The Ferroxidases

Cartwright and Wintrobe and their co-workers suggested a link between copper deficiency and anemia in mammals 50 years ago (see Lahey et al., 1952). Cartwright subsequently demonstrated that this copper-dependent anemia was unresponsive to iron supplementation but was corrected on administration of ceruloplasmin (see Lee et al., 1968). The molecular basis of this link was indicated by Osaki and Friedan, who characterized the ferroxidase activity of ceruloplasmin and kinetically demonstrated that Cp could play a critical role in catalyzing trafficking of the potentially cytotoxic Fe(II) in the plasma to apoA f (see Frieden and 

1974 Osaki, 1966 Osaki et al., 1966). Using the kinetic values given above, they estimated that without the ferroxidase activity of Cp in the plasma 80% of the iron released from erythrocyte turnover would accumulate as non-Tf-bound Fe(ll) and thereby would be unavailable for reabsorption by the reticuloendothelial system. Furthermore, this free Fe(II) could catalyze the formation of reactive oxygen species via the Fenton reaction. This, in turn, could lead to a subsequent organismal pathophysiology (Miyajima et al., 1996 Nakano, 1993). This inference has been strikingly confirmed by research over the past 6 years in both yeasts and mammals this research has directly tested the hypothesis that multicopper oxidase-dependent ferroxidase activity is essential to eukaryotic iron homeostasis (Askwith et al., 1996 Harris et al., 1995 1998 Wessling-Resnick, 1999). 

Based on present sequence data, known or likely ferroxidase enzymes can be identihed in several eukaryotes. These enzymes are listed in Table 11. All are multicopper oxidases, by sequence homology at least. In mammals, they include ceruloplasmin and, most likely, hephaestin (Hp), although only mouse Hp (mHp) has been characterized at this time (Vulpe et al., 1999). The alignments in Fig. 5A show that mHp is essentially 

Kingdom/Order Organism Protein Accession No. evaluated 

L lISHHBaWfBe vHiiG rffil I PRplSsGp3-H IISitgaLiiRGE[BBi l tEWMIk sISsB 

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Although channel mutations and chemical modifications reduce rates of iron oxidation and uptake, they do not completely abolish the ferroxidase activity of... 

The stoichiometries of both oxygen consumption and of proton release subsequent to Fe(III) hydrolysis have been determined by using a combined oximeter and pH stat (Yang et ah, 1998). The overall reaction at the ferroxidase centre is postulated to be ... 

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). 

Hence, the overall reaction for iron oxidation and hydrolysis at the ferroxidase centre, followed by further hydrolysis and migration to the core nucleation sites (equation 7) is ... 

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. 

The stoichiometry of 2Fe(II)/02, and the structure of the ferroxidase iron site suggest that the first step after iron (II) binding would be transfer of two electrons, one from each Fe(II), to a dioxygen molecule bound at the same site, to give a formal peroxodiferric intermediate formally this represents dioxygen binding, followed by Fe(II) oxidation (Equations (19.2) and (19.3)) ... 

The peroxo intermediate would then undergo iron (III) hydrolysis (Equations (19.4) and (19.5)) to give first the p-oxobridged Fe(III) dimer and then upon addition of another two molecules of H20, a protein-[Fe20(0H)2] species at the ferroxidase centre ... 

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. 

While core formation during hydrolysis of Fe(III) produces electrically neutral ferri-hydrite, it also produces protons two per Fe(II) oxidized and hydrolysed, whether due to iron oxidation and hydrolysis at the ferroxidase centre, followed by further hydrolysis and migration to the core nucleation sites or by direct Fe(II) oxidation and hydrolysis on the mineral surface of the growing core. These protons must either be evacuated from the cavity or else their charges must be neutralized by incoming anions, and it... 

Kauko, A., Pullianen, A.T., Haataja, S., Mayer-Klauke, W., Finne, J. and Papageorghiou, A.C. (2006) Iron incorporation in Streptococcus suis Dps-like peroxide resistance protein Dpr requires mobility in the ferroxidase center and leads to the formation of a ferrihydrite-like core, J. Mol. Biol., 364, 97-109. 

The L chain subunit appears to be involved mostly in mineralization of the core but modulates the ferroxidase activity of the H chain subunit as well. 

One of the significant differences between the multicopper oxidases as a group and the ferroxidases is that only the latter efficiently catalyze the... 

Fig. 4. Analysis of the kinetic constants of the ferroxidase reaction catalyzed by soluble Fet3p. Fe(II) oxidation (A) and O2 consumption (B) were measured continuously and the residual substrate concentration was plotted with respect to time according to the integrated form of the Michealis-Menten equation as indicated in each panel. Fe(II) oxidation was followed by the appearance of Fe(III) at 315 nm while O2 consumption was determinedby the use of an O2 electrode. The [Fet3p] =0.2 fcM in 0.1 M MES buffer, pH 6.0, at 25°C. The curve in each panel is a linear least-squares fit of the data to...

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