Reactivity of ferritin and the structure of ferritin-derived ferrihydrite

Background

In nature or in the laboratory, the roughly spherical interior of the ferritin protein is well suited for the formation and storage of a variety of nanosized metal oxy-hydroxide compounds which hold promise for a range of applications. However, the linkages between ferritin reactivity and the structure and physicochemical properties of the nanoparticle core, either native or reconstituted, remain only partly understood.

Scope of review

Here we review studies, including those from our laboratory, which have investigated the structure of ferritin-derived ferrihydrite and reactivity of ferritin, both native and reconstituted. Selected proposed structure models for ferrihydrite are discussed along with the structural and genetic relationships that exist among several different forms of ferrihydrite. With regard to reactivity, the review will emphasize studies that have investigated the (photo)reactivity of ferritin and ferritin-derived materials with environmentally relevant gaseous and aqueous species.

Major conclusions

The inorganic core formed from apoferritin reconstituted with varied amounts of Fe has the same structural topology as the inorganically derived ferrihydrite that is an important component of many environmental and soil systems. Reactivity of ferritin toward aqueous species resulting from the photoexcitation of the inorganic core of the protein shows promise for driving redox reactions relevant to environmental chemistry.

General significance

Ferritin-derived ferrihydrite is effectively maintained in a relatively unaggregated state, which improves reactivity and opens the possibility of future applications in environmental remediation. Advances in our understanding of the structure, composition, and disorder in synthetic, inorganically derived ferrihydrite are shedding new light on the reactivity and stability of ferrihydrite derived artificially from ferritin.     

Hydrogen ion interactions of horse spleen ferritin and apoferritin.

The interactions of horse spleen ferritin and its derivative apoferritin with H+ ions were studied by potentiometric and spectrophotometric titration; to aid in data analysis, heats of ionization over a limited pH range and amide content were also determined. Per apoferritin subunit, all tyrosine and cysteine side chains, two of the nine lysine side chains and at least three of the six histidine side chains were found not to titrate; a preliminary but self-consistent analysis of the titration data is proposed. The titration curve of ferritin was identical with that of apoferritin in the pH range 5.5 to 3. In addition, under the conditions used, the reactivities of ferritin histidines to bromoacetate and of ferritin lysines to formaldehyde were identical with those in apoferritin. Above pH 8, a time-dependent titration of the ferritin core occurs which prevents comparison of the titration curves of the two proteins in this region. However, in the pH regions 5.5 to 7.5, two extra groups per subunit titrate reversibly in ferritin relative to apoferritin. Moreover, although the isoionic points of ferritin and apoferritin are identical in water, the isoionic point of ferritin is 0.5 pH unit lower than that of apoferritin in 0.16 to 1 M KCl. The different effects of KCl and NaCl on the two proteins indicate the presence of cation binding sites in ferritin that are absent in apoferritin and possibly also the presence of anion binding sites in apoferritin that are occupied in ferritin by anions of the core. The difference between the isoionic points of the two proteins in KCl has been interpreted to indicate the presence of approximately 2 phosphate residues per ferritin subunit which serve as cation binding sites and which are negatively charged at the isoionic point in KCl. These phosphates may also represent the additional residues that titrate in ferritin between pH 5.5 and 7.5, or may interact with positively charged residues on the inner surface of the ferritin shell, or both.     

Ferritin: design and formation of an iron-storage molecule

Although essential for most forms of life, too much iron is harmful. To cope with these antagonistic phenomena an iron-storage molecule, ferritin, has evolved. The structure of horse spleen apoferritin, which has recently been refined, consists of 24 symmetrically related subunits forming a near-spherical hollow shell. In ferritin the central cavity is occupied by an iron core of 'ferrihydrite', a geologically ephemeral mineral found in hot or cold springs and in mine workings, or produced in the laboratory by heating solutions of ferric salts. Ferritin itself forms most readily from apoferritin, in the presence of dioxygen, from FeII, not FeIII. Access to its interior is through small intersubunit channels, and the protein influences both the rate of FeII-oxidation and the form of oxide produced.     

