The binuclear CuA site

Cytochrome c oxidase is the terminal oxidase in both prokaryotic and eukaryotic cells and is responsible for the generation of cellular energy via oxidative phosphorylation [127]. It couples the catalytic four-electron reduction of O2 to 

During electron transfer, the Cua site alternates between the fully reduced and the mixed-valence (Cu +Cu ) forms. Interestingly, the unpaired electron in the mixed-valence form seems to be delocalised between the two copper ions. Several theoretical investigations of the electronic structure and spectrum of the Cua dimer have been published [138-144]. In similarity to the blue copper proteins, it has been suggested that the structure and the properties of the Cua site is determined by protein strain. More precisely, it has been proposed [136] that Cua in its natural state is similar to an inorganic model studied by Tolman and coworkers [145]. This complex has a long Cu-Cu bond (293 pm) and short axial interactions (-212 pm). The protein is said to enforce weaker axial interactions, which is conpensated by shorter bonds to the other ligands and the formation of a Cu-Cu bond. This should allow the protein to modulate the reduction potential of the site [136,146]. 

It has been noted that some inorganic models of the Cua site have an appreciably longer Cu-Cu distance (-290 pm) [145]. This is accompanied by a change in the electronic state In the proteins there is a a antibonding interaction between 

The two electronic states differ in the localisation of the unpaired electron in the o state, the electron is delocalised over the whole system, whereas in the jc state, the electron is more localised to one copper. Our calculations reproduce this movement of the electron in the system wiA a long Cu-Cu bond, Ae elec- 

Oxidation states Electronic State Cu-Cu Distances (pm) CU-Scvs Cu-Nuis Cu Smci Cu-O 

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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... 

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.

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. ... 

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... 

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. 

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... 

Fig. 11. A schematic illustration of subunits I (SUI) and II (SUII) of cytochrome c and ubiquinol oxidases. The proton pathways used for proton pumping and for the delivery of protons to the O2 binding site (thick black lines) are called the D- and K-pathways (named after the conserved residues Asp for the D-pathway and Lys for the K-pathway). Dashed lines depict the electron path from the substrate to the O2 binding site. In cytochrome c oxidases, electrons are delivered to the copper A center (Cua) from the substrate, cytochrome c, and then passed onto the low-spin heme (heme a) and finally to the binuclear center (heme 3-Cub). In ubiquinol oxidases, electrons are delivered directly to the low-spin heme (heme b) by the substrate, ubiquinol, and then to the binuclear center (heme 03-CuB).

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). 

When the frilly reduced enzyme is allowed to react with O2, a more complicated sequence of events ensues (Figure 4), since now electrons are available also in the heme a and Cua centers. After formation of the O2 adduct (compound A), the next step is a ca. 30- j,s event which involves electron transfer from the low spin heme to the binuclear site. As reported by Morgan et al., the optical spectrum of the state formed is indistingishable from that of Pm if the absorption change owing to oxidation of heme a is accounted for. In contrast, Einarsdottir et al. have claimed that the spectrum of this Pr state formed from the fully reduced enzyme is different from that of Pm, and that Pr does not represent a stracturally... 

The electron originally at Cua becomes equilibrated approx. equally between heme a and Cua with the same kinetics as the F state is formed (Figure 4). This electron is finally transferred to the binuclear site, together with uptake of another proton via the D-pathway, and the O state is formed in ca. 2 3 ms. Different forms of the O state that differ in spectroscopic and kinetic parameters have long been described in the literature, but the stmctural basis for these differences remains unclear. Most recently Verkhovsky et al. reported a distinct difference in function, where reduction of the enzyme in state O was coupled to proton translocation only when snch rednction followed immediately after oxidation of the rednced enzyme by O2. The structure of the binuclear... 

Yoshikawa et al made the important statement that the role of heme a may be the biggest mystery in the stmcture and function of cytochrome c oxidase . It is indeed not at all obvious why all heme-copper oxidases have a low-spin heme group very close to the binuclear heme-copper site, especially since direct electron transfer from Cua to that site (if it occurred) would take place across almost the same distance. In the cytochrome c oxidases such direct electron transfer is effectively prevented, perhaps due to the bound Mg, the coordination sphere of which lies on the shortest path between... 

Cua and heme 3. The Mg site is indeed lacking from the quinol oxidases, which also lack Coa, and where electrons from a bound quinol on the back of the low-spin heme are transferred via that heme gronp to the binuclear site. ... 

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. 

The conversion P F (Figure 7) is accompanied by an electron equilibration between Cua and cytochrome a, and its rate is pH-dependent [60] and displays a kinetic isotope effect in D2O [60b]. Consequently, it has been suggested [51, 60a,c] that the formation of F is controlled by the protonation of the binuclear site. The conclusion that F really is a ferryl ion intermediate is supported by resonance-Raman measurements [61]. 

N20-reductase shows no marked sequence homology to the cytochrome oxidases [328]. They only share the CuA-dimer structure [280]. This could be the result of a translocation of the gene sequence encoding the binding site of the binuclear center, thus leading to the same sequence in two totally unrelated groups of proteins. 

Figure 24. 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-03/CuB site is also shown. Calculations were based on the Beratan and Onuchic model (6, 7). Coordinates were taken from the PDB, code IQLE. (See eolor...

The COX structures support Cua as the initial electron acceptor from cytochrome r, which is then transferred to heme a. Cua is closer to heme a (19.5 A, compared to heme a-, 22.1 A). Heme a, as it is close to heme 3, would then transfer electrons to the binuclear heme 3—Cub site. Both hemes are perpendicular to the membrane with interplanar angles of 104-108°, the shortest distance between the two hemes is 4.5 A, the iron-to-iron distance is 13.2 A. Thus, a direct electron transfer might be possible. The terminal electron acceptor, O2, is expected to bind between Fe s and Cub-... 

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