Multicopper ascorbate oxidase

Intramolecular ET between distinct copper centers is part of the catalytic cycles of many copper-containing redox enzymes, such as the multicopper oxidases, ascorbate oxidase, and ceruloplasmin, as well as the copper-containing nitrite reductases. Examination of internal LRET in these proteins is of considerable interest as it may also provide insights into the evolution of selected ET pathways in particular, whether and how the enzymes have evolved in order to optimize catalytic functions. With the increase in the number of known high-resolution 3D structures of transition metal containing redox enzymes, studies of structure-reactivity relationships have become feasible and indeed many have been carried out during the last two decades. 

S / V CONTENTS Preface, Robert W. Hay. Structure and Function of Manganese-Containing Biomolecules, David C. Weather-bum. Repertories of Metal Ions as Lewis Acid Catalysts in Organic Reactions, Junghan Suh. The Multicopper-Enzyme Ascorbate Oxidase, Albrecht Messerschmidt. The Bioinorganic Chemistry of Aluminum, Tomas Kiss and Etelka Farkas. The Role of Nitric Oxide in Animal Physiology, Anthony R. Butler, Frederick Flitney and Peter Rhodes. Index. 

Copper oxidases Blue oxidases (multicopper oxidases) Laccase Ascorbate oxidase Ceruloplasmin... 

The hemocyanlns which cooperatively bind dioxygen are found in two invertebrate phyla arthropod and mollusc. The mollusc hemocyanlns additionally exhibit catalase activity. Tyrosinase, which also reversibly binds dioxygen and dlsmutates peroxide, is a monooxygenase, using the dloxygen to hydroxylate monophenols to ortho-diphenols and to further oxidize this product to the quinone. Finally, the multicopper oxidases (laccase, ceruloplasmin and ascorbate oxidase) also contain coupled binuclear copper sites in combination with other copper centers and these catalyze the four electron reduction of dloxygen to water. 

Blue Multicopper Oxidases. These include laccases, ascorbate oxidase, and ceruloplasmin [22,61], which along with cytochrome c oxidase (CcO with Fe and Cu) can couple the one-electron oxidation of substrates (e.g., ascorbate, diamines, monophenols Fe2+ for ceruloplasmin cytochrome c, for CcO) to the full reduction of dioxygen to water (i.e., 02 + 4c + H+ —> 2H20). 

Some proteins contain more than one copper site, and are therefore among the most complicated and least understood of all. The active site known as type 4 is usually composed of a type 2 and a type 3 active site, together forming a trinuclear cluster. In some cases, such proteins also contain at least one type 1 site and are in this case termed multicopper oxidases, or blue oxidases [3], Representatives of this class are laccase (polyphenol oxidase) [7-9], ascorbate oxidase (Figure 5.Id) [10], and ceruloplasmin [11], which catalyze a range of organic oxidation reactions. 

The multicopper oxidases (laccase, ascorbate oxidase, and ceruloplasmin) catalyze a four-electron reduction of dioxygen to water (285-287). Consistent with the four-electron stoichiometry, the enzymes contain four copper ions. One of the copper ions is type I, causing an intensely blue color of the proteins, thus the enzymes of this family are referred to as blue oxidases. They also contain a monomeric copper site that exhibits normal spectroscopic features, whereas the other two copper... 

AO = Ascorbate oxidase (h)Cp = (human) Ceruloplasmin CT = Charge transfer Hp = Hephaestin GPl = Glycosyl-phosphatidylinositol Lac = Laccase MCO = Multicopper oxidase T1(2,3)D = Type 1 depleted (and/or type 2 or type 3) Tf = Transferrin. 

The presence of a single type 2 center in ascorbate oxidase is not consistent with the proposed concept of a quaternary structure composed of two identical subunits afi (25). On the other hand, all the multicopper oxidases described in the literature (5-8) have only one type 2 center per active molecule. Additional copper with type 2 characteristics can be bound by the macromolecule during isolation and purification (19). A close examination of the EPR spectra presented by Lee and Dawson (9) indicates the presence of so-called nonspecific copper with large hyperfine splittings at g. As expected, the ratio A330/A610 is approximately 1.5-2 for these preparations (estimated from Figure 2 in Ref. 9). 

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. 

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. 

