Cytochrome oxidase Binuclear center

Figme 6 X-ray structure of cytochrome oxidase binuclear center from bovine hertrt. 

FIGURE 21.19 The binuclear center of cytochrome oxidase. A ligand, L (probably a cysteine S), is shown bridging the Cng and Fe L metal sites. 

Li W, Palmer G. 1993. Spectroscopic characterization of the interaction of azide and thiocyanate with the binuclear center of cytochrome oxidase evidence for multiple ligand sites. Biochem 32(7) 1833-1843. 

Figure 7.48 Two cytochrome c oxidase Cua center model compounds (A) delocalized core with two i-l,3-(KN KO)-ureate bridges as reported in reference 165-and (B) a dithiolate-bridged mixed-valence binuclear copper ion complex as re in reference 166.

Nitric oxide binds to the Cu(II) ion in the binuclear center of fully oxidized cytochrome oxidase (Brudvig et al., 1980). The binding of NO creates an even spin copper center and effectively breaks the spin coupling between the heme and copper metal ions. As a result, the high-spin heme EPR signal is visible at g = 6. 

FIGURE 19-13 Critical subunits of cytochrome oxidase (Complex IV). The bovine complex is shown here (PDB ID 10CC). (a) The core of Complex IV, with three subunits. Subunit I (yellow) has two heme groups, a and a3 (red), and a copper ion, CuB (green sphere). Heme a3 and CuB form a binuclear Fe-Cu center. Subunit II (blue) contains two Cu ions (green spheres) complexed with the —SH groups of two Cys residues in a binuclear center, CuA, that resembles the 2Fe-2S centers of iron-sulfur proteins. This binuclear center and the cytochrome... 

Cytochrome bo ubiquinol oxidase from E. coli is a four-subunit heme-copper oxidase that catalyzes the four-electron reduction of O2 to water and functions as a proton pump (Puustinen et al., 1991 Fig. 11). All redox centers are located within the largest subunit (subunit I), with a low spin protoheme (heme b) acting as the electron donor to a binuclear center that is composed of an O-type heme (heme of) and a copper ion... 

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).

In summary, an energetically feasible reaction mechanism for 0-0 bond cleavage in cytochrome oxidase has been found. The requirements of this mechanism are that there is a water molecule available at the binuclear center and that one of the residues at the binuclear center is protonated. Both these requirements are in accordance with experimental observations [58, 59]. The calculations together with the X-ray structure suggest that... 

Two copper ions form the CuA-center, which is identical to that in the cytochrome oxidases [41,300,301]. The other two copper ions also form a binuclear structure [42]. 

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. 

Cytochrome c oxidase (COX) is the terminal enzyme in the respiratory system of most aerobic organisms and catalyzes the four electron transfer from c-type cytochromes to dioxygen (115, 116). The A-type COX enzyme has three different redox-active metal centers A mixed-valence copper pair forming the so-called Cua center, a low-spin heme-a site, and a binuclear center formed by heme-fl3 and Cub. The Cua functions as the primary electron acceptor, from which electrons are transferred via heme-a to the heme-fl3/CuB center, where O2 is reduced to water. In the B-type COX heme-u is replaced by a heme-fo center. The intramolecular electron-transfer reactions are coupled to proton translocation across the membrane in which the enzyme resides (117-123) by a mechanism that is under active investigation (119, 124—126). The resulting electrochemical proton gradient is used by ATP synthase to generate ATP. 

Structure and Mechanism. Cytochrome-c oxidase catalyzes the four-electron reduction of molecular oxygen to water and couples these redox processes to proton transfer across the mitochondrial membrane.As depicted in Fig. 24, the enzyme is structurally complex and contains four metal centers which are redox active, two copper ions and two heme a groups. One copper ion, Cug, and one of the heme a groups, cytochrome <13, (cyt a ), form a binuclear center which binds dioxygen. Electrons are ... 

Fig. 25. Reaction sequence for the oxidation of fully reduced cytochrome oxidase by dioxygen. Only the iron and copper ions of the binuclear center are shown. (From Varotsis et at. )...

Condensatfon of 57 with trans-urocanic acid chloride (62) furnished the chelated porphyrin In this case the imidazole was attached at the C-4 rather than the more common N-1 position to allow deprotonation to the imidazolate. After iron insertion and deprotonation addition of Cu(acac)2 yielded the p-imidazolato binuclear complex 64 (Scheme 24), a potential model for the [Cu5 /Cyta ] center of cytochrome oxidase. 

Complex IV, or cytochrome c oxidase, was the first of the mitochondrial electron transport complexes to have its molecular stmcture and the internal path of electron transfer revealed by X-ray crystallography. The catalytic core of the complex consists of two subunits. Subunit II contains a binuclear copper center (Cua) that is directly responsible for the oxidation of cytochrome c. From there electrons are passed to haem a and then to the adjacent binuclear center that consists of haem 03 and another copper ion (Cub), which are all held within subunit I (Fig. 13.1.4). Oxygen is bound and reduced between Cub and the iron of haem 03, and access paths for protons from the inside of the membrane and for oxygen from within the membrane have been defined from several crystal stmctures available for bovine and bacterial enzymes. In addition to the protons taken up for the reduction of oxygen, translocation of further protons across the membrane is coupled to electron transfer by a mechanism that is not yet understood (reviewed in Refs. [71, 72]). 

Figure 8.25. Complex IV cytochrome c oxidase. Stereo view of subunits I and II given in vine representation to show a total of eight His residues coordinated to redox sites. (A) O2 side of heme aj-Cus binuclear center with three coordinated His residues and Cua with two coordinated His residues. (B)...

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. 

Tire most studied of all copper-containing oxidases is cytochrome c oxidase of mitochondria. This multisubunit membrane-embedded enzyme accepts four electrons from cytochrome c and uses them to reduce 02 to 2 H20. It is also a proton pump. Its structure and functions are considered in Chapter 18. However, it is appropriate to mention here that the essential catalytic centers consist of two molecules of heme a (a and a3) and three Cu+ ions. In the fully oxidized enzyme two metal centers, one Cu2+ (of the two-copper center CuA) and one Fe3+ (heme a), can be detected by EPR spectroscopy. The other Cu2+ (CuB) and heme a3 exist as an EPR-silent exchange-coupled pair just as do the two copper ions of hemocyanin and of other type 3 binuclear copper centers. 

---