesses a particular ferroxidase center with an extreme low activity and exhibits unusual characteristics, indicating that it could be a member of a quite different subfamily of ferritins. Hereby, the crystal structure of the ferritin from Ureaplasma urealyticum (Uurf) is presented, Mpef and Uurf have very similar properties, though they display very low sequence similarity. Thus, ferritins from Mycoplasma with these unique properties do not belong to any known subfamily, but they should rather be placed in a novel ferritin subfamily, which we term Mycoplasma Ferritin (Mfr).

Structural Insights Into the Effects of Interactions With Iron and Copper Ions on Ferritin From the Blood Clam Tegillarca granosa.

In addition to its role as an iron storage protein, ferritin can function as a major detoxification component in the innate immune defense, and Cu2+ ions can also play crucial antibacterial roles in the blood clam, Tegillarca granosa. However, the mechanism of interaction between iron and copper in recombinant Tegillarca granosa ferritin (TgFer) remains to be investigated. In this study, we investigated the crystal structure of TgFer and examined the effects of Fe2+ and Cu2+ ions on the TgFer structure and catalytic activity. The crystal structure revealed that TgFer presented a typically 4–3–2 symmetry in a cage-like, spherical shell composed of 24 identical subunits, featuring highly conserved organization in both the ferroxidase center and the 3-fold channel. Structural and biochemical analyses indicated that the 4-fold channel of TgFer could be serviced as potential binding sites of metal ions. Cu2+ ions appear to bind preferentially with the 3-fold channel as well as ferroxidase site over Fe2+ ions, possibly inhibiting the ferroxidase activity of TgFer. Our results present a structural and functional characterization of TgFer, providing mechanistic insight into the interactions between TgFer and both Fe2+ and Cu2+ ions.

Structural characterization of the Myxococcus xanthus encapsulin and ferritin-like cargo system gives insight into its iron storage mechanism

Encapsulins are bacterial organelle-like cages involved in various aspects of metabolism, especially protection from oxidative stress. They can serve as vehicles for a wide range of medical applications. Encapsulin shell proteins are structurally similar to HK97 bacteriophage capsid protein and their function depends on the encapsulated cargos. The Myxococcus xanthus encapsulin system comprises EncA and three cargos: EncB, EncC, and EncD. EncB and EncC are similar to bacterial ferritins that can oxidize Fe+2 to less toxic Fe+3. We analyzed EncA, EncB, and EncC by cryo-EM and X-ray crystallography. Cryo-EM shows that EncA cages can have T= 3 and T= 1 symmetry and that EncA T= 1 has a unique protomer arrangement. Also, we define EncB and EncC binding sites on EncA. X-ray crystallography of EncB and EncC reveals conformational changes at the ferroxidase center and additional metal binding sites, suggesting a mechanism for Fe oxidation and storage within the encapsulin shell.

Crystallographic characterization of a marine invertebrate ferritin from the sea cucumber Apostichopus japonicus.

Ferritin is considered to be an ubiquitous and conserved iron‐binding protein that plays a crucial role in iron storage, detoxification, and immune response. Although ferritin is of critical importance for almost all kingdoms of life, there is a lack of knowledge about its role in the marine invertebrate sea cucumber (Apostichopus japonicus). In this study, we characterized the first crystal structure of A. japonicus ferritin (AjFER) at 2.75 Å resolution. The structure of AjFER shows a 4‐3‐2 symmetry cage‐like hollow shell composed of 24 subunits, mostly similar to the structural characteristics of other known ferritin species, including the conserved ferroxidase center and 3‐fold channel. The 3‐fold channel consisting of three 3‐fold negative amino acid rings suggests a potential pathway in which metal ions can be first captured by Asp120 from the outside environment, attracted by His116 and Cys128 when entering the channel, and then transferred by Glu138 from the 3‐fold channel to the ferroxidase site. Overall, the presented crystal structure of AjFER may provide insights into the potential mechanism of the metal transport pathway for related marine invertebrate ferritins.

Key carboxylate residues for iron transit through the prokaryotic ferritin SynFtn.

Ferritins are proteins forming 24meric rhombic dodecahedral cages that play a key role in iron storage and detoxification in all cell types. Their function requires the transport of Fe2+ from the exterior of the protein to buried di-iron catalytic sites, known as ferroxidase centres, where Fe2+ is oxidized to form Fe3+-oxo precursors of the ferritin mineral core. The route of iron transit through animal ferritins is well understood: the Fe2+ substrate enters the protein via channels at the threefold axes and conserved carboxylates on the inner surface of the protein cage have been shown to contribute to transient binding sites that guide Fe2+ to the ferroxidase centres. The routes of iron transit through prokaryotic ferritins are less well studied but for some, at least, there is evidence that channels at the twofold axes are the major route for Fe2+ uptake. SynFtn, isolated from the cyanobacterium Synechococcus CC9311, is an atypical prokaryotic ferritin that was recently shown to take up Fe2+ via its threefold channels. However, the transfer site carboxylate residues conserved in animal ferritins are absent, meaning that the route taken from the site of iron entry into SynFtn to the catalytic centre is yet to be defined. Here, we report the use of a combination of site-directed mutagenesis, absorbance-monitored activity assays and protein crystallography to probe the effect of substitution of two residues potentially involved in this pathway. Both Glu141 and Asp65 play a role in guiding the Fe2+ substrate to the ferroxidase centre. In the absence of Asp65, routes for Fe2+ to, and Fe3+ exit from, the ferroxidase centre are affected resulting in inefficient formation of the mineral core. These observations further define the iron transit route in what may be the first characterized example of a new class of ferritins peculiar to cyanobacteria.