Figure 4-3 Homology between Fet3p and the multicopper oxidases. Comparison was done using the ClustalW alignment program. Underlined sequence represents multicopper oxidase domains. Bold sequences represent hydrophobic regions. Only one representative member of each family was used in the comparison. Laccase is from Thanatephorus cucunteris, Ctenbank accession number 2833189. AO is an ascorbate oxidase from Cucumis sativus (cucumber), Genbank accession number 114270. PCOA is the copper resistance protein PCOA from...

Copper-dioxygen compounds or interactions of higher nuclearity exist in the multicopper oxidases (Cu 02 = 3 l) such as in ascorbate oxidase (AO), laccase, and ceruloplasmin, all of which couple the four electron reduction of O2 to water with the oxidation of a substrate (7). A number of AO protein X-ray studies are available, including a derivative with an end-on... 

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... 

Multicopper oxidases are typically active in the catalytic one-electron oxidation of a variety of diphenolic, polyphenolic, enediolic, and aminophe-nolic substrates 1,53,166,167). The mechanism of these reactions is complex and, as discussed in Section I, it involves a sequence of four one-electron oxidations of substrate molecules. The radical products of these reactions undergo dismutation, as shown in Scheme 21 for the oxidation of ascorbate to semidehydroascorbate radical 168,169). The substrate binds to the enzymes close to type 1 Cu, whereas the trinuclear cluster is only accessible to dioxygen, or other small molecules. This situation is clearly difficult to reproduce in a model system and for this reason the type of model oxidation reactions that have been studied so far using synthetic trinuclear copper complexes is more related to the activity of type 3 Cu enzymes than multicopper oxidases. Nevertheless, such trinuclear complexes open new perspectives in stereoselective catalysis, because one of the metal centers... 

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. 

Nakamura et al. 191) have carried out a fairly comprehensive study of ascorbate oxidase from cucumber Cucumis sativus) peel. The Cu + was shown to undergo cychc reduction and oxidation and the pH-activity curve was demonstrated to be bell-shaped with a broad maximum between pH 5.5 and 7. The velocity of the reaction depended on both ascorbate and O 2 concentrations with ifm02 lmM and = mM at 25 °C. Azide was found to inhibit uncompetitively and Ki = 0.2 mM. Ascorbate oxidase appears to be substantially similar in its structure-function relationships to all the other known multicopper oxidases. 

Multicopper oxidases and cytochrome oxidases are the only proteins known to catalyze the four-electron reduction of O2 to H2O. The multicopper oxidases possess a distinctive subdomain structure (706). Laccase, ascorbate oxidase, and Fet3 have three domains while human ceruloplasmin has six. In all the multicopper oxidases there is significant internal homology among the subdomains suggesting they aU arose from a common gene ancestor by gene duplication (707, 706). 

Multicopper Oxidases (Blue Copper Oxidases) Ascorbate Oxidase, Ceruloplasmin, and Laccase. The multicopper oxidases (MCOs) are important enzymes, which are found in many plants (lignin formation), fungi (lignin degradation and detoxification), bacteria, as well as humans (ferroxidase activity) (13). MCOs catalyze the four-electron reduction of O2 to two waters with the electrons coming firom one-electron oxidation of four substrate molecules. The latter are organic reductants for ascorbate oxidase (AO) (32) and laccase (Lc) (130), and a metal ion (ferrous ion) for ceruloplasmin (Cp) (33) (Scheme 9). 

Blue multicopper oxidases (BMCOs) such as laccase, ceruloplasmin, bilirubin oxidase (BOx), and ascorbate oxidase (AOx) have been extensively investigated as cathodic biocatalysts for DET-based biodevices [44]. BMCOs have a catalytic center consisting of four coppers a type 1 (Tl) Cu site, which accepts electrons from the substrate and from the electrode surface, and a type 2/type 3 (T2/T3) cluster, where O2 is reduced directly to water. High redox potential laccases and BOx, with redox potential up to 780 and 670 mV versus normal hydrogen electrode (NHE), respectively [44,45], can be used to create efficient biocathodes with current densities up to a few mA cm . In 2012, Shleev and coworkers used the DET ability of these enzymes to create several completely DET-based BFCs [42]. The enzymes have also been used in different MET-based approaches [46,47] specifically, Heller and coworkers... 

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