Enzymes with Trinuclear Active Sites

Figure 17.3 Anatomy of a redox enzyme representation of the X-ray crystallographic structure of Trametes versicolor laccase III (PDB file IKYA) [Bertrand et al., 2002]. The protein is represented in green lines and the Cu atoms are shown as gold spheres. Sugar moieties attached to the surface of the protein are shown in red. A molecule of 2,5-xyhdine that co-crystallized with the protein (shown in stick form in elemental colors) is thought to occupy the broad-specificity hydrophobic binding pocket where organic substrates ate oxidized by the enzyme. Electrons from substrate oxidation are passed to the mononuclear blue Cu center and then to the trinuclear Cu active site where O2 is reduced to H2O. (See color insert.)...

Laccase contains four copper atoms and catalyzes the four-electron reduction of dioxygen to water. X-Ray absorption edge spectroscopy has been used to determine the oxidation states of copper in Rhus vernicifera laccase, following the reaction of the reduced enzyme with dioxygen (202). This study included the incorporation of mercury(II) in the Type 1 copper site (see Section IV,B). The results demonstrate that the Type 2/Type 3 trinuclear copper site, as found in ascorbate oxidase (103), represents the minimal active site required for the multielectron reduction of dioxygen. 

Copper (I) complexes exhibit catalytic activity for the four-electron (4-e) reduction of O2 to water. Natural occurring enzymes like Cu-containing fungal laccase reduce O2 directly to water very efficiently at very positive potentials, not far from the thermodynamic standard potential of the O2/H2O couple. These enzymes involve a trinuclear Cu active site [149-153]. For this reason some authors have investigated the catalytic activity of Cu(I) complexes for ORR, in particular Cu phenanthrolines confined on graphite or glassy carbon surfaces [154-169], with the aim of achieving the total reduction of O2 via the transfer of four-electrons. 

The active site of MCOs consists of a minimum of four Cu atoms, referred to as type 1 (Tl, blue copper), type 2 (T2, normal copper) and type 3 (T3, coupled dinuclear center). While AO is considered a homodimer of Lc, Cp has two additional Tl copper centers. The Tl site in the four-copper enzymes is the site of the substrate oxidation, and it is characterized by an intense S(cysteine)-to-Cu(II) charge-transition at 610 nm (thus, these are blue copper oxidases). The trinuclear cluster (TNC) is formed with the T2 and T3 sites, and the reduction of O2 to water is catalyzed here. The TNC is 13 A away from Tl site and coupled electronically through a Tl-Cys-His-T3 electron-transfer pathway (Fig. 13) (131). 

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