Ferroxidase site

The initial stages of iron incorporation requires the ferroxidase sites of the protein. Thereafter the inner surface of the protein shell provides a surface which supplies ligands that can partially coordinate iron but which leave some coordination spheres available for mineral phase anions, thereby enabling the biomineralization process to proceed, with formation of one or more polynuclear ferrihydrite crystallites. Iron is transferred from the ferroxidase sites to the core nucleation sites by the net reaction (Yang et ah, 1998) ... 

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). 

It is unfortunately the case that when we incubate apoferritin with a certain number of iron atoms (for example as ferrous ammonium sulfate), the product, after elimination of non-protein-bound iron, does not have a homogeneous distribution of iron molecules which were able to (i) take up iron rapidly through the three fold channels, (ii) quickly transfer it and form a diiron centre on a ferroxidase site, and (iii) to transfer the iron inward to a nucleation site, where (iv) it will begin to catalyse iron oxidation on the surface of the growing crystallite, will accumulate iron much more rapidly, and in much greater amounts than molecules in which steps (i), (ii) and (iii) are slower, for whatever reasons (perhaps most importantly subunit composition, and the disposition of subunits of the two types H and L, one with regard to the other). This polydispersity makes the analysis of the process of iron uptake extremely difficult. 

The mammalian protein has an enzymatic activity and catalyzes the oxidation of Fe2+ at ferroxidase sites present only in H subunits [88], This reaction may proceed through a di-ferric-p-peroxo species, which rapidly decomposes, eventually forming a ferric oxyhydroxide mineral core via an inorganic hydrolysis polymerization [89]. 

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. 

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. 

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). 

Iron is oxidized for incorporation into the mineralized core by either a protein enzymatic mechanism involving a putative dinuclear Fe ferroxidase site on the H chain subunit or a mineral surface mechanism. The net stoichiometric reactions for the two kinetic pathways are given by the following equations ... 

Fig. 16. Models for the membrane topology and orientation for the FetSp/Ftrlp and Fet5p/Fthlp complexes in yeast membranes. (Top) FetSp/Ftrlp are pictured in the yeast plasma membrane oriented to the extracellular space. The (R)EXXE motifs that may be involved in iron uptake and the transmembrane domain required for correct assembly are indicated. These roles have been suggested by mutagenesis studies (Stearman et al., 1996). (Bottom) Model of Fet5p/Fthlp complex proposed for the membrane of the yeast vacuole oriented to the lumen (inside) of the vacuole. In this model, the EXXE motifs in Fthlp are not oriented toward the ferroxidase site on Fet5p (Urbanowski and Piper, 1999).

The relative specificity that laccases have for bulky aromatic (poly) phenols and amines is due not only to the H-bond Network. The type 1 site associated with these H-bonds lies at the base of a shallow cavity that is lined with nonpolar side chains, or the nonpolar aliphatic carbon skeleton of a polar one, for example, Q and K. The binding cavities of two laccases (superimposed) are shown in Figure 9(b). As discussed below, those MCOs that exhibit a strong specificity for Fe + as substrate the ferroxidases - have a substrate binding site that differs in two respects from the laccase one the ferroxidase site is spatially more constrained and it... 

Fig 12. Schematic diagram of the ferroxidase center of human rHF (a), and the equivalent region of horse light-chain ferritin (b) showing the ferroxidase site replaced by a salt bridge. 

The ferroxidase site in reduced bacteiioferritin from Desulfovibrio desulfuricans (DdBfr) with iron—ligand distances in angstroms is shown in Figure 6.6a as determined by crystallography and, in Figure 6.6b, adjusted to... 

Fe(II) must then move from the 12 A long channel, and traverse a further distance of about 8 A along a hydrophilic pathway from the inner side of the three-fold channel to the ferroxidase site, and a putative pathway for Fe(II) is shown in Figure 19.6. The diiron ferroxidase centre is located in the central region of the four-helix subunit bundle and the coordination geometry of the ferroxidase centre of human H-chain ferritin is shown in Figure 19.7. Detailed analysis of the ferroxidase reaction in H-chain ferritin has allowed the identification of a number of intermediates, which are illustrated in Figure 19.8. 

Iron minerals are nucleated on the inner surface of ferritin, at clusters of conserved carboxylate residues [45]. Substitution of Glu residues with Ala in L-type ferritin slows the rate of mineralization and decreases the pattern of bound water, which is highly conserved [46]. The nucleation sites are constant in all known ferritin sequences, which contrasts with the presence of the ferroxidase site only in H-type ferritins [1, 2, 47]. 

Starting with dimer completely lacking non-heme iron, the time-course for the oxidation of added Fe " " was biphasic, as seen also with the 24-mer (Figure 14-6). In the case of wild-type E. coli Bfr the faster initial phase of oxidation has been shown to be associated with the activity of the ferroxidase site [19,20] and not to be significantly influenced by the presence or absence of heme [18]. We have carried out less detailed experiments with R. capsulatus wild-type Bfr than those used for E. 

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