Structure of Bovine Heart Cytochrome c Oxidase

FIGURE 21.14 All electrophoresis gel showing the complex subunit structure of bovine heart cytochrome c oxidase. The three largest subunits, I, II, and III, are coded for by mitochondrial DNA. The others are encoded by unclear DNA. (Photo kindly provided by Professor Roderick Capaldi)... 

An X-ray structure of bovine heart cytochrome c oxidase at 2.8-A resolution showed eight phospholipid molecules, including five phospha-tidylethanolamines and three phosphatidylglycerols (Tsukihara et al.. 

In the X-ray structure of bovine heart cytochrome c oxidase in the fully oxidized state at 2.8 resolution, two hydrogen bond networks connecting the molecular surface on the matrix side to that on the intermembrane surface were identified (Tsukihara et ah, 1996). Both networks contain possible hydrogen bond structures, as defined by the criteria stated above, but do not include elements of structure comprising the O2 reduction site. However, small conformational changes induced by changes in the redox... 

Figure 10 Schematic view of the polymetallic active site of bovine heart cytochrome c oxidase, deduced from its x-ray structure [49a].

X-ray structures of 2.8-A resolution of bovine heart cytochrome c oxidase with the metals in the fully oxidized state were reported in 1995 (Tsukihara et al., 1995). The X-ray structure of cytochrome c oxidase from Paracoccus denitrificans in the fuUy oxidized azide-bound state at 2.8-A resolution was also published in the same week (Iwata et al., 1995). The structure and location of the metal sites of the two enzymes are astonishingly similar at that resolution. Later, the resolution of the bovine enzyme structure was improved to 2.3A (Yoshikawa et al., 1998). However, resolution of the Paracoccus enzyme has been improved to 2.7-A resolution (Ostermeier et al., 1997). Recently another bacterial ba3-type oxidase at 2.3-A resolution (Soulimane et al., 2000) and Escherichia coli quinol oxidase at 3.5-A resolution were reported (Abramson et al., 2000). X-ray structures of the protein and its redox-active metal sites are discussed in terms of the bovine enzyme below. 

Recently, a sodium ion has been detected in the X-ray structures of the fully oxidized and reduced forms of bovine heart cytochrome c oxidase at 2.3 and 2.35 resolutions, respectively (Yoshikawa et al., 1998). In the bacterial enzyme, a site corresponding to the Na site in the bovine enzyme is occupied by a Ca + ion (Ostermeier et al., 1997). A comparison of the coordination geometry for the bacterial versus the mammalian species shows slight differences, and this observation is consistent with differences in coordination chemistry associated with each of the two metals. Prior to the determination of the X-ray structures, the existence of these metal sites had never been proposed. The presence of these metals in X-ray structure strongly indicates that the metals are intrinsic components of the enzyme. However, as in the case of the Mg and Zn ions, the physiological functions of Na and Ca are not understood. 

Amino acid sequence analysis and determination of subunit composition are painstaking but these steps are usually necessary before further structural investigations are undertaken. It should not be forgotten that chemical composition and amino acid sequencing provided a foundation for recent structure-function findings in the cytochrome oxidase field. The complete amino acid sequence and a successful prediction of the number of a-helices greatly contributed to the successful and rapid crystallographic analysis of bovine heart cytochrome c oxidase at 2.8 resolution, four years ago (Tsukihara et al., 1995 Tsukihara et al., 1996). 

In the currently accepted theories on membrane protein crystallization, discussed above, the only role of the detergent is to stabilize the membrane protein in aqueous solution. However, the effect of detergent structure on the crystallization of bovine heart cytochrome c oxidase. 

Figure 4 shows a C backbone trace of the asymmetric unit of the orthorhombic crystal form of bovine heart cytochrome c oxidase (Tsukihara et al., 1996). The structure revealed that cytochrome c oxidase is a... 

Shinzawa-Itoh, K., Aoyama, H., Muramoto, K., Terada, H., Kurauchi, T., Tadehara, Y., Yamasaki, A., Sugimura, T., Kurono, S., Tsujimoto, K., Mizushima, T., Yamashita, E., Tsukihara, T. and Yoshikawa, S., Structures and physiological roles of 13 integral lipids of bovine heart cytochrome c oxidase, Embo J 26 (2007) 1713-1725. 

Tsukihara T, Aoyama H, Yamashita E, Tomizaki T, Yamaguchi H, Shinzawaitoh K, Nakashima R, Yaono R and Yoshikawa S 1995 Structures of metai sites of oxidized bovine heart cytochrome c oxidase at 2.8 angstrom Science 269 1069-74... 

More recently, Yoshikawa,Tsukihara, and co-workers published a study of fully oxidized (PDB 1V54) and fully reduced (PDB 1V55) bovine heart cytochrome c oxidase structures. " In this study, they identified an aspartate residue, asp51, which undergoes a substantial change in position between the oxidized and reduced structures (see inset in Figure 7.41A). 

Tsukihara, T. et al. (1995). Structures of Metal Sites of Oxidized Bovine Heart Cytochrome c Oxidase at 2.8 A. Science 269 1069. 

Bovine heart cytochrome c oxidase is in a dimer state in the asymmetric unit of the crystal as shown in Fig. 7 (see color insert) (Tsukihara et al., 1996). Thirteen different subunits were identified in each monomer in the X-ray structure of the fully oxidized enzyme at 2.8-A resolution. The top view from the intermembrane side indicates a fairly strong interaction between the two monomers. The middle portion of the side view is readily identified as the transmembrane region by the large cluster of a-helices. This part was composed mainly of 28 a-helices as had been predicted by the amino acid sequences. The Ga backbone traces show that most of the a-helices are not arranged stricdy perpendicularly to the membrane surfaces, in contrast to the prediction by the amino acid sequences. Thus, most of a-helices in the X-ray structure are longer than those predicted by the amino acid sequences. The three largest subunits, subunits I, II, and III, form a core portion and the other 10 nuclear-encoded subunits surround the core as shown in Figs. 7C and 7D. In the X-ray structure at 2.8-A resolution, 3560 of 3606 amino acid residues were identified in the asymmetric unit composed of a dimer. Only 23 of 1803 amino acid residues per monomer were not detectable in the electron density map. Most of the undetectable residues are in the N- and C-terminals, which are exposed to the bulk water phase. 

Figure 2. Structure of metal centers and ligands in bovine heart cytochrome c oxidase as rendered by PyMOL (original coordinates were published in Ref 4).

Figure 7.39 Schematic diagram for cytochrome c oxidase. Distances taken from bovine heart X-ray crystallographic structure (PDB 20CC). Entry and exit channels for dioxygen, protons, and water are schematic only. (Adapted with permission from Figure 1 of reference 138. Copyright 2004 American Chemical Society.)...

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