Neutron spin echo studies on ferritin: free-particle diffusion and interacting solutions

The dynamics of proteins are often studied by means of quasielastic neutron scattering (QENS), for example by time-of-flight methods. The spatial dimensions (10-20 nm) present in protein solutions are accessible by neutron scattering. In this article, a systematic study of diffusive dynamics of ferritin and apoferritin (=ferritin without iron core) is presented. Apoferritin consists of a spherical shell built of 24 protein units and carries net negative charge at pH 5. We have studied diffusive dynamics of ferritin solutions by neutron spin echo (NSE). We pay attention to an important feature of this technique compared to other QENS methods, which being the usage of a broad wavelength band. Using a more sophisticated fit function than usually used in NSE, we find as expected in low concentrated systems that the diffusion coefficient approaches the free-particle value of apoferritin and coincides with the diameter of the apoferritin shell (12.2 nm). In interacting solutions, the NSE results reveal that the dynamic picture of this complex liquid is dominated by slowing down of the dynamics. In low-salt solutions, a structure factor peak appears due to ordering of the ferritin molecules on the length scale of several intermolecular distances. We discuss the usage of different NSE fit functions for interacting solutions near the structure factor peak. Comparison of the dependence of elastic and dynamic data on the scattering vector value shows the influence of indirect interactions on the dynamic picture, irrespective of the way of data analysis, which being necessary due to the broad wavelength spectrum.     

The monomers and oligomers of ferritin and apoferritin: association and dissociation.

We have reinvestigated the association and dissociation of ferritin and apoferritin in phosphate buffer (pH 7.2, I = 0.05). When oligomer-enriched solutions of horse spleen ferritin were mixed with more concentrated, but unenriched solutions of horse spleen apoferritin, there was dissociation of the ferritin oligomers, as determined by polyacrylamide gel electrophoresis and from iron/protein ratios. Some evidence was also obtained for association of monomers in the mixture of ferritin and apoferritin after pelleting and redissolution of pellets in minimal volumes of the phosphate buffer. Monomer-enriched, biosynthetically labeled rat liver ferritin was pelleted, redissolved in minimal volumes of phosphate buffer, and separated by polyacrylamide gel electrophoresis; the fractions were isolated and counted. The results revealed that an association of monomers of the rat liver ferritin had taken place which doubled the concentration of dimers. However, our results also indicate that association by concentration was limited to a fraction of monomers.     

Fe2+ binding to apo and holo mammalian ferritin

The binding of Fe2+ to both apo and holo mammalian ferritin has been investigated under anaerobic conditions as a function of pH. In the pH range 6.0-7.5, 8.0 +/- 0.5 Fe2+ ions bind to each apoferritin molecule, but above pH 7.5, a pH-dependent Fe2+ binding profile is observed with up to 80 Fe2+ ions binding at pH 10.0. This Fe2+ binding is reversible and is accompanied by up to two H+ being released per Fe2+ bound at pH 10.0. The Fe2+ binding to apoferritin probably occurs in the 3-fold channels. A much larger and more complex pH-dependent Fe2+ binding stoichiometry was observed for holoferritin with up to 300 Fe2+ ions binding at pH 10.0. This pH-dependent Fe2+ binding was interpreted as Fe2+ interaction at the FeOOH mineral surface with displacement of H+ from -OH or phosphate surface groups by the incoming Fe2+ ions. Mossbauer spectroscopic measurements using 57Fe-labeled Fe2+ under anaerobic conditions showed that 57Fe2+ binding to holoferritin was accompanied by electron transfer to the core, yielding 57Fe3+, presumably bound to the mineral surface. Removal of added iron by Fe2+-specific chelating agents yielded 57Fe2+, demonstrating the reversibility of this electron-transfer process. The Fe2+ bound to apo- and holoferritin is readily converted to Fe3+ by exposure to O2 and strongly retained by the respective ferritin species.     

Uptake of iron by apoferritin from a ferric dihydrolipoate complex

A study was made on the uptake of iron by horse spleen apoferritin, by using as an iron source the same ferric dihydrolipoate complex which represents the major product in the anaerobic removal of ferritin-bound iron by dihydrolipoate at neutral pH. The ferric dihydrolipoate complex was chemically synthesized and used as an iron donor to apoferritin. Iron uptake was studied, at slightly alkaline pH and in anaerobic conditions, as a function of the concentration of both the iron donor and apoferritin. Isolation of ferritin from mixtures of ferric dihydrolipoate and apoferritin, and subsequent identification of the oxidation state of ferritin-bound iron, showed that the first metal atoms were taken up in the ferrous form and that this early step was accompanied by accumulation of ferric iron. Total iron uptake increased with the molar ratio of complex to apoprotein and ranged over 25-40% of the iron being supplied. The amount of ferrous iron found inside the protein did not exceed 50-60 mol iron/mol ferritin after a 48-h incubation. At this time, ferric iron represented a significant fraction of the iron found in the isolated ferritin. Analytical and spectroscopic data indicated that fractional rates and equilibria for disassembly of the ferric complex in the presence of apoferritin were independent of the concentration of the protein and of the complex itself.     

