Galactose oxidase active site

Figure 6 Galactose oxidase active site with its coordinated redox active cofactor.

Fig. 22. A possible peroxide ghost in the galactose oxidase active site. Two crystallographically well-dehned solvent molecules (HOH 294 and HOH 703, PDB IGOG numbering) lie along the face of the active site metal complex at the base of the substrate access channel in the resting enzyme.

Fig. 24. The duncamine chelate (dnc), a structural model for the galactose oxidase active site.

Figure 3. Optimized structures for galactose oxidase active site before (A) and after (B) the proton transfer from the substrate to the axial tyrosine.

Interest in this class of coordination compounds was sparked and fueled by the discovery that radical cofactors such as tyrosyl radicals play an important role in a rapidly growing number of metalloproteins. Thus, in 1972 Ehrenberg and Reichard (1) discovered that the R2 subunit of ribonucleotide reductase, a non-heme metal-loprotein, contains an uncoordinated, very stable tyrosyl radical in its active site. In contrast, Whittaker and Whittaker (2) showed that the active site of the copper containing enzyme galactose oxidase (GO) contains a radical cofactor where a Cu(II) ion is coordinated to a tyrosyl radical. 

The function of the metal site in the oxygen-dependent radical enzymes galactose oxidase, amine oxidases, ribonucleotide reductase, and cytochrome c oxidase is inter alia to bind 02 in their reduced forms and undergo the appropriate redox chemistry to generate a metal-bound, activated oxygen species of variable nature. 

These systems are also described as normal copper proteins due to their conventional ESR features. In the oxidized state, their color is light blue (almost undetectable) due to weak d-d transitions of the single Cu ion. The coordination sphere around Cu, which has either square planar or distorted tetrahedral geometry, contains four ligands with N and/or 0 donor atoms [ 12, 22]. Representative examples of proteins with this active site structure (see Fig. 1) and their respective catalytic function include galactose oxidase (1) (oxidation of primary alcohols) [23,24], phenylalanine hydroxylase (hydroxy-lation of aromatic substrates) [25,26], dopamine- 6-hydroxylase (C-Hbond activation of benzylic substrates) [27] and CuZn superoxide dismutase (disproportionation of 02 superoxide anion) [28,29]. 

Fig.1 Structures of the active sites of galactose oxidase, catechol oxidase, and ascorbate oxidase metalloenzymes...

In contrast to the active site of galactose oxidase, to pre-catalyst 13, and to the system reported by Stack et al., the proposed catalytic species 15 does not imdergo reduction to Cu intermediates, as the oxidation equivalents needed for the catalysis are provided for solely by the phenoxyl radical Hgands. Since the conversion of alcohols into aldehydes is a two-electron oxidation process, only a dinuclear Cu species with two phenoxyl ligands is thought to be active. Furthermore, concentrated H2O2 is formed as byproduct in the reaction instead of H2O, as in the system described by Marko et al. [159]. 

There are numerous reports on the chemical synthesis of models for the active site of galactose oxidase both in the reduced Cu(l) and the oxidized Cu(II) form. We mention only a selection in which EPR is at least used to characterize the complex either on the phenoxy radical or on the copper part, typically in conjunction with X-ray data.48,49 50 A review on structural, spectroscopic and redox aspects of galactose oxidase models is available.51 More important with respect to EPR is the report on the 3-tensor calculation of the thioether substituted tyrosyl radical by ab initio methods but this is borderline to the aspects treated in this review since the copper ion is not involved.52... 

Figure 16-29 Drawing of the active site of galactose oxidase showing both the Cu(II) atom and the neighboring free radical on tyrosine 272, which has been modified by addition of the thiol of cysteine 228 and oxidation. See Halfen et al.557 Based on a crystal structure of Ito et al.558...

The CuA center has an unusual structure.130-132 It was thought to be a single atom of copper until the three-dimensional structure revealed a dimetal center, whose structure follows. The CuB-cytochrome a3 center is also unusual. A histidine ring is covalently attached to tyrosine.133-1353 Like the tyrosine in the active site of galactose oxidase (Figs. 16-29,16-30), which carries a covalently joined cysteine, that of cytochrome oxidase may be a site of tyrosyl radical formation.135... 

Figure 5.1 Schematic representations of selected active sites of the copper proteins plastocyanin [56] (type 1, a) galactose oxidase [57] (type 2, b) oxy hemocyanin [58] (type 3, c) ascorbate oxidase [10] (type 4, or multicopper site, d) nitrous oxide reductase [59] (CuA site, e) cytochrome c oxidase [15]...

Rothlisberger U, P Carloni, K Doclo, M Parrinello (2000) A comparative study of galactose oxidase and active site analogs based on QM/MM Car Parrinello simulations. J. Biol. Inorg. Chem. 5 (2) 236-250... 

Galactose oxidase can illustrate how ligands, geometry, and active site groups together provide the basis for the structure-function properties of a metal active site. Figure 10 summarizes mutual interactions... 

This approach was used to examine the redox chemistry of the Cu site in galactose oxidase (41), which had been proposed to contain an unusual Cu(III) center (52). The lack of a significant Cu K-edge energy shift between the oxidized and reduced forms of the protein demonstrated that the redox chemistry was not metal-centered and implicated another redox active site. The crystal structure of the protein subsequently revealed a novel thioether composed of a cysteine and a tyro-sinate ligand of the Cu site that is likely to be involved in the redox process (53). 

The generation, stability, and function of tyrosyl radicals in ribonucleotide reductase, PGH synthase, and galactose oxidase continue to be active areas of research. The difficulties encountered in preparing and handling these proteins, as well as in probing the physical properties and reactivity of their metal-phenoxyl radical active sites, make the preparation and investigation of stable phenoxyl radical metal model complexes an attractive goal. 

Transition metal ions with organic radicals exist in the active sites of metalloproteins. The best understood example is galactose oxidase, which features a single Cu(II) ion coordinated to a modified tyrosyl radical. Many combined experimental and theoretical studies have focused on electronic properties of metal complexes with redox active ligands, yet reactivity beyond characterization has been limited. We will demonstrate the influence of the metal complex redox state on H2 activation by anilino-phenolate noninnocent ligands. 

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