Ferroxidase enzymes

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... 

Hassett, R. E, Yuan, D. S., and Kosman, D. J. (1998). Spectral and kinetic properties of the Fet3 protein from Saccharomyces cerevisiae, a multinuclear copper ferroxidase enzyme. J. Biol. Chem. 273, 23274-23282. 

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. 

Copper is a component of many enzymes including amine oxidase, lysyl oxidase, ferroxidase, cytochrome oxidase, dopamine P-hydroxylase, superoxide dismutase and tyrosinase. This latter enzyme is present in melanocytes and is important in formation of melanin controlling the colour of skin, hair and eyes. Deficiency of tyrosinase in skin leads to albinism. Cu " ion plays an important role in collagen formation. 

Copper-dependent enzymes, ASCORBATE OXIDASE CATECHOL OXIDASE FERROXIDASE GAACTOSE OXIDASE ACCASE... 

The multi-copper oxidases include laccase, ceruloplasmin, and ascorbate oxidase. Laccase can be found in tree sap and in fungi ascorbate oxidase, in cucumber and related plants and ceruloplasmin, in vertebrate blood serum. Laccases catalyze oxidation of phenolic compounds to radicals with a concomitant 4e reduction of O2 to water, and it is thought that this process may be important in the breakdown of lignin. Ceruloplasmin, whose real biological function is either quite varied or unknown, also catalyzes oxidation of a variety of substrates, again via a 4e reduction of O2 to water. Ferroxidase activity has been demonstrated for it, as has SOD activity. Ascorbate oxidase catalyzes the oxidation of ascorbate, again via a 4e reduction of O2 to water. Excellent reviews of these three systems can be found in Volume 111 of Copper Proteins and Copper Enzymes (Lontie, 1984). 

A copper-binding protein, ceruloplasmin, which is a blood serum protein, has been demonstrated in milk by immunodiffusion techniques (Hanson et al. 1967 Poulik and Weiss 1975). It may be the enzyme ferroxidase (EC 1.16.3.1). 

Fig. 1. Schematic overview of copper trafficking and homeostasis inside the yeast cell. The actions of Mad and Ace 1, copper-dependent metalloregulatory transcription factors, control the production of copper import [copper transporter (Ctr) and reductase (Fre)] and detoxification/sequestration [metallothionein (MT)] machineries, respectively. Three chaperone-mediated delivery pathways are shown. Atxl shuttles Cu(I) to the secretory pathway P-type ATPase Ccc2 (right). CCS delivers Cu(I) to the cytoplasmic enzyme copper-zinc superoxide dismutase (SOD) (left). Coxl7 shuttles Cu(I) to cytochrome c oxidase (CCO) in the mitochondria (bottom). Mitochondrial proteins Scol and Sco2 may also play a role in copper delivery to the CuA and CuB sites of CCO. Copper metabolism and iron metabolism are linked through the actions of Fet3, a copper-containing ferroxidase required to bring iron into the cell (lower right) (see text).

Ceruloplasmin is a multifunctional enzyme capable of oxidizing phenols and aromatic amines (Musci et al., 1999). It can also efficiently oxidize Fe(II) to Fe(III), which is currently considered its main in vivo biological function. The ferroxidase activity of this enzyme was hrst reported in 1960 (Curzon and O Reilly, 1960) and it was later suggested that such activity is important for loading iron into the transferrin, since it binds only Fe(III) (Osaki, 1966). Recent studies on ceruloplasmin knockout mice demonstrated that they indeed exhibit a severe impairment of... 

A number of copper requiring enzymes are located at the cell surface or are exported into the extracellular milieu. Examples of such secretory Cu-enzymes include copper requiring ferroxidases that fimction in iron transport (e g. ceruloplasmin, CP), enzymes for neurotransmission (peptidyl amidating enzyme and dopamine hydroxylase), an extracellular superoxide dismutase (SOD) that fimctions in antioxidant defense and enzymes for formation of connective tissue (lysyl oxidase), and pigments (tyrosinase) (reviewed in ). En route to their designated location, each of these enzymes passes through a specialized compartment of the late Golgi where copper insertion takes place. 

Iron entering the bloodstream from the gastrointestinal tract is thought to be present as Fe , and must be oxidized to Fe before binding to transferrin, which then delivers iron to many different types of cell. The non-enzymic route for oxidation of Fe in serum appears to be too slow for the formation of iron(III) transferrin. As noted in Section 62.1.8.5.1, the copper protein ceruloplasmin has ferroxidase activity, being responsible for the oxidation of Fe" to Fe . It is well known that deficiency of copper influences iron metabolism in animals, in accord with this role for ceruloplasmin. 

Iron Metabolism. Copper-containing enzymes— namely ferroxidase I (ceruloplasmin) and ferroxidase II, and the recently described hephaestin in the enterocyte—oxidize ferrous iron to ferric iron. This allows incorporation of Fe into transferrin and eventually into hemoglobin. Ferroxidase II is a yellow protein, the importance of which in iron metabolism is not as well characterized as that of ceruloplasmin. 

Ferroxidases are enzymes that oxidize ferrous to ferric iron in the presence of oxygen according to the formula ... 

The high-affinity pathway involves oxidation of Fe to Fe by the ferroxidase FET3 and subsequent transport of Fe " " across the plasma membrane by the permease FTRl. FET3p is a member of the family of multicopper oxidases, which include ascorbate oxidase, laccase, and ceruloplasmin (see Chapter 14), and does not become functional until it is loaded with copper intracellularly through the activities of the copper chaperone ATX Ip and the copper transporter CCC2p. It appears that Fe " " produced by FET3 is transferred directly to FTRl, and does not equilibrate with the bulk phase, as is illustrated in Fig. 7.13. This is almost certainly achieved by the classic metabolite-channeling mechanism, a common feature of multifunctional enzymes. 

The iron in meats is in the form of heme, which is readily absorbed. The non-heme iron in plants is not as readily absorbed, in part because plants often contain oxalates, phytates, tannins, and other phenolic compounds that chelate or form insoluble precipitates with iron, preventing its absorption. Conversely, vitamin C (ascorbic acid) increases the uptake of non-heme iron from the digestive tract. The uptake of iron is also increased in times of need by mechanisms that are not yet understood. Iron is absorbed in the ferrous (Fe ) state (Fig. 44.6), but is oxidized to the ferric state by a ferroxidase known as ceruloplasmin (a copper-containing enzyme) for transport through the body. 

Numerous observations in our laboratory suggested the possible presence of another non-Cp ferroxidase activity in normal human serum. The key experimental tool for this study was the fact that there always appeared to be a residual nonazide-sensitive ferroxidase activity in human serum and even in Cohn IV-I fractions. Based on this, a new ferroxidase activity has been identified in our laboratory by Topham et al. (27). As shown in Table HI, this new enzyme diflFers from Cp in a number of key properties. The K s are in the /xM range but the new ferroxidase has only one. Copper appears to be present but in a diflFerent ratio, and the estimated molecular weight appears to be much larger. The new enzyme seems to reside in the same fraction as Cohn IV-I but can be separated by appropriate DEAE-Sephadex chromatography. We have not fully exhausted the possible substrate range of the new enzyme except that we know that it has no PPD oxidase, mono-, or di-amine oxi-... 

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