Plastocyanin, domain structure

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. 

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... 

Crystallographic analysis has provided us with a detailed structure of hCp on the other hand, essentially all of the structure-function analyses have been done on FetSp. Also, except for the copper site structural homology, the two proteins are quite different. hCp is composed of six plastocyanin-like domains (plastocyanin is a type 1 copper-containing protein) that are arranged in a trigonal array (Zaitseva et al., 1996). One result of this domain replication is a conformational fold that produces a distinct, negatively charged patch on the protein surface adjacent to the catalytically active type 1 Cu(II). This copper atom is in domain 6. (Domains 2 and 4 contain type 1-like copper sites that do not participate in the ferroxidase reaction.) Lindley et al. (1997) have proposed that this... 

The first class is cupredoxins—single-domain blue copper proteins composed of only one BCB domain. These proteins include plastocyanin, azurin, pseudoazurin, amicyanin, auracyanins, rusticyanin, halocyanin, and sulfocyanin (see Section IV). Plantacyanin of the phytocyanin family (Section V), subunit II of the cytochrome c oxidase, and the recently characterized nitrosocyanin also fall into this class. The last two are single BCB domain polypeptides closely related structurally to cupredoxins, but harboring, respectively, a binuclear copper site known as CuA and a novel type of copper-binding site called red (see Sections IX and X). 

Fig. 5. The complex of cytochrome/and plastocyanin as determined by paramagnetic NMR (PDB Accession Code 2PCF). The solution structure of Spinacia oleracea plastocyanin was determined by NMR, while cytochrome f was modeled from the previous crystal structure of the soluble domain of Brassica rapa cytochrome / (PDB Code ICTM), with only the contacts between the two proteins determined by NMR. The distance shown is between the heme Fe atom in cytochrome / and the eN of the His-87 copper ligand in plastocyanin.

In contrast, spinach plastocyanin binds to the soluble domain of its physiological partner cytochrome/ in a single orientation, indicating a short electron transfer path between the metal ions (Ubbink et al., 1998). A low-resolution structural model of the plastocyanin-cytochrome/complex was obtained by including paramagnetic constraints (derived from H and chemical shift differences) in molecular dynamics simulations where the structures of the two partners were kept rigid (Ubbink et al.,... 

At this writing, the three-dimensional sttuctures of eight different naturally occurring type 1 copper proteins are known. These include the cupredoxins plastocyanin at 1.33 A resolution (pdb code 1 PTC), azurin at 1.8 A (pdb code 2AZA), pseudoazurin at 1.55 A (pdb code IPAZ), amicyanin at 1.3 A (pdb code lAAC), auracyanin at 1.55 A (pdb code IQHQ), rusticyanin at 1.9 A (pdb code IRCY), and the phytocyanins cucumber basic protein at 1.8 A (pdb code2CBP), and stellacyanin at 1.6 A (pdb code IJER) Atomic coordinates for these and all other single-domain type 1 copper proteins are available from the Research Collaboratory for Structural Bioinformatics (RCSB) Protein Data Bank (PDB) and can be accessed online at www.rcsb.org/pdb/. 

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 subsequent measurement of the structure of crystals formed from the 252-residue, lumen-side domain at 1.96 A by Martinez et revealed the presence of an L-shaped array of five water molecules embedded near the heme, as shown in Fig. 6 (D), left, and in an enlarged view for the water chain and its heme environment in Fig. 6 (D), right. The water chain extends in two directions from ligand His-25. The longer branch extends 11 A in the direction of Lys-66, which is known to be in the basic patch at the top of the domain and has been implicated in the interaction with the electron acceptor, plastocyanin. The authors suggested that the water chain may function as an exit port on the lumen side for protons translocated by the Cyt b(f complex. 

Structure of the copper centers. The mononuclear type 1 copper center (CU1) is located in domain 3 and is coordinated by His 445 (N1), His 512 (N1), Cys 507 (S), and Met 517 (S). These ligands form a distorted trigonal pyramid, similar to the type 1 copper center of plastocyanins (Table 11). In the latter, as in the... 

Fig. 39 a, b. Phylogenetic trees of the structural domains of a laccase and ascorbate oxidase b ceruloplasmin. Plastocyanin is the reference protein in both cases. Lac/Aox 1-3 N- to C-ter-minal domains of laccase/ascorbate oxidase. Cpn 1-6 N- to C-terminal domains of ceruloplasmin. According to Ryden and Hunt 1993 [71]... 

Type 1 copper proteins are the class of proteins for which cupredoxins were originally named. Type 1 copper proteins include both proteins with known electron transfer function (e.g., plastocyanin and rusticyanin), and proteins whose biological functions have not been determined conclusively (e.g., stellacyanin and plantacyanin). Although these proteins with unknown function cannot be called cupredoxins by the strict functional definition, they have been classified as cupredoxins because they share the same overall structural fold and metal-binding sites as cupredoxins. In addition, many multidomain proteins, such as laccase, ascorbate oxidase, and ceruloplasmin, contain multiple metal centers, one of which is a type 1 copper. Those cupredoxin centers are also included here. Finally, both the Cua center in cytochrome c oxidase (CcO) and nitrous oxide reductase (N2OR), and the red copper center in nitrocyanin will be discussed in this chapter because their metal centers are structurally related to the type 1 copper center and the protein domain that contains both centers share the same overall structural motif as those of cupredoxins. The Cua center also functions as an electron transfer agent. Like ferredoxins, which contain either dinuclear or tetranuclear iron-sulfur centers, cupredoxins may include either the mononuclear or the dinuclear copper center in their metal-binding sites. 

The multicopper NiR is a homotrimer, in which each monomer contains two domains.On the other hand, AO and Lc contain three domains while human Cp contains six domains. Each domain of these enzymes has a typical cupredoxin fold. The blue copper center resides in the first domain of NiR, the third domain of AO and Lc, and the second, fourth, and sixth domains of human Cp. The blue copper centers in domain 1 of NiR (see Structure C in Figure 2), domain 3 of AO and in domains 4 and 6 of human Cp are quite similar to that in plastocyanin, consisting of Cu (NHis)2ScysSMet in a flattened tetrahedral geometry. While the distance of Cu -S (Met) is shorter for the blue copper center in NiR (2.62-2.64 A) than in plastocyanin, the distances in AO and Cp (2.8-3.0 A) are unusually longer (see Table 4). Further-... 

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