Ascorbate oxidase copper binding

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. 

Figure 10-11. The structure of the active site in ascorbate oxidase. The enzyme contains four copper centres. Three of these form a triangular reaction site. Why are four copper ions needed How does the substrate bind ...

The low-temperature MCD and absorption titration studies (Figure 10) have determined that azide binds to both the type 2 and type 3 centers with similar binding constants. A series of chemical perturbations and stoichiometry studies have shown that these effects are associated with the same azide. This demonstrates that one N3 bridges between the type 2 and type 3 centers in laccase. These and other results from MCD spectroscopy first defined the presence of a trinuclear copper cluster active site in biology (89). At higher azide concentration, a second azide binds to the trinuclear site in laccase. Messerschmidt et al. have determined from X-ray crystallography that a trinuclear copper cluster site is also present in ascorbate oxidase (87, 92) and have obtained a crystal structure for a two-azide-bound derivative (87). It appears that some differences exist between the two-azide-bound laccase and ascorbate oxidase derivatives, and it will be important to spectroscopically correlate between these sites. 

Ascorbate oxidase is a tetramer each subunit has 552 amino acids and contains 4 copper ions, the type-I blue copper center and the adjacent trinuclear center (arranged as a type-n center and a type-in dinuclear center) separated by /S-sheets (Figure 20) °. Ascorbate is oxidized to dehydroascorbate by dioxygen however, it is not bound directly to the metal center to be oxidized, but is proposed to bind near the type-I Cu site which may facihtate electron transfer to oxygen, presumably in the tri-Cu cluster site. Since humans cannot synthesize ascorbic acid, conservation of this important compound is highly regulated. For example, the oxidized ascorbate can be transported into red blood... 

Laccases also become inhibited at higher pH values. Tree laccase is inhibited above pH 6.5. It appears that at pH 7.4 50% of the enzyme molecules are inhibited due to the binding of OH to the type-2 copper. The nature of this OH binding to the resting form of ascorbate oxidase will be demonstrated below. 

The reaction of nitric oxide with laccase (76) and ascorbate oxidase (147) has been studied as well. Nitric oxide fully reduces fungal and tree laccase when it is added to the oxidized enzyme under anaerobic conditions. In addition the binding of one NO molecule to laccase can be detected. This is characterized by a new EPR signal and has been described as coordinated with the type-2 copper (76). Only the reduction of the type-1 copper has been observed when NO has been added to ascorbate oxidase under anaerobic conditions. 

The reaction of dioxygen with laccase or ascorbate oxidase was reviewed in Section IX and in Messerschmidt et al. (74), where the possible binding modes of dioxygen to binuclear and trinuclear copper centers are discussed. A novel mode of dioxygen binding to a binuclear copper complex was found in a compound synthesized by Kitajima et al. (165). The complex contains peroxide in the mode, i.e., side-on between... 

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