Ray Structure of Ascorbate Oxidase

There have been several attempts to crystallize laccase from different species by various groups without any encouraging results, and these are therefore not documented in the literature. The main reason for this failure is very likely the heterogeneity introduced in the glycosyla-tion of the laccase. 

The first crystals of ascorbate oxidase were grown by Ladenstein et al. (85) in a final 1.2 M sodium-potassium phosphate buffer, pH 7.0. They were orthorhombic, space group P2i2,2i with a = 190.7 k, b = 125.2 A,c = 112.3 A, and two molecules (four subunits) per asymmetric unit. Some years later Bolognesi et al. (86) obtained a different crystal form of ascorbate oxidase with 2-methyl-2,4-pentane-diol as precipitant. The crystals were orthorhombic as well, space group P2j2j2 with a = 106.7 A, b = 105.1 A, c = 113.5 A, and one molecule (two subunits) per asymmetric unit. In both cases, the protein material was prepared from the peels of green zucchini squash (C.pepo medullosa). The preliminary three-dimensional X-ray structure of ascorbate oxidse, based on 

The subunits are arranged in the crystals as homotetramers with D2 symmetry. The structure of a subunit is shown schematically in Fig. 1 (87). Each subunit of 552 amino acid residues has a globular shape with dimensions of 49 x 53 x 65 A and is built up of three domains arranged sequentially on the polypeptide chain, tightly associated in space. The folding of all three domains is of a similar jS-barrel type. It is distantly related to the small blue copper proteins, for example, plastocyanin or azurin. Domain 1 is made up of two four-stranded jS-sheets (Fig. lb), which form a jS-sandwich structure. Domain 2 consists of a six-stranded and a five-stranded jS-sheet. Finally, domain 3 is built up of two five-stranded jS-sheets that form the jS-barrel structure and a four-stranded j8-sheet that is an extension at the N-terminal part of this domain. A topology diagram of ascorbate oxidase for all three domains and of the related structures of plastocyanin and azurin is shown in Fig. 2. Ascorbate oxidase contains seven helices. Domain 2 has a short a-helix (aj) between strands A2 and B2. Domain 3 exhibits five short a-helices that are located between strands D3 and E3 (a ), 13 and J3 (a ), and M3 and N3 (a ) as well as at the C terminus (ag and a ). Helix 2 connects domain 2 and domain 3. 

A comparison of the different variants of the jS-barrel domain structure in Fig. 1 shows that domain 1 of ascorbate oxidase has the simplest /3-barrel with only two four-stranded /8-sheets. Plastocyanin and azurin are quite similar but between strands 4 (El) and 6 (FI) they have insertions of one strand (plastocyanin) or one strand and an a-helix (azurin). Domain 2 has one additional strand H2 in sheet D next to strand E2 (sheet B and strand El in domain 1) and two additional strands, F2 and G2, in sheet C next to strand 12 (sheet A and strand FI in domain 1). Domain 3 resembles domain 2 except for the insertion of the short a-helices and the addition of the four-stranded /8-sheet at its N terminus. 

The mononuclear copper site is located in domain 3 and the trinuclear copper species is bound between domain 1 and domain 3 (see Fig. la). The copper site geometries will be discussed later. 

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Occurrence, Sequences, and Biological Function Molecular and Spectroscopic Properties X-Ray Structure of Ascorbate Oxidase... 

This chapter will concentrate mainly on structural and functional aspects of these enzymes with the major emphasis on ascorbate oxidase and laccase. Significant progress has been achieved in the last 10 years the determination of amino acid sequences of all three enzymes, each from several sources, and the X-ray structure of ascorbate oxidase. The new information forms the basis of a much deeper understanding of the function of the enzymes as will be demonstrated in this chapter. 

It turns out from the recently determined X-ray structure of ascorbate oxidase (73, 74) that the nonblue EPR-active type-2 copper together... 