Lability of iron at the dinuclear centres of ferritin studied by competition with four chelators

Ferritin molecules contain 24 polypeptide chains folded as four-helix bundles and arranged as a hollow shell capable of storing up to 4500 Fe(III) atoms. H chains contain ferroxidase centres which lie within the bundle, about 12?Å (1.2?nm) from the outside surface and 8?Å from the inner surface of the protein shell. Catalysis of Fe(II) oxidation precedes storage of Fe(III) as ferrihydrite, with the formation of μ-oxo-bridged Fe(III) dimers as intermediates. Factors influencing the movement of μ-oxo-bridged Fe(III) from the ferroxidase centre to the ferritin cavity are uncertain. Assistance by small chelators is one possibility. The aim of this investigation was to determine whether iron at the dinuclear centres of three ferritins (human H chain homopolymer, HuHF, the non-haem ferritin of Escherichia coli, EcFTN, and horse spleen ferritin, HoSF) is accessible to chelators. Forty-eight Fe(II) atoms/molecule were added to the apoferritins followed, 2?min later, by the addition of chelator (1,10-phenanthroline, 2,2-bipyridine, desferrioxamine or 3,4-dihydroxybenzaldehyde). Iron species were analysed by Mössbauer spectroscopy or visible absorbance. Competition between chelators and apoferritin for Fe(II) was also investigated. The main conclusions of the study are that: (1) dinuclear iron and iron in small iron-cores in HuHF and EcFTN is mobilisable by all four chelators; (2) the chelators penetrate the shell; (3) 3,4-dihydroxybenzaldehyde is the most efficient in mobilising Fe(III) but the least successful in competing for Fe(II); (4) Fe(III) is more readily released from EcFTN than from HuHF; (5) 2,2′-bipyridine aids the movement of Fe(III) from ferroxidase centre to core.     

Subunit dimers in sheep spleen apoferritin. The effect on iron storage

Ferritin with high and low iron content, 2000 and 790 iron atoms/molecule, was isolated from the spleens of copper-poisoned and control lambs, respectively. Differences in the iron content in vivo were reflected in the properties of the apoferritin protein shells, since the apoprotein from the low iron ferritin took up iron relatively more slowly (0.52 +/- 0.09) and released it more rapidly (1.68 +/- 0.06) in vitro. Although the two types of apoferritin were indistinguishable in terms of surface charge (pI range 4.98-5.43) and in consisting of both heavy and light subunits, the subunit interactions differed markedly; 40-50% of the subunits of low iron ferritin were in dimers stable to reduction and carboxylmethylation, 4% mercaptoethanol, 8% sodium dodecyl sulfate, and 100 degrees C for 30 min, 70% formic acid, and 30% methanol. Subunit dimers were also observed in liver ferritin from mouse and neonatal pig and were enriched in a low iron fraction of horse spleen ferritin. Based on cyanogen bromide fragmentation and NH2-terminal analysis, the natural and chemically cross-linked subunit dimers had two peptides in common; natural subunit dimers also appeared to have a second region cross-linked, suggesting the possibility of both intra- and intersubunit links in the natural dimers. In sheep spleen ferritin, both heavy and light subunits appeared to participate in subunit dimerization. Natural subunit dimers were enriched in low iron ferritin fractions of all ferritin preparations tested (linear correlation = 0.94) and can explain, at least in part, the previously observed effects of iron core size on the apoferritin shell. Whether the subunit cross-links represent part of the subunit assembly process subsequently cleaved by iron (or copper) or whether the cross-links form after iron core formation in vivo has yet to determined. In either case, it is clear that such post-translational variations can affect iron uptake and release and emphasize the importance of the protein shell in determining the iron storage properties of ferritin.     

M?ssbauer spectroscopic investigation of structure-function relations in ferritins.

