Redox reactions galactose oxidase

Galactose oxidase is an extracellular enzyme secreted by the fungus Dactylium den-droides. It is monomeric (M = 68000), contains a single copper site and catalyses the oxidation of a wide range of primary alcohols to the corresponding aldehydes. The two-electron transfer reaction RCH2OH - RCHO + 2H+ + 2e does not utilise a Cu(III)/Cu(I) couple, but a second redox site, involving a tyrosine radical which mediates the transfer of the second electron. 

The simple coordination chemistry characteristic of the majority of protein-metal interactions is replaced in certain cases by irreversible covalent modifications of the protein mediated by the metal ion. These modifications are essential for the function and are templated by the structure of the protein, as no other proteins are required for the reaction to occur. These self-processing reactions result in the biogenesis of redox cofactors in some enzymes (amine oxidases, galactose oxidase, cytochrome c oxidase) and activation of hydrolytic sites in others (nitrile hydratase). The active sites of all of these enzymes are bifunctional, directing not only the catalytic turnover reaction of the mature enzyme but the modification steps required for maturation. 

The principle of potential-dependent selectivity control of a galactose oxidase membrane (Johnson et al., 1982, 1985) using galactose, raffi-nose, and dihydroxyacetone as substrates has been discussed in Section 2.2.2. The change of the redox potential influences all reactions in the same direction. However, since the activity is different towards different substrates the degree of conversion of a particular substrate can be affected by the applied potential. At first the better substrates, e.g. raffinose or galactose, can be measured and at optimal potential all substrates are detected. 

Type A PCET reactions describe amino acid radical generation steps in many enzymes, since the electron and proton transfer from the same site as a hydrogen atom [188]. Similarly, substrate activation at C-H bonds typically occurs via a Type A configuration at oxidized cofactors such as those in lipoxygenase [47, 48] galactose oxidase [189-191] and ribonucleotide reductase (Y oxidation at the di-iron cofactor, vide infra) [192]. Here, the HATs are more akin to the transition metal mediated reactions of Section 17.3.1 since the final site of the electron and proton are on site differentiated at Ae (redox cofactor) and Ap (a ligand). 

Since both alcoholic oxidation and O2 reduction are two-electron processes, the catalytic reaction is conceptually equivalent to a transfer of the elements of dihydrogen between the two substrates. Biological hydrogen transfer generally involves specialized organic redox factors (e.g., flavins, nicotinamide, quinones), with well-characterized reaction mechanisms. Galactose oxidase does not contain any of these conventional redox factors and instead utilizes a very different type of active site, a free radical-coupled copper complex, to perform this chemistry. The new type of active site structure implies that the reaction follows a novel biochemical redox mechanisms based on free radicals and the two-electron reactivity of the metalloradical complex. 

Mechanism. Since its discovery in 1959 (2), galactose oxidase has attracted considerable interest in the scientific community because of its enigmatic catalytic mechanism. Various aspects of early research have been discussed in two excellent reviews (J, 4), The most puzzling property of this enzyme has been the ability of the monomeric protein to carry out two-electron redox reactions without any apparent involvement of a cofactor. Although a number of elegant explanations of this phenomena have been proposed (3, 5), the mechanism still remains elusive. On the... 

Besides the large group of hydrolytic enzymes, metal ions are often present in enzymes, which catalyze redox processes. Nature provides a large number of oxidoreductases, which catalyze diverse reactions. Many of them are copper enzymes that use O2 as the ultimate oxidant. A prominent example for such a type-2 copper enzyme is galactose oxidase. The structure of galactose oxidase and its mechanism... 

Galactose oxidase (GO) is a mononuclear radical-coupled copper enzyme which catalyzes the oxidation of primary alcohols using molecular oxygen, producing aldehydes and H2O2. Galactose oxidase can be present in three oxidative states (6,7) The active oxidized form with a tyrosine radical in the active site, the reduced form which results from the two electron redox reaction by which a primaiy alcohol is converted to an aldehyde (Cu, tyrosine), and an intermediate semi-form containing tyrosine. The three oxidation states can be defined as in equation I. 

Two phenoxyl radical complexes [Cu (2 )N03] and [Zn (2 )N03] oxidize benzyl alcohol to benzaldehyde and have been studied as models for the enzyme galactose oxidase (GO). GO contains a dipeptide unit (3) in which a tyrosine residue is covalently bound to an adjacent cysteine residue and which is similar to (2), the tyrosyl radical in (3) also being bound to the Cu centre (see Figure 1). Second-order kinetics were observed with respect to [Zn°(2 )N03]+ and there was no evidence of redox reaction at the zinc site, suggesting that a dimeric form of the complex is active however, the reaction of [Cu H2 )N03]+ with benzyl alcohol is first order in the metal complex and [Cu (2H)]+ is identified as a product, suggesting a formal 2e /2H+ mechanism in which the monomeric form coordinates the alcohol in the manner believed to operate for G0. 2... 

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