Galactose oxidase radical sites

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. 

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... 

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 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. 

Galactose oxidase exhibits a mononuclear copper-active site, which is flanked by a tyrosinyl radical. In a single step, a two-electron oxidation of alcohols can be performed by this enzyme, where one electron is extracted by copper and the other by the tyrosine residue [19]. 

The involvement of a free radical in the active site of galactose oxidase is not obvious and was, in fact, overlooked for many years. As mentioned earher (Section V, C), the Cu(II) ion strongly interacts with the radical in the active enzyme (AGO), and the normal spectroscopic signatures of a free radical are absent. The strong interactions between the radical and the metal ion result in an EPR-silent complex and an unusual absorption spectrum that is clearly not a simple superposition of spectra for Cu(II) and a free radical (see below). However, a distinctive and unusual free radical EPR signal is consistently present as a minority species in EPR spectrum of oxidant-treated GAOX. This same feature is produced when... 

Babcock, G. T., El-Deeb, M. K., Sandusky, P. O., Whittaker, M. M., and Whittaker, J. W., 1992, Electron paramagnetic resonance and electron nuclear double resonance spectroscopies of the radical site in galactose oxidase and of thioether-substituted phenol model compounds, J. Am. Chem. Soc. 114 372793734. 

Whittaker, J. W., 1994, The free radical-coupled copper active site in galactose oxidase. Metal Ions in Biology, Editors Sigel, H. and Sigel, A., Marcel Dekker publishers, 30 3159 360. 

Zurita, D., Gautier-Luneau, L, MEnage, S., Pierre, J.-L., and Saint-Aman, E., 1997, A First Model for the Oxidized Active Form of the Active Site of Galactose Oxidase a Free Radical Copper Complex, J. Biol. Inorg. Chem. 2 46n55. 

Galactose oxidase is a monomer composed of a single polypeptide chain (Mr = 68 kD), with two disnlfide bridges, and a single copper ion that is essential for catalytic activity. A second redox center in the active site is provided by a tyrosyl radical (Tyr272). The enzyme as isolated is a mixture of active and inactive forms. The inactive form of the enzyme has a typical Type 2 EPR signal... 

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). 

Galactose oxidase, as well as the other non-blue oxidases, avoids the necessity of an external cofactor by creating an internal cofactor via modification of a residue of its own peptide chain. The amino acid residue which is modified in all cases is a tyrosine. While the amine oxidases produce TOPA quinone, galactose oxidase forms a thioether bond between the tyrosine and a cysteine residue. This modified tyrosine is the site which carries the free radical. 

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