Galactose oxidase complexity

Fig. 15. EPR spectra for galactose oxidase complexes, (a) Oxidized enzyme (AGO) prepared by treating native galactose oxidase with K3Fe(CN)0. (b) Radical-free lAGO complex, prepared by treating native galactose oxidase with K4Fe(CN)0. Instrumental parameters microwave power, 10 [xW microwave frequency, 9.223 GHz modulation amplitude, 5 G temperature, 30 K.

A number of complexes of copper with 1,1-dithiolenes are known they are interesting, inasmuch as they form (1) polynuclear species, e.g., [Cu4(i-mnt)3]2 . Recently, a copper(III) complex of 1,1-dicarboeth-oxy-2-ethylenedithiolate (DED ) was prepared (375) by oxidation of aqueous solutions of K2[Cu(DED)2] with a 10-15% excess of Cu(II) or H202, and of (BzPh3P)2[Cu(DED)2] with I2. The possibility of this system as a model for the Cu "/Cu. system in n-galactose oxidase has been pointed out. Lewis and Miller (113) also prepared M[Cu(S2C CHN02)2] (M = Cu, or Zn) and Cu[Cu S2C C(CN)2 2], and found that they are effective insecticides. 

Copper(II) complexes with phenoxo ligands have attracted great interest, in order to develop basic coordination chemistry for their possible use as models for tyrosinase activity (dimeric complexes) and fungal enzyme galactose oxidase (GO) (monomeric complexes). The latter enzyme catalyzes the two-electron oxidation of primary alcohols with dioxygen to yield aldehyde and... 

Fialova, R, Namdjou, D.-J., Ettrich, R. et al. (2005) Combined application of galactose oxidase and beta-iV-acetylhexosaminidase in the synthesis of complex immuno-active, V-acclyl-D-galaclosamiHides. Advanced Synthesis and Catalysis, 347 (7 + 8), 997-1006. 

E S R.. Fenton. Transferrin Complexes with Non-Physiological and Toxic Metals, David M. Taylor. Transferrins, Edward N. Baker. Galactose Oxidase, Peter Knowles and Nobutoshi Ito. Chemistry of Aqua Ions of Biological Importance, David T. Richens. From a Structural Perspective Structure and Function of Manganese - Containing Biomolecules, David C. Weatherburn, Index. Volume 3,1996,304 pp. 109.50/ 70.00 ISBN 1-55938-642-8... 

Interest is mounting in this state, promoted once again by its possible implication in biological systems. Galactose oxidase, for example, is a copper enzyme which catalyses the oxidation of galactose to the corresponding aldehyde. The tervalent oxidation state may be prepared from Cu(II) by chemical, anodic and radical oxidation. Cu(III) complexes of peptides and macrocycles have been most studied, particularly from a mechanistic viewpoint. The oxidation of I" by Cu(III)-deprotonated peptide complexes and by imine-oxime complexes have a similar rate law... 

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... 

The paramagnetic copper present in the non-blue oxidases such as galactose oxidase and amine oxidases, and also in the blue oxidases, has d-d and ESR spectra typical of coordination complexes of copper(II). 

However, the Schiff base complex lacks the stability towards reduction by CN" that characterizes the Cu( II) in galactose oxidase. While the enzyme binds a single CN" even at large CN" excess (22), the Cu(II) in the model is reduced by the ligand. To assess the underlying structural components which stabilize the enzymic Cu(II) towards reduction by CN", a five-coordinate model (Figure 2) having square bipyramidal symmetry was prepared (23). (The conditions and system procedures... 

All anions which bind to the Cu(II) in galactose oxidase lower the gzz and Azz values (22). This is consistent with (but not required for) a blue shift in the d-d transitions (32, 33, 34). Fe(CN)63" is the only anion among the limited ones we have studied which produces a red shift in the optical bands (Figure 4). At 1 1, 5 1, or 100 1 molar ratios of Fe(CN)63" to enzyme the same difference absorbance spectrum is obtained, and it is consistent with complex formation between galactose oxidase and the anion. Namely, the positive difference peaks at 455, 830,... 

Hamilton and co-workers (27, 28) have suggested Cu(III) as a probable intermediate in the reaction catalyzed by galactose oxidase. Papers by Kosman and co-workers (29, 30) seem at variance with this interpretation. Regardless of the outcome of this dispute, we hope that our evidence for the existence and properties of Cu(III)—peptide complexes will encourage more investigations of the presence of trivalent copper in biological systems. Our work shows that this oxidation state is readily attained under biological conditions. 

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 hinds a single copper ion within Domain 11 on the axis of the wheel. The active site (Fig. 5) is unhke any other biological copper complex, an appropriate distinction for this remarkable enzyme. To explore the site in more detail, the protein environment of the mononuclear copper center may be separated into (A) direcdy coordinated metal hgands (hrst shell, inner sphere interactions) and (B) the extended active site environment (the second shell or outer coordination sphere). 

Fig. 7. Inner sphere of the galactose oxidase copper-binding site. Geometric details of the ligand arrangement in the aquo complex are indicated in the figure. (Based on protein coordinates PDB ID IGOG.)...

Fig. 9. Redox-active amino acid residues related to tyrosine, (a) Tyrosine, the redox center in ribonucleotide reductase, prostaglandin H synthase, and the photosynthetic oxygen evolving complex, (b) 2,4,5-Trihydroxyphenylalanine, the redox cofactor of the quinoprotein amine oxidase, (c) Tyrosine-cysteine (Tyr-Cys), the redox cofactor of galactose oxidase.

Fig. 11. Optical absorption spectra for galactose oxidase. (A) Redox-activated (AGO) complex. (B) Reductively inactivated (lAGO) complex. (C) Fully reduced (RGO) complex.

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