Ascorbate oxidase domains

Fig. 10. (a) Copper site in domain 1 of ascorbate oxidase, (b) Ribbon drawing of tbe ascorbate oxidase domain 1 backbone, (c and d) Schematic of ascorbate oxidase domain 1 topology. 

Fig. 11. (a) Ribbon drawing of the ascorbate oxidase domain 2 backbone, (b and c) Schematic of ascorbate oxidase domain 2 topology. 

Fig. 2. The binding of Cu s in ascorbate oxidase. A section of the polypeptide (plus side chains), and position of the type 1 Cu in a plastocyanin like domain (to the right) are shown. The type 3 Cu s are attached at what corresponds to the remote site. Residues HisS07 and CysSOS are analogous to Tyr83 and Cys84 in plastocyanin...

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... 

With the structure of ascorbate oxidase in hand, a new structurally based alignment of the sequences of ascorbate oxidase, laccase, and ceruloplasmin has been performed (Messerschmidt and Huber, 1990). In brief, while gene triplication for ceruloplasmin is still revelant, its sequence can be further subdivided into two domains per unit of triplicated sequence, or six domains in total. Each of these sequences bears some resemblance to each of the three domains of ascorbate oxidase, as does each of the two domains in laccase. The coppers of the trinuclear site of ceruloplasmin then are predicted to be bound between domains 1 and 6, with a type I site also lying in both domains 6 and 4 (see Huber, 1990). The relative orientation of each of these domains is not predicted by this alignment, but it turns out that the structure of nitrite reductase may shed some light on this (see Section V,C). 

Other [3-Sheet Proteins. Influenza neuraminidase contains a very unusual [I-sheet propellor-like structure. Ascorbate oxidase (Figure 12. le) is a large / -sheet protein that contains parallel / -sheet domains, and interleukin-1/1 may be described as containing both a barrel and a propeller. 

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... 

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 second class consists of multidomain blue copper proteins composed of exclusively two or more BCB domains and includes nitrite reductase (Section IV, E), multicopper blue oxidases such as laccase, ascorbate oxidase, ceruloplasmin, and hephaestin (Section VII), and some sequences found in extreme halophilic archaea (see Section V, E). 

Multicopper blue oxidases are synthesized as a single polypeptide chain, which is composed of three BCB domains in the case of laccases (LC) and ascorbate oxidases (AO) and six such domains in ceruloplasmin (CP) and hephaestin (HP). Structurally they are arranged in a triangular manner. These enzymes, along with heme-copper oxidases (cytochrome c oxidases and quinol-oxidases) and a cyanide-resistant alternative oxidase found in mitochondria of plants and fungi, are the only known enzymes capable of catalyzing four-electron reduction of dioxygen to water. In the... 

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. 

Fig. 2. A topology/packing diagram of the domains of the ascorbate oxidase monomer compared with plastocyanin and azurin. Each /3-strand is represented by a triangle whose apex points up or down depending on whether the strand is viewed from the C or N terminus, a-helices are represented by circles.

Fig. 3. Main-chain hydrogen bonding scheme of the seven /3-sheets in ascorbate oxidase. (a) Domain 1. (b) Domain 2 wide /3-bulge x— 264, 1 —> 238, 2 — 239. (c) Domain 3 classic /3-bulge x — 492, 1 - 457, 2 - 458. The amino acid residues are indicated by the one-letter code.

The mononuclear copper site is located in domain 3 and has the four canonical type-1 copper ligands (His, Cys, His, and Met) also found in plastocyanin and azurin. It is coordinated to the NDl atoms of His 445 and 512, the SG atom of the Cys 507, and the SD atom of Met 517 in a distorted trigonal pyramidal geometry. The SD atom is at the long apex (see Fig. 4). Bond lengths of the type-1 copper for both subunits are displayed in Table III. They are compared with oxidized poplar plastocyanin (95) and azurin from Pseudomonas aeruginosa (96). Figure 5 shows an overlay of the type-1 copper site in azurin, plastocyanin, and ascorbate oxidase. 

Fig. 8. (a) Drawing of the trimer of nitrite reductase from Achromobacter cycloclastes. (b) Drawing of the interface between domain 1 (subunit A) and domain 2 of the adjacent symmetry-related molecule (subunit C) of nitrite reductase from A. cycloclastes. (c) Drawing of domain 1 and 3 of ascorbate oxidase. The type-1 copper is in domain 3 and the trinuclear copper center is between domain 1 and domain 3. The domains have an orientation similar to that of the corresponding domains of the nitrite reductase shown in b. The figure was produced by the RIBBON Program (S7). 

The similarity matrix calculated in Messerschmidt and Huber (202) indicates clearly the six-domain structure of ceruloplasmin and three-domain structures for laccase and ascorbate oxidase. The internal triplication within the ceruloplasmin amino-acid sequence is reflected by values of about 60% difference. Comparison of both the N-terminal domains and the C-terminal domains of the blue oxidases indicates, respectively, a relationship that is closer and relevant values for percent difference that are significantly lower than those for other comparisons. This might reflect the requirements for the trinuclear copper site. The lowest values of about 70 to 73% difference are observed for both N-terminal and C-terminal domains of laccase and ascorbate oxidase, showing that the two oxidases are more closely related to ceruloplasmin than either of them. 

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. 

A structurally based amino-acid sequence alignment strongly suggests a three-domain structure for laccase, closely related to ascorbate oxidase, and a six-domain structure for ceruloplasmin. These domains demonstrate homology with the small blue copper proteins. The relationship suggests that laccase, like ascorbate oxidase, has a mononuclear blue copper in domain 3 and a trinuclear copper between domain 1 and domain 3, and ceruloplasmin has mononuclear copper ions in domains 2, 4, and 6 and a trinuclear copper between domain 1 and domain 6. 

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