Ferritin plays an important role in iron metabolism and our aim is to understand the mechanisms by which iron is sequestered within its protein shell as the mineral ferrihydrite. We present M?ssbauer spectroscopic data on recombinant human and horse spleen ferritin from which we draw the following conclusions: (1) that apoferritin catalyses Fe(II) oxidation as a first step in ferrihydrite deposition, (2) that the catalysis of Fe(II) oxidation is associated with residues situated within H chains, at the postulated 'ferroxidase centre' and not in the 3-fold inter-subunit channels previously suggested as the initial Fe(II) binding and oxidation site; (3) that both isolated Fe(III) and Fe(III) mu-oxo-bridged dimers found previously by M?ssbauer spectroscopy to be intermediates in iron-core formation in horse spleen ferritin, are located on H chains; and (4) that these dimers form at ferroxidase centres. The importance of the ferroxidase centre is suggested by the conservation of its ligands in many ferritins from vertebrates, invertebrates and plants. Nevertheless iron-core formation does occur in those ferritins that lack ferroxidase centres even though the initial Fe(II) oxidation is relatively slow. We compare the early stages of core formation in such variants and in horse spleen ferritin in which only 10-15% of its chains are of the H type. We discuss our findings in relation to the physiological role of isoferritins in iron storage processes.     

HIV-1 capsid protein forms spherical (immature-like) and tubular (mature-like) particles in vitro: structure switching by pH-induced conformational changes.

The viral genome and replicative enzymes of the human immunodeficiency virus are encased in a shell consisting of assembled mature capsid protein (CA). The core shell is a stable, effective protective barrier, but is also poised for dissolution on cue to allow transmission of the viral genome into its new host. In this study, static light scattering (SLS) and dynamic light scattering (DLS) were used to examine the entire range of the CA protein response to an environmental cue (pH). The CA protein assembled tubular structures as previously reported but also was capable of assembling spheres, depending on the pH of the protein solution. The switch from formation of one to the other occurred within a very narrow physiological pH range (i.e., pH 7.0 to pH 6.8). Below this range, only dimers were detected. Above this range, the previously described tubular structures were detected. The ability of the CA protein to form a spherical structure that is detectable by DLS but not by electron microscopy indicates that some assemblages are inherently sensitive to perturbation. The dimers in equilibrium with these assemblages exhibited distinct conformations: Dimers in equilibrium with the spherical form exhibited a compact conformation. Dimers in equilibrium with the rod-like form had an extended conformation. Thus, the CA protein possesses the inherent ability to form metastable structures, the morphology of which is regulated by an environmentally-sensitive molecular switch. Such metastable structures may exist as transient intermediates during the assembly and/or disassembly of the virus core.     

Iron (III) can be transferred between ferritin molecules.

The iron-storage molecule ferritin can sequester up to 4500 Fe atoms as the mineral ferrihydrite. The iron-core is gradually built up when FeII is added to apoferritin and allowed to oxidize. Here we present evidence, from M?ssbauer spectroscopic measurements, for the surprising result that iron atoms that are not incorporated into mature ferrihydrite particles, can be transferred between molecules. Experiments were done with both horse spleen ferritin and recombinant human ferritin. M?ssbauer spectroscopy responds only to 57Fe and not to 56Fe and can distinguish chemically different species of iron. In our experiments a small number of 57FeII atoms were added to two equivalent apoferritin solutions and allowed to oxidize (1-5 min or 6 h). Either ferritin containing a small iron-core composed of 56Fe, or an equal volume of NaCl solution, was added and the mixture frozen in liquid nitrogen to stop the reaction at a chosen time. Spectra of the ferritin solution to which only NaCl was added showed a mixture of species including 57FeIII in solitary and dinuclear sites. In the samples to which 150 56FeIII-ferritin had been added the spectra showed that all, or almost all, of the 57FeIII was in large clusters. In these solutions 57FeIII initially present as intermediate species must have migrated to molecules containing large clusters. Such migration must now be taken into account in any model of ferritin iron-core formation.     

