Copper catalytic mechanism

Fungal laccases (benzenediokoxygen oxidoreductase, EC 1.10.3.2) belong to the multicopper blue phenoloxidases. They comprise glycosylated proteins expressed in multiple forms and variable molecular weight, ranging from 59 to 110 kDa. Laccase is expressed as multiple constitutive and induced isoenzymes [30, 64]. The enzyme contains four copper atoms (Cu), in different states of oxidation (I, II, III) [65], which play an important role in the catalytic mechanism. Laccase oxidizes different compounds while reducing O2 to H20, a total reduction of four electrons. 

M. Mure, S.A. Mills, J.P. Klinman, Catalytic mechanism of the topa quinone containing copper amine oxidases. Biochemistry 41 (2002) 9269-9278. 

A copper-centered mechanism for the Cu-TEMPO-catalyzed aerobic oxidation of alcohols was proposed by Sheldon and co-workers, wherein the active catalytic Cu" species is generated by oxidation of a Cu species with TEMPO, in the presence of alcohol, with formation of TEMPOH (Scheme 3) [146]. The resulting Cu" species is then capable of oxidizing the alcoholate to the aldehyde or ketone species. Regeneration of the TEMPO radical species was achieved by rapid oxidation of TEMPOH with O2. 

Scheme 3 Proposed catalytic mechanism for the copper catalyzed oxidation of alcohols with TEMPO as oxidant...

Cu has a bifunctional role in copper-quinoprotein amine oxidases, catalyzing the formation of TPQ from the specific tyrosy l residue in the precursor protein and playing a role in the catalytic mechanism, most probably with 02 reacting with Cu(I) in the oxidative part of the cycle. The recent discovery of LTQ suggests that other combinations of quinone cofactors may be found in the future. 

Copper(l) and Copper(ll) Complexes with [22]pr4pz Unraveling Catalytic Mechanisms... 

Other metals, such as copper, nickel, or silver, have been used as electrode materials in connection with specific applications, such as the detection of amino acids or carbohydrates in alkaline media (copper and nickel) and cyanide or sulfur compounds (silver). Unlike platinum or gold electrodes, these electrodes offer a stable response for carbohydrates at constant potentials, through the formation of high-valence oxyhydroxide species formed in situ on the surface and believed to act as redox mediators (40,41). Bismuth film electrodes (preplated or in situ plated ones) have been shown to be an attractive alternative to mercury films used for stripping voltammetry of trace metals (42,43). Alloy electrodes (e.g., platinum-ruthenium, nickel-titanium) are also being used for addressing adsorption or corrosion effects of one of their components. The bifunctional catalytic mechanism of alloy electrodes (such as Pt-Ru or Pt-Sn ones) has been particularly useful for fuel cell applications (44). 

Copper has a direct role in the catalytic mechanism. Complete removal of both copper and zinc destroys the activity of the enzyme which can be restored by addition of cupric ion but not by other metals (12). Pulse radiolytic methods have been used to generate superoxide ion and to follow the enzyme kinetics (24, 25). The reaction is second order with a rate constant of 2.37 X 109 M"1 sec-1 at 25°, independent of pH over the range 4.8-9.5. The following two-step mechanism has been proposed to account for the enzyme activity ... 

The structure at the copper center is well defined. The bridge between copper and zinc is broken in all reduced isoenzymes examined to date. His-63 is protonated on reduction. This proton is within the van der Waals distance of copper(I) and is strategically located to be involved in the catalytic mechanism, presumably by interacting with O2 An H bond between His-63 and Og could be responsible for the attraction of an electron from copper (I) to Og, with the subsequent formation of HOg. 

Scheme 4 Proposed catalytic mechanism of PHM and D/3M showing the reactive ternary complex. Proposed structure of the intermediate formed after reaction of Cub(H)-02 with substrate to form a substrate-derived free radical and Cub(11)-OOH. This illustrates a possible pathway for electron transfer from QiaCI) to Cub(H)-OOH throngh the solvent-filled cleft and the changes in copper ligation that accompany oxidation. With the exception of reactive intermediates, the water molecules complexed to the copper sites have been omitted. (Ref 27, Reproduced by permission of American Society for Biochemistry and Molecular Biology)...

These data have led to the development of a catalytic mechanism, shown in Scheme 6, that has been further refined by kinetic isotope effect (KIE) experiments. Substrate binds to Cu(II), replacing bound solvent. The metal coordination facilitates the deprotonation of the substrate hydroxyl group. The proton is transferred to Tyr495, which dissociates from copper. The temperature and pH dependence of the visible absorption and circular dichroism spectra indicate that galactose oxidase exists as an equilibrium of the Tyr495-Cu(II) form (TyroN) and the protonated Tyr495 state. 

In contrast to the sMMO, the pMMO has been only poorly characterized. This enzyme is extremely unstable and has never been purified. However, induction experiments suggest that three major membrane polypeptides are involved in this enzyme and that at least one contains copper (6) (Table II). The pMMO has a more restricted substrate range, and oxidizes only straight-chain alkanes and alkenes. This enzyme has been reported to have a lower apparent Km (half-saturation constant or Michaelis constant) for methane than the sMMO (7, 8), and so the two enzymes differ with respect to components, kinetics, and, presumably, catalytic mechanism. 

The postulated catalytic mechanism of amine oxidases starts from the qui-none form of the cofactor (Fig. 17). The distal oxygen atom is replaced by an amino group in a transamination reaction. The amine is then re-oxidized by molecular oxygen to the original quinone. The copper ion is not involved directly in catalysis but is only a cofactor in the synthesis of TOPA quinone (Fig. 18). 

Fig. 27. The catalytic mechanism of Cu, Zn-superoxide dismutase Oj displaces the axial water molecule of Cu2+ reducing it to Cu+. Protonation of the Cu02 complex liberates 02 and dissolves the ligand bond between His61 and copper. A second -Oj binds to copper, oxidizing it to Cu2+. Protonation produces H202, whereas deprotonation of His61 reestablishes the Cu-His61 bond, releasing H202. From Getzoff et al. 1983 [229] with permission...

Dioxygen binds to Cub with an end-on r geometry in the precatalytic complex (Figure 19). This geometry is compatible with dioxygen or superoxide bound to copper, but not with Cu-peroxo species. The catalytic mechanism of PHM will be discussed in the next section because the reaction schemes of PHM and D/3M are very similar. 

The catalytic mechanism of tyrosinase was first studied in detail by Solomon et al Solomon proposed a mechanism for both the cresolase and catecholase activities of tyrosinase (Figure 25). This mechanism suggests the oxy state to be the starting point of cresolase activity (inner circle). This state is present in the resting form of tyrosinase in a proportion of about 15% (85% met state). A monophenol substrate binds to the oxy state and is monooxygenated to o-diphenol. This diphenol subsequently binds to the copper center of met tyrosinase in a... 

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