Ferroxidase reaction

In vitro experiments with Cp and Fet3p have shown dehnitively that both enzymes have a strong substrate specihcity toward Fe(II) that is not shared by the other well-characterized multicopper oxidases. Furthermore, several independent analyses have demonstrated that the formation of Tf Fe(III) in a mixture of apo iand Fe(II) is strongly catalyzed by Cp. At the other end of the experimental spectrum are the well-established correlations between organismal abnormalities in iron handling in 

Some of the functional characteristics of these mutant proteins are reviewed below. However, irrespective of other outcomes of these experiments, they uniformly demonstrate that if the FetSp in the plasma membrane is inactive as a ferroxidase, the cell exhibits no Ftrlp-dependent iron uptake. To appreciate the significance of this result, one needs to appreciate the steps involved in iron (and copper) uptake in yeast (Askwith et al., 1996 Hassett and Kosman, 1995 Kosman, 1993 Wesshng-Resnick, 1999). This is diagrammed in Fig. 14. 

The first key element in the uptake of these two metal ions is that the substrate is the lower valent state species, Cu(I) in the case of copper and Fe(II) in the case of iron (Dancis et al., 1990, 1992 Hassett and Kosman, 1995 Kosman, 1993). Normally, these reduced valence species are provided by the action of plasma membrane metal reductases, an activity in yeast provided predominantly by the product of the FREl gene (Dancis et al., 1992). However, Fe(II) [or Cu(I)] provided exogenously to the cell is equally competent for uptake and, in most experimental regimes, is added directly or generated in situ by the addition of a strong reductant like ascorbate or dithionite. Cu(I) is the direct substrate for uptake, through the Ctrlp copper permease in most yeast strains (Dancis et al., 1994). However, the presence of Fe(II), although required, alone is not 

These constants indicate that the ferroxidase reaction catalyzed by Fet3p might be rate-limiting in iron uptake. The value of Acat for Fe uptake, 36 min, is based on the assumption that there are 1000 

Kinetic Constants for the Fet3p Ferroxidase Reaction in Vitro and Fe Uptake in Vivo 

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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 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. 

Hence, the overall reaction for iron oxidation and hydrolysis at the ferroxidase centre, followed by further hydrolysis and migration to the core nucleation sites consists of two reactions, the protein-catalysed ferroxidase reaction itself and the Fe(II) plus H202 detoxification reaction (Equations (19.7) and (19.8), respectively) ... 

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. 

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...

Crystallographic analysis has provided us with a detailed structure of hCp on the other hand, essentially all of the structure-function analyses have been done on FetSp. Also, except for the copper site structural homology, the two proteins are quite different. hCp is composed of six plastocyanin-like domains (plastocyanin is a type 1 copper-containing protein) that are arranged in a trigonal array (Zaitseva et al., 1996). One result of this domain replication is a conformational fold that produces a distinct, negatively charged patch on the protein surface adjacent to the catalytically active type 1 Cu(II). This copper atom is in domain 6. (Domains 2 and 4 contain type 1-like copper sites that do not participate in the ferroxidase reaction.) Lindley et al. (1997) have proposed that this... 

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,... 

The overall ferroxidase reaction is therefore reaction (9) below ... 

Following reaction (9), the ferric iron remains at the ferroxidase center as a /x-oxo bridged species instead of moving into the core and thus regenerating the ferroxidase center. The addition of more than 48 ferrous ions per protein saturates the ferroxidase reaction and any further iron is oxidized within the core (phase 3). This process is significantly slower than phase 2. Core formation occurs according to reaction (10) below. 

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. 

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