M?ssbauer spectroscopic study of the initial stages of iron-core formation in horse spleen apoferritin: evidence for both isolated Fe(III) atoms and oxo-bridged Fe(III) dimers as early intermediates

Ferritin stores iron within a hollow protein shell as a polynuclear Fe(III) hydrous oxide core. Although iron uptake into ferritin has been studied previously, the early stages in the creation of the core need to be clarified. These are dealt with in this paper by using M?ssbauer spectroscopy, a technique that enables several types of Fe(II) and Fe(III) to be distinguished. Systematic M?ssbauer studies were performed on samples prepared by adding 57Fe(II) atoms to apoferritin as a function of pH (5.6-7.0), n [the number of Fe/molecule (4-480)], and tf (the time the samples were held at room temperature before freezing). The measurements made at 4.1 and 90 K showed that for samples with n less than or equal to 40 at pH greater than or equal to 6.25 all iron was trivalent at tf = 3 min. Four different Fe(III) species were identified: solitary Fe(III) atoms giving relaxation spectra, which can be identified with the species observed before by EPR and UV difference spectroscopy; oxo-bridged dimers giving doublet spectra with large splitting, observed for the first time in ferritin; small Fe(III) clusters giving doublets of smaller splitting and larger antiferromagnetically coupled Fe(III) clusters, similar to those found previously in larger ferritin iron cores, which, for samples with n greater than or equal to 40, gave magnetically split spectra at 4.1 K. Both solitary Fe(III) and dimers diminished with time, suggesting that they are intermediates in the formation of the iron core. Two kinds of divalent iron were distinguished for n = 480, which may correspond to bound and free Fe(II).     

On the mechanism of horse spleen apoferritin assembly: a sedimentation velocity and circular dichroism study

The apoferritin shell is known to assemble spontaneously from its subunits obtained at acid pH upon neutralization. The reassembly of apoferritin from horse spleen has been followed by means of sedimentation velocity and circular dichroism experiments as a function of the pH and the nature of the assembly buffer in order to obtain information on the assembly pathway. In all the buffer systems tested the subunits sediment as a single peak of varying sedimentation and diffusion coefficients, and shell assembly starts at pH values around 3.5. In dilute glycine-acetate buffers the subunits are essentially dimeric up to this pH value. Therefore, the dimeric building blocks of the apoferritin shell that are apparent in the X-ray structure represent the first assembly intermediates. When the pH is increased to 4.0-4.3, the weight-average sedimentation velocity of the subunits increases to 3.6-4.7 S, respectively, and the subunit population becomes heterogeneous. Concomitantly, significant changes in the circular dichroism properties of the aromatic residues take place. On the basis of the X-ray structure, where aromatic residues appear to be located at or near the fourfold symmetry axes, these data suggest that assembly proceeds from dimers through tetramers and octamers. In the pH range 4.5-6.5 the reassembly process cannot be followed due to reversible precipitation of the subunits near their isoelectric point; at neutral pH values essentially quantitative reassembly is obtained.(ABSTRACT TRUNCATED AT 250 WORDS)     

Crystal structure of a secreted insect ferritin reveals a symmetrical arrangement of heavy and light chains

Ferritins are iron storage proteins made of 24 subunits forming a hollow spherical shell. Vertebrate ferritins contain varying ratios of heavy (H) and light (L) chains; however, known ferritin structures include only one type of chain and have octahedral symmetry. Here, we report the 1.9A structure of a secreted insect ferritin from Trichoplusia ni, which reveals equal numbers of H and L chains arranged with tetrahedral symmetry. The H/L-chain interface includes complementary features responsible for ordered assembly of the subunits. The H chain contains a ferroxidase active site resembling that of vertebrate H chains with an endogenous, bound iron atom. The L chain lacks the residues that form a putative iron core nucleation site in vertebrate L chains. Instead, a possible nucleation site is observed at the L chain 3-fold pore. The structure also reveals inter- and intrasubunit disulfide bonds, mostly in the extended N-terminal regions unique to insect ferritins. The symmetrical arrangement of H and L chains and the disulfide crosslinks reflect adaptations of insect ferritin to its role as a secreted protein.     

Hydrogen-tritium exchange by apoferritin and ferritin

The out-exchange kinetics of tritium from apoferritin, ferritin of various iron contents, and apoferritin subunits were examined. The exchange kinetics indicated no detectable conformational differences in the tetracosamer with and without hydrous ferric oxide in the internal cavity of the molecule. The data for apoferritin subunits were markedly different from those for the tetracosameric state. The exchange kinetics for apoferritin were consistent with a rapid exchange of water between the internal cavity of the protein and the bulk solvent outside the protein shell.     