Conservative and Atypical Ferritins of Sponges.

Ferritins comprise a conservative family of proteins found in all species and play an essential role in resistance to redox stress, immune response, and cell differentiation. Sponges (Porifera) are the oldest Metazoa that show unique plasticity and regenerative potential. Here, we characterize the ferritins of two cold-water sponges using proteomics, spectral microscopy, and bioinformatic analysis. The recently duplicated conservative HdF1a/b and atypical HdF2 genes were found in the Halisarca dujardini genome. Multiple related transcripts of HpF1 were identified in the Halichondria panicea transcriptome. Expression of HdF1a/b was much higher than that of HdF2 in all annual seasons and regulated differently during the sponge dissociation/reaggregation. The presence of the MRE and HRE motifs in the HdF1 and HdF2 promotor regions and the IRE motif in mRNAs of HdF1 and HpF indicates that sponge ferritins expression depends on the cellular iron and oxygen levels. The gel electrophoresis combined with specific staining and mass spectrometry confirmed the presence of ferric ions and ferritins in multi-subunit complexes. The 3D modeling predicts the iron-binding capacity of HdF1 and HpF1 at the ferroxidase center and the absence of iron-binding in atypical HdF2. Interestingly, atypical ferritins lacking iron-binding capacity were found in genomes of many invertebrate species. Their function deserves further research.

Ferritin with Atypical Ferroxidase Centers Takes B-Channels as the Pathway for Fe2+ Uptake from Mycoplasma

Here, we present a 1.9 Å resolution crystal structure of Mycoplasma Penetrans ferritin, which reveals that its ferroxidase center is located on the inner surface of ferritin but not buried within the four-helix of each subunit. Such a ferroxidase center exhibits a lower iron oxidation activity as compared to the reported ferritin. More importantly, we found that Fe2+ enters into the center via the rarely reported B-channels rather than the normal 3- or 4-fold channels. All these findings may provide the structural bases to explore the new iron oxidation mechanism adopted by this special ferritin, which is beneficial for understanding the relationship between the structure and function of ferritin.

Iron Oxidation in Escherichia coli Bacterioferritin Ferroxidase Centre, a Site Designed to React Rapidly with H2 O2 but Slowly with O2.

Both O2 and H2O2 can oxidize iron at the ferroxidase center (FC) of Escherichia coli bacterioferritin (EcBfr) but mechanistic details of the two reactions need clarification. UV/Vis, EPR, and Mössbauer spectroscopies have been used to follow the reactions when apo‐EcBfr, pre‐loaded anaerobically with Fe2+, was exposed to O2 or H2O2. We show that O2 binds di‐Fe2+ FC reversibly, two Fe2+ ions are oxidized in concert and a H2O2 molecule is formed and released to the solution. This peroxide molecule further oxidizes another di‐Fe2+ FC, at a rate circa 1000 faster than O2, ensuring an overall 1:4 stoichiometry of iron oxidation by O2. Initially formed Fe3+ can further react with H2O2 (producing protein bound radicals) but relaxes within seconds to an H2O2‐unreactive di‐Fe3+ form. The data obtained suggest that the primary role of EcBfr in vivo may be to detoxify H2O2 rather than sequester iron.

Iron Oxidation in Escherichia coli Bacterioferritin Ferroxidase Centre, a Site Designed to React Rapidly with H2O2 but Slowly with O2.

Both O2 and H2O2 can oxidize iron at the ferroxidase center (FC) of Escherichia coli bacterioferritin (EcBfr) but mechanistic details of the two reactions need clarification. UV/Vis, EPR, and Mössbauer spectroscopies have been used to follow the reactions when apo-EcBfr, pre-loaded anaerobically with Fe2+, was exposed to O2 or H2O2. We show that O2 binds di-Fe2+ FC reversibly, two Fe2+ ions are oxidized in concert and a H2O2 molecule is formed and released to the solution. This peroxide molecule further oxidizes another di-Fe2+ FC, at a rate circa 1000 faster than O2, ensuring an overall 1:4 stoichiometry of iron oxidation by O2. Initially formed Fe3+ can further react with H2O2 (producing protein bound radicals) but relaxes within seconds to an H2O2-unreactive di-Fe3+ form. The data obtained suggest that the primary role of EcBfr in vivo may be to detoxify H2O2 rather than sequester iron.

Electron Transfer from Haem to the Di-Iron Ferroxidase Centre in Bacterioferritin.

The iron redox cycle in ferritins is not completely understood. Bacterioferritins are distinct from other ferritins in that they contain haem groups. It is acknowledged that the two iron motifs in bacterioferritins, the di‐nuclear ferroxidase centre and the haem B group, play key roles in two opposing processes, iron sequestration and iron mobilisation, respectively, and the two redox processes are independent. Herein, we show that in Escherichia coli bacterioferritin, there is an electron transfer pathway from the haem to the ferroxidase centre suggesting a new role(s) haem might play in bacterioferritins.