Ferritin ferroxidase

The detection of a peroxodiferric intermediate in the ferritin ferroxidase reaction establishes the ferritin ferroxidase site as being very similar to the sites in the 02-activating (/x-carboxylato)diiron enzymes. However, in ferritins, the peroxodiferric intermediate forms diferric oxo or hydroxo precursors, which are transferred to biomineralization sites with release of hydrogen peroxide. 

Oxygen consumption measurements [56-58] suggest distinct oxidation reactions for ferritin ferroxidase and mineralization processes. For the recombinant human and frog H ferritins, ferroxidase activity dominates with low iron loading (less than 50 Fe/ferritin 24-mer) and a Fe/Oa ratio of 2 is observed with H2O2 being the principle reduction product. The catalytic reaction was suggested to follow Eq. (16.3). 

The ferroxidase center, important for rapid oxidation of Fe to Fe, was discovered relatively recently in the history of research into the metal sites in ferritins. Ferroxidase activity within H subunits appears to occur at a dinuclear site situated within a four-helix bundle and resembling the dinuclear centers found in ribonucleotide reductase, methane monooxygenase, fatty acid desaturases, and ruberythrin (Chapter 8.11). In bacterioferritins, for which protein crystal structures have been reported for ferritin from Escherichia col and Rhodobacter capsulatus the overall motif of a shell of 24 subunits with relative masses of about 18,500 Da is preserved but there are also 12 protoporphyrin IX heme groups present with unknown function which might have a role in connecting the dimer units and are buried within the shell between identical subunits related by twofold symmetry. In these bacterioferritins the subunits are all identical and contain both ftrroxidase and nucleation sites. 

Figure 3 The suggested coordination environments of the iron centres in ferritin ferroxidase sites for (a) bacterioferritin reduced form, 2 x Fe(II) (b) bacterioferritin oxidized form, 2 x Fe(III) (c) human ferritin (Fl-chain) both oxidized and reduced forms (d) E. coli ferritin (ecFTN) oxidized and reduced forms and...

Broxmeyer, H., Cooper, S. Arosio, P, Mutated recombinant human heavy-chain ferrittins and myelosuppression intdviinovivo A link between ferritin ferroxidase activity and biological function. PNAS, 1991.88 p. 770-774. 

Ferritin, bacterioferritin Vertebrates, bacteria Ferroxidase ( ) EX34EX2HX4lEX36E ... 

It has been proposed that Glu-61 could alternately act as a ligand to the ferroxidase site and to the nucleation site, and hence serve as a go-between to move iron (eventually in both directions) from one site to another (Lawson et al, 1991). What is clear is that modification of both the ferroxidase centre and the nucleation centre leads to ferritins which do not oxidize or incorporate iron (Wade et al, 1991 Sun et al, 1993). 

We can briefly conclude that the mineralization process of iron in ferritin cores is a difficult process to follow experimentally. While we believe that iron is delivered for storage within the protein cavity as Fe(II), and that an oxidation step occurs in the formation of the ferritin iron core, it is not clear what percentage of iron oxidation occurs on the growing surface of the mineral and what at the catalytic ferroxidase... 

Tatur, J. and Hagen, W.R. 2005. The dinuclear iron-oxo ferroxidase center of Pyrococcus furiosus ferritin is a stable prosthetic group with unexpectedly high reduction potentials. FEBS Letters 579 4729 1732. 

Theil, E.C. and Huynh, B.H. (1998) Direct spectroscopic and kinetic evidence for the involvement of a peroxodiferric intermediate during the ferroxidase reaction in fast ferritin mineralization. Biochemistry, 37, 9871-9876. 

Iron is stored in these proteins in the ferric form, but is taken up as Fe2+, which is oxidized by ferroxidase sites (a more detailed account of iron incorporation into ferritins is given later in this chapter). As we point out in Chapter 13, ferritins are members of the much larger diiron protein family. After oxidation, the Fe3+ migrates to the interior cavity of the protein to form an amorphous ferric phosphate core. Whereas the ferritins in bacteria appear to fulfil the classical role of iron-storage proteins, the physiological role of bacterioferritins is less clear. In E. coli it seems unlikely that bacterioferritin plays a major role in iron storage. 

Plants contain phytoferritins, which accumulate in non-green plastids1 in conditions of iron loading. They are targeted to the plastids by a putative-transit peptide at their N-terminal extremity, and possess the specific residues for ferroxidase activity and iron nucleation, found in mammalian H-type or L-type ferritin subunits. 

About a quarter of the total body iron is stored in macrophages and hepatocytes as a reserve, which can be readily mobilized for red blood cell formation (erythropoiesis). This storage iron is mostly in the form of ferritin, like bacterioferritin a 24-subunit protein in the form of a spherical protein shell enclosing a cavity within which up to 4500 atoms of iron can be stored, essentially as the mineral ferrihydrite. Despite the water insolubility of ferrihydrite, it is kept in a solution within the protein shell, such that one can easily prepare mammalian ferritin solutions that contain 1 M ferric iron (i.e. 56 mg/ml). Mammalian ferritins, unlike most bacterial and plant ferritins, have the particularity that they are heteropolymers, made up of two subunit types, H and L. Whereas H-subunits have a ferroxidase activity, catalysing the oxidation of two Fe2+ atoms to Fe3+, L-subunits appear to be involved in the nucleation of the mineral iron core once this has formed an initial critical mass, further iron oxidation and deposition in the biomineral takes place on the surface of the ferrihydrite crystallite itself (see a further discussion in Chapter 19). 

Iron incorporation into mammalian ferritins is thought to involve the following steps (Crichton, 2001) (1) Uptake of Fe2+ into the protein shell, most probably through the hydrophilic three fold channels. (2) Oxidation of ferrous iron by the dinuclear ferroxidase... 

As we pointed out earlier, the H subunit catalyses the ferroxidase reaction, which occurs at all levels of iron loading, but decreases with increasing amounts of iron added (48-800 Fe/ protein). Reaction (19.8) catalysed by both FI- and L-chain ferritins, occurs largely at intermediate iron loadings of 100-500 Fe/protein. Once nucleation has taken place, the role of the protein is to maintain the growing ferrihydrite core within the confines of the protein shell, thus maintaining the insoluble ferric oxyhydroxide in a water-soluble form. 

Fe(II) penetrates inside the spherical shell by the hydrophilic channels. After an oxidation on ferroxidase sites, located on H subunits, Fe(III) iron ions migrate to a nucleation site, situated on L subunits, where a crystal of hydrated iron oxide grows. Up to 4500 Fe(III) can be stored inside this mineral phase (31). The number of iron atoms contained in the ferritin molecule is called the loading factor (LF). 

Figure 16-4 The dinuclear iron center or ferroxidase center of human ferritin based on the structure of a terbium(III) derivative.73 Courtesy of Pauline Harrison.

Once absorbed, iron becomes part of the cellular iron pool, either stored as ferritin or transported across the basolateral membrane of the enterocyte into the circulation by an iron transporter called ferroportin 1. Hephaestin, a basolateral membrane ferroxidase, oxidizes the ferrous iron back to its ferric form, thus completing the absorption process (Harrison and Bacon, 2003). 

Moenne-Loccoz, P., Krebs, C., Herlihy, K., Edmondson, D. E., Theil, E. C., Huynh, B. H., and Loehr, T., 1999, The ferroxidase reaction of ferritin reveals a diferric p-1,2 bridging peroxide intermediate in common with other 02-activating non-heme diiron proteins,... 

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