Reconstituted and native iron-cores of bacterioferritin and ferritin

The structural and magnetic properties of the iron-cores of reconstituted horse spleen ferritin and Azotobacter vinelandii bacterioferritin have been investigated by high-resolution transmission electron microscopy, electron diffraction and Mossbauer spectroscopy. The structural properties of native horse spleen ferritin, native Az. vinelandii, and native and reconstituted Pseudomonas aeruginosa bacterioferritins have also been determined. Reconstitution in the absence of inorganic phosphate at pH 7.0 showed sigmoidal behaviour in each protein but was approximately 30% faster in initial rate for the Az. vinelandii protein when compared with horse spleen apoferritin. The presence of Zn2+ reduced the initial rate of Fe(II) oxidation in Az. vinelandii to 22% of the control rate. The iron-cores of the reconstituted bacterioferritins adopt defect ferrihydrite structures and are more highly ordered than their native counterparts, which are both amorphous. However, the blocking temperature for reconstituted Az. vinelandii (22.2 K) is almost identical to that for the native protein (20 K). Particle size measurements indicate that the reconstituted Az. vinelandii cores are smaller in median diameter than the native cores and this reduction in particle volume (V) offsets the increased magnetocrystalline contribution to the magnetic anisotropy constant (K) in such a way that the magnetic anisotropy barrier (KV), and hence the blocking temperature, is similar for both proteins. Reconstituted horse spleen ferritin exhibits a similar blocking temperature (38 K) to that determined for the native protein, although it is structurally more disordered. The possibility of introducing structural and compositional modifications in both horse ferritin and bacterioferritins by in-vitro reconstitution suggests that these proteins do not function primarily as a crystallochemical-specific interface for core development in vivo.     

Monomer and oligomer of type I collagen: molecular properties and fibril assembly

Type I collagen purified from calf skin was further separated into monomeric and oligomeric fractions and characterized with gel electrophoresis and measurement of solution viscosity. The thermal stabilities of the triple-helical structure of the collagen molecules of these preparations and the fibrils assembled therefrom were determined with differential UV spectroscopy and scanning microcalorimetry. The monomeric collagen was reduced with NaBH4-, and the kinetics and equilibrium of the reversible fibril assembly-disassembly were examined in detail. Fibril assembly and disassembly of the collagen induced by slow scans of temperature showed hysteresis. The assembly curve was very sharp whereas the disassembly curve was gradual. Equilibrium centrifugation showed the collagen disassembled from the fibrils to be predominantly monomers. However, unlike the unassembled collagen, the collagen disassembled from fibrils by cooling showed no lag phase in subsequent cycles of fibril assembly. The thermodynamic parameters of fibril growth were derived from a fibril disassembly curve. Fibril growth was weaker for the NaBH4-reduced monomeric collagen than the native crude collagen, perhaps due to the removal of oligomers and the changes in the molecular structure brought by the reduction. The results corroborated the strongly cooperative mechanism for the fibril assembly proposed in the preceding paper.     

X-ray structure of recombinant horse L-chain apoferritin at 2.0 Å resolution: implications for stability and function

The X-ray structure of recombinant horse L-chain (rL) apoferritin, solved at 2.0?Å resolution with a final R factor of 17.9%, gives evidence that the residue at position 93 in the sequence is a proline and not a leucine, as found in earlier sequencing studies. The structure is isomorphous with other apoferritin structures, and we thus draw particular attention to those structural features which can be related to the stability and function of the protein. Analysis of hydrogen bonding and salt bridge interactions shows that dimers and tetramers are the most stable molecular entities within the protein shell: a result confirming earlier biophysical experiments. The stability of horse rL apoferritin to both dissociation into subunits at acidic pH values and to complete unfolding in guanidine chloride solutions is compared with that of other apoferritins. This emphasizes the role played by the salt bridge in the stability of this protein family. The horse rL apoferritin is significantly more resistant to denaturation than horse spleen ferritin, which in turn is more resistant than any human rH apoferritins, even those for which a salt bridge is restored. Finally, this structure determination not only establishes that a preformed pocket exists in L-chain apoferritin, at a site known to be able to bind porphyrin, but also underlines the particular function of a cluster of glutamic acids (E53, E56, E57 and E60) located at the entrance of this porphyrin-binding pocket.