The Blue Copper Oxidases

Blue copper oxidase (BCO) is a classification of oxidoreductase enzymes that contain at least one blue or T1 copper and a T2AT3 trinuclear cluster. These enzymes typically give rise to a characteristic blue color as a result of a strong absorption band around 

600 nm due to the T1 Cu. Many BCOs are known and have been isolated from plant, animal, and fungal sources alike. Some of the more widely studied BCOs include lacease, aseorbate oxidase, ceruloplasmin, phenoxazinone synthase, BOx, dihydro-geodin oxidase, sulochrin oxidase, and FET3 (from Saccharomyces cerevi-siae) [35,36]. These enzymes all have a common catalytic ability to oxidize a substrate that results in a reduced Cu state, which subsequently reduces O2. The native state of most BCOs is fiilly oxidized that is, each of the four Cu ions is divalent. Therefore, for die frill reduction of O2 to H2O (Equation 15.2), the enzyme must oxidize four substrate molecules. 

BCOs exhibit variable substrate specificity plant and fungal laccases, for example, typieally exhibit wide substrate specificity and can oxidize a variety of aminophenols, diphenols, and aryl diamines. The mechanism for these enzymes is consistent with Mareus theory [37], and suggests that oxidation occurs in the outer sphere and there is likely no specific binding pocket Conversely, some BCOs such as ascorbate oxidase (specifieity toward L-ascorbate) [38,39] and ceruloplasmin (specificity toward Fe +) [40,41] are often stereospecilic and highly substrate specific [42]. 

Substrate-specific BCOs, when reacting with substrates under natural conditions, follow Michaelis-Menten kinetics with reasonably high catalytic activity [42]. The substrate-enzyme reaction, or reduction of the Cu sites, is the rate-determining step for the overall enzymatic reaction [37], whereas the oxygen reduction reaction (ORR) occurs much more rapidly. This type of enzymatic reaction would not participate in any electrocatalytic electron transfer unless the substrate is actually the electrode. Therefore, the goal for BFCs is to configure the enzyme by a method that allows the electrode to assume the role of substrate so that the electrons can travel through an external circuit. 

Despite the broad substrate specificity of many laccases, the primary catalytic reaction of interest for BFC research is the ORR. Hence, the focus turns to the Cu sites of BCOs, such as laccase. Much is already known about the functionality of each of the types of Cu for example, it is known that ORR is initiated by reducing the T1 Cu (near the surface) [25]. The resulting electron tunnels down a His-Cys-His tripeptide to the trinuclear Cu center (TNC) [44 6]. As the activation site for ORR, the redox potential of the T1 site determines the redox potential for ORR. The actual ORR, 

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Various spectroscopic methods have been used to probe the nature of the copper centers in the members of the blue copper oxidase family of proteins (e.g. see ref. 13). Prior to the X-ray determination of the structure of ascorbate oxidase in 1989, similarities in the EPR and UV-vis absorption spectra for the blue multi-copper oxidases including laccase and ceruloplasmin had been observed [14] and a number of general conclusions made for the copper centers in ceruloplasmin as shown in Table 1 [13,15]. It was known that six copper atoms were nondialyzable and not available to chelation directly by dithiocarbamate and these coppers were assumed to be tightly bound and/or buried in the protein. Two of the coppers have absorbance maxima around 610 nm and these were interpreted as blue type I coppers with cysteine and histidine ligands, and responsible for the pronounced color of the protein. However, they are not equivalent and one of them, thought to be involved in enzymatic activity, is reduced and reoxidized at a faster rate than the second (e.g. see ref. 16). There was general concurrence that there are two type HI... 

RELATIONSHIP WITH OTHER COPPER OXIDASES 5.1 The "Blue" Copper Oxidase Family... 

This discussion of copper-containing enzymes has focused on structure and function information for Type I blue copper proteins azurin and plastocyanin, Type III hemocyanin, and Type II superoxide dismutase s structure and mechanism of activity. Information on spectral properties for some metalloproteins and their model compounds has been included in Tables 5.2, 5.3, and 5.7. One model system for Type I copper proteins39 and one for Type II centers40 have been discussed. Many others can be found in the literature. A more complete discussion, including mechanistic detail, about hemocyanin and tyrosinase model systems has been included. Models for the blue copper oxidases laccase and ascorbate oxidases have not been discussed. Students are referred to the references listed in the reference section for discussion of some other model systems. Many more are to be found in literature searches.50... 

Copper oxidases are widely distributed in nature, and enzymes from plants, microbes, and mammals have been characterized (104,105). The blue copper oxidases, which include laccases, ascorbate oxidases, and ceruloplasmin, are of particular interest in alkaloid transformations. The principle differences in specificity of these copper oxidases are due to the protein structures as well as to the distribution and environment of copper(II) ions within the enzymes (106). While an in vivo role in metabolism of alkaloids has not been established for these enzymes, copper oxidases have been used in vitro for various alkaloid transformations. 

In the discussion of the biochemistry of copper in Section 62.1.8 it was noted that three types of copper exist in copper enzymes. These are type 1 ( blue copper centres) type 2 ( normal copper centres) and type 3 (which occur as coupled pairs). All three classes are present in the blue copper oxidases laccase, ascorbate oxidase and ceruloplasmin. Laccase contains four copper ions per molecule, and the other two contain eight copper ions per molecule. In all cases oxidation of substrate is linked to the four-electron reduction of dioxygen to water. Unlike cytochrome oxidase, these are water-soluble enzymes, and so are convenient systems for studying the problems of multielectron redox reactions. The type 3 pair of copper centres constitutes the 02-reducing sites in these enzymes, and provides a two-electron pathway to peroxide, bypassing the formation of superoxide. Laccase also contains one type 1 and one type 2 centre. While ascorbate oxidase contains eight copper ions per molecule, so far ESR and analysis data have led to the identification of type 1 (two), type 2 (two) and type 3 (four) copper centres. 