Various spectroscopic methods have been used to probe the nature of the copper centers in the members of the blue copper oxidase family of proteins (e.g. see ref. 13). Prior to the X-ray determination of the structure of ascorbate oxidase in 1989, similarities in the EPR and UV-vis absorption spectra for the blue multi-copper oxidases including laccase and ceruloplasmin had been observed [14] and a number of general conclusions made for the copper centers in ceruloplasmin as shown in Table 1 [13,15]. It was known that six copper atoms were nondialyzable and not available to chelation directly by dithiocarbamate and these coppers were assumed to be tightly bound and/or buried in the protein. Two of the coppers have absorbance maxima around 610 nm and these were interpreted as blue type I coppers with cysteine and histidine ligands, and responsible for the pronounced color of the protein. However, they are not equivalent and one of them, thought to be involved in enzymatic activity, is reduced and reoxidized at a faster rate than the second (e.g. see ref. 16). There was general concurrence that there are two type HI... 

The type 1-3 terminology to distinguish different Cu protein active sites remains extremely useful. Sub-groupings are appearing however in all three categories particularly in the case of the binuclear (EPR inactive) type 3 centers. Thus, in the recently determined X-ray crystal structure of ascorbate oxidase the type 3 and type 2 centers are present as a single trimer unit [. A discrete binuclear type 3 center is, however, retained in hemocyanin [6]. 

While there is at present no full understanding as to why plastocyanin should require two sites for reaction, there is now much evidence detailing this two-site reactivity. Moreover, the recent X-ray crystal structure of ascorbate oxidase (which has 4 Cu atoms per molecule) has indicated a plastocyanin-like domain, with the two type 3 Cu s (in close proximity with the type 2 Cu) located at the remote site. Fig. 2 [5]. Since electrons are transferred, from the type 1 Cu to O2 bound at the type 3 center this structure defines two very similar through-bond routes for biological electron transfer. 

The recent X-ray crystal structure of ascorbate oxidase [6] has indicated the relative positions of type 1, 2 and 3 Cu centers. The type 1 center is in a plastocyanin like domain, and is the primary acceptor of electrons from substrate. The shortest pathway for electron transfer from the type 1 to type 3 Cu s is the bifurcated path via Cys508 and either His 507 or His509. The two histidines are part of the plastocyanin-like domain, and serve also to coordinate the type 3 Cu s, Fig. 2. The His507 to Cys508 bonding is similar to that of Tyr83... 

This progress is mainly due to the determination of the amino-acid sequences for all members of this group and the X-ray crystal structure of ascorbate oxidase. The three-dimensional structure of ascorbate oxidase showed the nature and spatial arrangement of the copper centers and the three-domain structure. However, modern spectroscopic techniques (e.g., low-temperature MCD and ENDOR) made invaluable contributions as well. 

The X-ray crystal structure of ascorbate oxidase (21) defines a route for electron transfer from the T3qie 1 Cu center to the Cus site via connecting Cys and His residues. The Type 1 domain has structural... 

Messerschmidt, A., Landenstein, R., Huber, R., Bolognesi, M., Avigliano, L., Petruzzelli, R., Rossi, A. and Finazzi Agro, A. 1992. Refined crystal structure of ascorbate oxidase at 1.9 A resolution. Journal of Molecular Biology 224, 179-205. Miki, K., Ezoe, T., Masui, A., Yoshisaka, T., Mimuro, M., Fujiwara-Arasaki, T. and Kasai, N, 1990, Crystallization and preliminary X-ray diffraction studies of C-phycocyanin from a red alga, Porphyra tenera. Journal of Biochemistry 108, 646-649. Molecular Probes. Handbook of fluorescent probes and research chemicals. 1992-1994. 

Messerschmidt, A., Luecke, H., and Huber, R. (1993). X-ray structures and mechanistic implications of three functional derivatives of ascorbate oxidase from zucchini./. Mol. Biol 230, 997-1014. 

E. X-Ray Structure of the Azide Form of Ascorbate Oxidase The Catalytic Mechanism... 

X-ray crystal structures of four functional derivatives of ascorbate oxidase were determined (149,150). The results of these investigations and implications for the catalytic mechanism of the blue oxidases will be outlined in the next section. 

