Crystallization ascorbate oxidase

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 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... 

Two crystalline forms of ascorbate oxidase from zucchini (Messerschmidt et al., 1989) have been analysed at 2.5 A resolution and a model of the polypeptide chain and the copper ions and their ligands has been prepared. The crystal forms M2 and Ml contain a dimer of 140000 Mr and a tetramer of 280000 Mr in the asymmetric unit. Each subunit of about 550 amino acid residues has a globular shape with dimensions of 49 A x 53 A x 65 A. The subunit has three domains arranged sequentially... 

The crystal structure of the resting form of ascorbate oxidase from zucchini has recently been refined to 1.9 A resolution (Messerschmidt et al., 1992) the subunits of 552 residues (70000 Mr) are arranged as tetramers with D2 symmetry. Asp 92 is the attachment site for one of the two N-linked sugar moieties, which has the defined elec-... 

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. 

Preliminary observation of additional electron density at this fourth coordination position of Cu-2 upon soaking crystals with N02 is consistent with this idea. Thus, from the structural data it would appear that Cu-1 is a type 1 center that functions to transfer electrons to the catalytic Cu-2 ion (See Note Added in Proof). It has been suggested, largely on the basis of electronic structural considerations (27, 28), that the Cys-136-His-135 link between Cu-1 and Cu-2 is a possible conduit for electron transfer between the two sites. An analogous dipeptide bridge between the type 1 center and the catalytic tricopper cluster in ascorbate oxidase (29, 30) may function similarly. Indeed, other close similarities between protein domains in ascorbate oxidase and NiR have been noted (17). 

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. 

Contact surface areas for the monomer-monomer interactions within the homotetramer present in both crystal forms were calculated using the algorithm of Lee and Richards (94). It is evident from these calculations (74) that the contact surface areas between the two dimers about the crystallographic dyad are by far the largest. Ascorbate oxidase... 

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. 

Crystals of native oxidized ascorbate oxidase were anaerobically dialyzed in microcells against 50 mM sodium phosphate buffer, pH 5.2, containing 25% (v/v) methylpentanediol (MPD), 1 mM EDTA, 2 mM dimethyl-glyoxime (DMG), and 5 mM ferrocyanide for 7 and 14 hr. 

Native crystals of ascorbate oxidase were soaked in harvesting buffer solution (50 mM sodium phosphate, 25% MPD, pH 5.5) containing... 

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... 

Laccases (p-diphenol O2 oxidoreductase EC 1.10.3.2) catalyze the oxidation of p-diphenols with the concurrent reduction of dioxygen to water. However, the actual substrate specificities of laccases are often quite broad and vary with the source of the enzyme [116,117]. Laccases are members of the blue copper oxidase enzyme family. Members of this family have four cupric (Cu +) ions where each of the known magnetic species (type 1, type 2, and type 3) is associated with a single polypeptide chain. In the blue copper oxidases the Cu + domain is highly conserved and, for some time, the crystallographic structure of ascorbate oxidase, another member of this class of enzymes, has provided a good model for the structure of the laccase active site [124,125]. The crystal structure of the Type-2 Cu depleted laccase from Coprinus cinereus at 2.2. A resolution has also been elucidated [126]. 

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. 

The blue oxidases-related enzymes include phenoxazinone synthase from S. antibioticus This enzyme is a copper-containing oxidase that catalyzes the coupling of 2-aminophenols to form the 2-aminophenoxazinone chromophore. This reaction constitutes the final step in the biosynthesis of the potent antineoplastic agent actinomycin. The crystal structure of the oxidized form phenoxazinone synthase from S. anibioticus has been determined. It has been solved in his hexameric form. One monomer is very similar to Iaccase or ascorbate oxidase but it contains a long loop, which connects two domains and stabilizes the hexameric structure. Bound... 

Messerschmidt, A., Rossi, A., Landenstein, R., Huber, R., Bolognesi, M., Gatti, G., Marchesini, A., Petruzzelli, R. and Finazzi Agro, A. 1989. X-ray crystal structure of the blue oxidase ascorbate oxidase from zucchini. Journal of Molecular Biology 206, 513 -529. 

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. 

Figure 9 Crystal structure of the active site of resting (oxidized) ascorbate oxidase from Cucurbita pepo...

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