Binuclear CuA sites

The covalent mixed-valent nature of the Cua site implied by its EPR spectrum is supported by excited-state speetroseopic data. The absorption and MCD spectra are dominated by three characteristie strong features two at 500 nm and the third at 750 nm [69,70]. The two bands near 500 nm ( 20000 cm ) originate from the Cys S t - Cu CT transitions, where x and y indicate eoordinates in the CU2S2 plane (Fig. 2a). The high intensity of these bands originates from flie covalent Cu-S bonds. This is supported by S K-edge XAS on an engineered Cua azurin construct 

The relative contributions of the direct Cu-Cu bond and the superexchange interactions to the Cua electronic structure have been evaluated via comparative studies on the Cua sites from Bacillus subtilis CcO, engineered azurin, and ami-cyanin vs. a mixed-valence model complex by Tolman and coworkers [69,73]. The model complex has two thiolate bridges and complete electron delocalization, evidenced by the characteristic EPR spectrum with a seven-line hyperfine splitting pattern (g gy, = 2.204, 2.046, 2.010, and = 35 x 10 cm , = 51 x 10  

---

M.H.M. Olsson U. Ryde (2000) Geometry, reduction potential, and reorganisation energy of the binuclear Cua site studied by theoretical methods , Proc. Natl. Acad. Sci. USA, submitted. 

Finally, the ET reactivity of the binuclear Cua site present in cytochrome c oxidase and nitrous oxide reductase illustrates an additional interesting... 

All HCOs also have a six-coordinate heme in the same subunit as the catalytic site. In addition aU cytochrome c oxidases have a binuclear Cua site and some also have a six-coordinate heme in the noncatalytic subunit (subunit n). These centers are absent in quinol oxidases. The six-coordinate heme(s) and Cua sites are responsible for electron-relay between the external electron... 

A new representative of a multicopper cluster in a protein is Cuz in nitrous oxide reductase. As was discussed above this enzyme contains a binuclear CuA centre as in COX. While the latter in addition has CuB in the form of a copper-heme group, N20 reductase has Cuz which is the site of dinitrogen formation from the substrate N20. Recently a central inorganic sulfide has been found as a ligand to copper and multiple forms of Cuz were detected in the enzyme from Paracoccus pantotrophus.134 More recently a tetranuclear copper cluster with X-S bridges was proposed as structure for Cuz..135... 

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. 11. The binuclear CuA (A) and tetranuclear CuZ (B) copper-binding sites of nitrous oxide reductase from Pseduomonas nautica (PDB Accession Code IQNl). The sulfur atom in the tetranuclear copper site is marked with an S.

COX 17 is a small cysteine rich protein that can bind copper in the form of a binuclear cuprous-thiolate cluster. The COX 17 protein has been localized to both the cytoplasm and mitochondria, and because of this dual location, COX 17 was proposed to shuttle copper ions between these two compartments. However, work by D. Winge has shown that only the mitochondrial form of COX 17 is needed for copper activation of cytochrome oxidase. The role of the cytoplasmic form is not understood. In any case, mitochondrial COX 17 is believed to capture IMS copper and then transfer the metal to a second set of accessory proteins for the Cua site of cytochrome oxidase SCOl and SC02. 

Figure 24 (Chapter 1). Electron-transfer pathway between Cua and heme-a in P. denitrificans COX. The path consists of 14 covalent bonds and two hydrogen bonds. The direct distance between to two metal ion centers is 2.0 nm. The binuclear heme-Os/Cua site is also shown. Calculations were based on the Beratan and Onuchic model (6, 7). Coordinates were taken from the PDB, code IQLE.

In a purely aqueous system, simple chelation of copper by a histidine residue shifts the pKa of the imidazole group by about 3 pH units. On the other hand, in a more hydrophobic site, as in the inner mitochondrial membrane site of Complex IV, it would be interesting to see whether pKa shifts would be sufficient to access the imidazolate state in order to pick up and release proton as the redox state of the relevant center changed. Another point of consideration in this case relates to the superfamily of heme-copper respiratory oxidases, wherein there are ubiquinol oxidases in which ubiquinol replaces the binuclear copper center. This raises the question as to whether the binuclear (Cua)2 center might be examined as replacement for ubiquinol as a site of proton release on oxidation. ... 

In addition to the Tl, T2, and T3 sites, recent studies on cytochrome c oxidase [23,24] and nitrous oxide reductase [25,26] have revealed new classes of eopper sites. These are the binuclear Cua and the tetranuclear Cuz sites, both of whieh have mixed-valent Slot = 1/2 ground states. (A Cub center is also found in the eyto-chrome c oxidase. It forms a binuclear heme a3-CuB active site where 4e reduction of O2 occurs. However, due to the lack of a distinctive spectral feature, studies on the Cub center have been limited. Interestingly, it has a covalently linked Tyr residue bound to a His ligand, which is believed to have an important role in flic reactivity [27])... 

The other copper-only binuclear centre to be considered is the CuA or purple copper complex. It is part of the terminal oxidase in mitochondrial respiration, cytochrome c oxidase (COX). Its EPR signature, a seven-line spectrum, has since long been known to be different from the classes type 1 to 3 and arises from two copper ions in a 1.5 valence (or mixed valence) state, first proposed from EPR-analysis of a similar center in nitrous oxide (N20) reductase. There is a close correspondence between the blue and purple states of copper since each of the two copper ions in CuA can be considered as being structurally related to the mononuclear blue site coordination. 

---