A tentative catalytic mechanism of ascorbate oxidase has been proposed based on the refined X-ray structure and on spectroscopic and mechanistic studies of ascorbate oxidase and the related laccase. The results of these studies have been discussed in detail (74). The X-ray structure determinations of the fully reduced and peroxide derivatives define two important intermediate states during the catalytic cycle. A proposal for the catalytic mechanism incorporating this new information is given in Messerschmidt et al. (150) and presented in Fig. 14. This scheme should be valid in principle also for laccase due to the close similarities of both blue oxidases. 

X-ray structures of functional derivatives of ascorbate oxidase provided pictures of intermediate states, which will probably be passed during the catalytic cycle. A catalytic mechanism that is based on the available mechanistic data and these new results has been proposed. 

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. 

The low-temperature MCD and absorption titration studies (Figure 10) have determined that azide binds to both the type 2 and type 3 centers with similar binding constants. A series of chemical perturbations and stoichiometry studies have shown that these effects are associated with the same azide. This demonstrates that one N3 bridges between the type 2 and type 3 centers in laccase. These and other results from MCD spectroscopy first defined the presence of a trinuclear copper cluster active site in biology (89). At higher azide concentration, a second azide binds to the trinuclear site in laccase. Messerschmidt et al. have determined from X-ray crystallography that a trinuclear copper cluster site is also present in ascorbate oxidase (87, 92) and have obtained a crystal structure for a two-azide-bound derivative (87). It appears that some differences exist between the two-azide-bound laccase and ascorbate oxidase derivatives, and it will be important to spectroscopically correlate between these sites. 

Thereafter, crystals were brought back to the aerobic 25% MPD solution, buffered with 50 mAf sodium phosphate, pH 5.5. This procedure is based on Avigliano et al. s (157) method of preparing T2D ascorbate oxidase in solution and was modified by Merli et al. (159) for use with ascorbate oxidase crystals. The 2.5-A-resolution X-ray structure analysis by difference-Fourier techniques and crystallographic refinement shows that about 1.3 copper ions per ascorbate oxidase monomer are removed. The copper is lost from all three copper sites of the trinuclear copper species, whereby the EPR-active type-2 copper is the most depleted (see Fig. 10). Type-1 copper is not affected. The EPR spectra from polycrystalline samples of the respective native and T2D ascorbate oxidase were recorded. The native spectrum exhibits the type-1 and type-2 EPR signals in a ratio of about 1 1, as expected from the crystal structure. The T2D spectrum reveals the characteristic resonances of the type-1 copper center, also observed for T2D ascorbate oxidase in frozen solution, and the complete disappearance of the spectroscopic type-2 copper. This observation indicates preferential formation of a Cu-depleted form with the holes equally distributed over all three copper sites. Each of these Cu-depleted species may represent an anti-ferromagnetically coupled copper pair that is EPR-silent and that could explain the disappearance of the type-2 EPR signal. 

The author thanks Professors R. Huber and R. Ladenstein and other colleagues involved in the X-ray structural work on ascorbate oxidase. These results would not have been possible without their expertise, cooperation, and support. Furthermore, he is indebted to Professor E. Adman for supplying the coordinates of nitrite reductase prior to their being deposited in the data bank. 

A Messerschmidt, A Rossi, R Ladenstein, R Huber, M Bolognesi, G Gatti, A Marchesini, R Petruzelli, A Finazzi-Agro. X-ray crystal structure of the blue oxidase ascorbate oxidase from zucchini. Analysis of the polypeptide fold and a model of the copper sites and ligands. J Mol Biol 206 513-529, 1989. 

Besides the blue type-1 copper sites, the blue oxidases contain a trinuclear copper center, which is located between the N- and C-terminal domains. It can be described spectroscopically as a coupled type-2/type-3 copper center. The atomic structure of the trinuclear copper site for oxidized ascorbate oxidase as derived from X-ray crystallography is displayed in Figure 6. The trinuclear cluster has eight histidine ligands symmetrically supplied from... 

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