Galactose oxidase structure

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... 

There are a number of excellent sources of information on copper proteins notable among them is the three-volume series Copper Proteins and Copper Enzymes (Lontie, 1984). A review of the state of structural knowledge in 1985 (Adman, 1985) included only the small blue copper proteins. A brief review of extended X-ray absorption fine structure (EXAFS) work on some of these proteins appeared in 1987 (Hasnain and Garner, 1987). A number of new structures have been solved by X-ray diffraction, and the structures of azurin and plastocyanin have been extended to higher resolution. The new structures include two additional type I proteins (pseudoazurin and cucumber basic blue protein), the type III copper protein hemocyanin, and the multi-copper blue oxidase ascorbate oxidase. Results are now available on a copper-containing nitrite reductase and galactose oxidase. 

In early 1990 it became apparent that the structure of galactose oxidase from Dactylium dendroides was about to emerge. A 2.5 A multiple isomor-phous replacement (MIR) map based on area detector data from a native and three derivative crystals yielded a polypeptide chain tracing. The refined structure at 1.9 A (R = 0.179) (Ito et al., 1991) shows that galactose oxidase consists of three domains, each of which is predominandy jS... 

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...

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... 

Nonblue. These include galactose oxidase (GO) and amine oxidases (e.g., plasma amine oxidase, diamine oxidase, lysyl oxidase), which produce dihydrogen peroxide by the two-electron reduction of 02 [33], For GO (stereospecific primary alcohol oxidation), spectroscopic studies by Whittaker [70,71] suggest that the two-electron oxidation carried out by a mononuclear copper center is aided by a stabilized ligand-protein radical (i.e., (L)Cu(I) + 02 —> (L +)Cu(lI) + H202), obviating the need to get to Cu(III) in the catalytic cycle. Protein x-ray structures [33,72] reveal a novel copper protein cofactor, which would seem... 

The classification introduced in this review (type I-type IV) should cover all structural types of copper sites known to date. For instance, based on this nomenclature, ascorbate oxidase contains type I and type IV, and nitrite reductase contains type I and type II (more precisely, type IIA). Galactose oxidase has a type IIB site. 

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... 

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). 

Figure 3. Sketch of the copper coordination in galactose oxidase, as determined by the 1.7 A resolution crystal structure.

Figure 4. Drawing of the stacking interaction between Trp 290 and the thioether bond formed by Cys 228 with Tyr 272 in galactose oxidase as determined from the crystal structure.

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