Structural Relationships among the Blue Copper Oxidases... 

Molecular as well as spectroscopic properties of representative members of the blue copper oxidases are summarized in Tables I (62-69) and II (70-72), respectively. Data from other spectroscopic techniques used on blue oxidases, such as fluorescence, CD, or resonance Raman spectroscopy, have not been included because the implications of most of these studies are not so striking. The interested reader is referred to the existing review articles (10, 18, 19, 26). 

Active sites containing two copper ions that are antiferromagneti-cally coupled in the oxidized state are often referred to as type 3 copper sites (129). It has recently become evident that these centers cannot all be considered alike and that in the blue copper oxidases the type 3 sites are in fact part of a tricopper cluster these will be considered in Section VA. The proteins containing dinuclear type 3 copper sites comprise hemocyanin and a number of oxygenase enzymes, of which the best known are tyrosinase and dopamine j8-hydroxylase. 

Multiple Types of Copper in One Enzyme. When the blue copper oxidases were first investigated, attention naturally centered on the copper responsible for the intense blue color and the highly unusual ESR signal. Since both the unique optical and ESR parameters of the Cu(II) disappeared when the proteins were denatured, any more normal Cu(II) detected by ESR in these proteins was most often attributed to denatured enzyme (60). However, as more extensive studies on homogeneous samples of the blue copper oxidases were carried out, it became clear that... 

The function of the diamagnetic copper in the blue copper oxidases is not clear. Anaerobic titrations of Polyporus laccase with a number of... 

Blanford, C.F., Foster, C.E., Heath, R.S., and Armstrong, F.A. (2008) Efficient electrocatalytic oxygen reduction by the blue copper oxidase, laccase, directly attached to chemically modified carbons. Faraday Discussions, 140, 319-335. 

Laccases (p-diphenol O2 oxidoreductase EC 1.10.3.2) catalyze the oxidation of p-diphenols with the concurrent reduction of dioxygen to water. However, the actual substrate specificities of laccases are often quite broad and vary with the source of the enzyme [116,117]. Laccases are members of the blue copper oxidase enzyme family. Members of this family have four cupric (Cu +) ions where each of the known magnetic species (type 1, type 2, and type 3) is associated with a single polypeptide chain. In the blue copper oxidases the Cu + domain is highly conserved and, for some time, the crystallographic structure of ascorbate oxidase, another member of this class of enzymes, has provided a good model for the structure of the laccase active site [124,125]. The crystal structure of the Type-2 Cu depleted laccase from Coprinus cinereus at 2.2. A resolution has also been elucidated [126]. 

The blue copper oxidases are similar to cytochrome oxidase in their ability to catalyze reduction of Oj to HjO. Catalysis is centered upon the protein-bound copper ions that can be differentiated into three classes according to their physical, chemical, and functional properties. They are designated Types 1, 2, and 3 copper . In the blue copper proteins (tree and fungal laccases, ceruloplasmin, ascorbate oxidase) these three classes of copper appear in varying amounts the laccases contain the minimum amounts of each (one each of Types 1 and 2 and two Type 3 coppers). 

The blue copper oxidases catalyze substrate oxidation by O2. The reduction potentials of the various protein-bound copper species suggest tht electron flow occurs from substrate to Types 1 and 2 copper and subsequently to the Type 3 center and O2. That this is indeed the pathway of electron flow has been demonstrated in rapid-mix, stopped-flow optical and rapid-freeze EPR measurements " . Substrate reduces Types 1 and 2 copper in a rapid initial phase (typical second-order rate constants are 10 M" s" ), although from the data presented it is not entirely clear whether substrate reduces... 

Figure 1. Proposed mechanism for the catalytic cycle and dioxygen reduction site structure in the blue copper oxidase, laccase (after ref. 19, with permission).

In addition to providing a binding site for the intermediate described above, Type 2 copper appears to mediate interactions between the metal components of the enzyme. Thus Type 2 depleted enzyme shows slow electron transfer between the remaining Type 1 and 3 copper species and the reoxidation of the reduced Type 2 depleted enzyme is slow ". Moreover, the redox potential of the Type 3 copper center depends on the redox and ligation state of the Type 2 center and the formation of a Type II copper-OH species apparently inactivates the protein. It appears, therefore, that deeper insight into the function and mechanism of the blue copper oxidases will come through a more fundamental understanding of the role and position of the Type 2 copper. 

Although completely adequate models for the metal centers in the blue copper oxidases remain to be developed, substantial progress has been made recently. This comes primarily from insight that has been gained into the in situ structures described above and the resulting sharper focus and more stringent boundary conditions which they impart. 

Models for the blue copper oxidase peroxide-level intermediate... 

Figure 8 Physiologically relevant reactions catalyzed by the blue copper oxidases (after Solomon and...

Spectroscopic, mechanistic, and X-ray structural insights (see Section 8.17.3.3) allow generalization of a number of features that apply to the entire family of the blue copper oxidases. 

Freeman JC, Nayar PG, Begley TP, Villafranca JJ. Stoichiometry and spectroscopic identity of copper centers in phenoxazinone synthase A new addition to the blue copper oxidase... 

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