[4] This free radical ligand is a covalently cross-linked cysteine and tyrosine side chains that is formed during post-translational modification.

[1][3][7] Although the oxidation reaction of D-galactose gives galactose oxidase its name, the coupled reduction of dioxygen to hydrogen peroxide is believed to have greater physiological significance in yeasts.

One histidine (His581) of Domain 3 serves as the ligand for copper, contributing to the metal-containing active site of the enzyme.

[1][8] It contains a single copper center that adopts square planar or square-based pyramidal coordination geometry.

[3][4] The copper in the active site of galactose oxidase is described as having a "distorted square pyramidal" coordination geometry.

In the fully oxidized form of galactose oxidase, the free radical couples to the copper(II) center antiferromagnetically, supported by EPR spectroscopic studies.

[3] Secondly, the indole ring of a tryptophan (Trp290) lies above and parallel to Tyrosine-Cysteine, behaving like a shield protecting the radical from the external solvent environment.

[1][3][4] Supporting evidence comes from that mutation of this tryptophan residue leads to a lower stability of the active form of galactose oxidase.

[3] This two-electron oxidation is achieved by the double-redox site: the copper(II) metal center and the free radical, each capable of accepting one electron from the substrate.

[4] The proton on the carbon to which the hydroxyl group used to be attached is then transferred to Tyr272 (serving as the hydrogen acceptor), coupled with the oxidation of the substrate.

[4] The proton subtraction step is rate determining and stereospecific since only the pro-S hydrogen on the alcohol carbon is removed (supported by studies of its kinetic isotope effect).

[3][4] The mechanism for this tyrosine-cysteine linkage is not thoroughly understood, but a few key events have been predicted:[1] copper(I) coordinates with Tyr272 and histidines at the (future) active site.

Two possible forms of the free radical, thiyl and phenoxyl, are possible;[3] addition of thiyl radical to phenol, or addition of phenoxyl radical to thiol, generates the covalent linkage between the sulfur atom of cysteine and the aromatic ring of tyrosine;[2] A second dioxygen molecule reacts with the copper center coordinated with cross-linked tyrosine-cysteine to generate radical-copper complex.

[3] Galactose oxidase has been utilized as a biocatalyst in the synthesis of aldehydes and carboxylic acids from primary alcohols.

[4] It appears that electron-sharing between the copper and the free radical is the crucial element in the success of synthesizing these compounds.