Multicopper oxidases characterization

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... 

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... 

Laccase, ascorbate oxidase, and ceruloplasmin are the classical members of the multicopper oxidase family also known as blue oxidases. Recently, a small number of bacterial members of this family have been characterized, including CueO from E. coli a spore-coat laccase (CotA) from Bacillus suhtilis and phenoxazinone synthase from Streptomyces antibioticus The catalyzed reaction of these enzymes except for phenoxazinone synthase is given in Equation (11). A comprehensive overview of the broad and active research on blue copper oxidases is presented in Messerschmidt. Recent results have been included in a review on the reduction of dioxygen by copper-containing enzymes. The nature and number of the different copper sites in blue oxidases has been described in the sections about the type-1 copper site and the trinuclear copper cluster. 

A great deal more is known for binuclear systems, which are better for comparison to O2 adducts within the trinuclear T2/T3 multicopper oxidase active site (see Figure 11). Mononuclear ligand-Cu(I) or dicopper(I) complexes with a binucleating ligand react with O2 to yield peroxo or hydroperoxo-dicopper(II) complexes. These products have been characterized, often via low-temperature spectroscopic studies (see Chapter 8.15). Kitajima s -peroxo (side-on peroxo) dicopper(II) adduct ... 

The blue multicopper oxidases constitute a heterogeneous family of enzymes from different sources (7). In addition to the well characterized members of this family, ascorbate oxidase (45,46), laccase (47,48), and ceruloplasmin (49,50), all from higher organisms, two other proteins have attracted much recent interest FetSp, which is involved in iron uptake in yeast (51), and CueO, which is required for copper homeostasis in Escherichia coli (52). The characteristic reactivity of these enzymes is the one-electron oxidation of four substrate equivalents coupled to the four-electron reduction of dioxygen to water (1). These processes occur at a catalytic unit constituted by four copper atoms classified according to their spectroscopic properties in... 

With the exception of a study carried out with a partially characterized multicopper oxidase isolated from tea leaves (85), there has been very little detailed work concerned with the steady state kinetic behavior of laccases. Early work on the transient kinetics indicated, however, that (1) enzyme bound Cu + was reduced by substrate and reoxidized by O2, and (2) substrate was oxidized in one-electron steps to give an intermediate free radical in the case of the two electron donating substrates such as quinol and ascorbic acid. The evidence obtained suggested that free radicals decayed via a non-enzymatic disproportionation reaction rather than by a further reduction of the enzyme (86—88). In the case of substrates such as ferrocyanide only one electron can be donated to the enzyme from each substrate molecule. It was clear then that the enzjmie was acting to couple the one-electron oxidation of substrate to the four-electron reduction of oxygen via redox cycles involving Cu. 

Largely due to the great difficulty associated with obtaining quantities of purified enzyme adequate for physical-chemical studies its characterization has not been carried to the same stage as that of ceruloplasmin and the laccases. Nevertheless, recent developments in the purification procedure (191, 192) have allowed measurements which signify a great similarity of the Cu-binding sites on this enzyme with those of other blue, multicopper oxidases. The properties of the enzyme are summarized in Table 2. 

Cole JL, BaUou DP, Solomon El. 1991. Spectroscopic characterization of the peroxide intermediate in the reduction of dioxygen catalyzed hy the multicopper oxidases. J Am Chem Soc 113 8544-8546. 

Ivnitski D, Khripin C, Luckarift HR, Johnson GR, Atanassov P. Surface characterization and direct bioelectrocatalysis of multicopper oxidases. Electrochim Acta 2010 55 7385-7393. 

PURIFICATION AND CHARACTERIZATION OF MULTICOPPER OXIDASES FOR ENZYME ELECTRODES... 

---