Cytochrome c oxidase, bovine heart

Our purification procedure (Yoshikawa et al., 1977) is essentially identical to the Okunuki method (Okunuki et al., 1958). Steps in the Okunki method include solubilization of the enzyme from the mitochondrial inner membrane, followed by fraetionation of the solubilized sample with ammonium sulfate in the presenee of eholate, and then followed by additional fractionations with ammonium in the presence of the non-ionic detergent, decyl maltoside. The nonionie detergents are not as effective as the ionic detergents for the solubilization of this enzyme. Namely, the most effective detergent for solubilization may not neeessarily be the best detergent for stabilization of the isolated protein in an aqueous solution. 

Reproducibility of the purifieation procedure for a protein is one of the most critical factors not only for the determination of the chemical composition of the protein but also for the optimization of the crystallization conditions. Based on our experienee, the most reproducible procedure 

As discussed below, crystallization of the enzyme is also an effeetive method for removing contaminating and denatured proteins. Crystallization has the potential to produce a preparation not only of high purity but also of extreme reproducibility in both composition and enzyme aetivity. An important property of crystallization is its inherent eapability to seleet for protein molecules that possess the same three dimensional strueture. This is in eontrast to other purification steps which are likely to induee some degree of denaturation. 

In order to elucidate the reaction mechanism of cytochrome c oxidase, the complete structure of the enzyme must be determined. The first step in this process is the complete determination of its composition. X-ray crystallographic analysis at high resolution was required in addition to chemical analysis for crystalline enzyme preparation. 

The involvement of copper ions as the prosthetic group of this enzyme was recognized as a result of chemical and spectroscopic (especially copper EPR) analyses of the purified preparation during the years 

Effect of Crystallization on the Metal Content of Bovine Heart Cytochrome c Oxidase  

The physiological roles of these metals have not been identified. Thus, the possibility exists that these metals are copurified contaminants. Table I indicates the amounts of metals insensitive to repeated crystallization this provides strong evidence that they are intrinsic components. This is the case for the amount of copper (3 Cu/enzyme), which is consistent with the X-ray structure as described below. 

An X-ray structure of the fully oxidized enzyme at 2.3-A resolution shows a sodium site near the intermembrane side as described below (Yoshikawa et al., 1998). Prior to publication of the X-ray structure, a sodium site had not been considered. This result shows that X-ray structural analysis is indispensable for determination of the metal content of proteins. 

---

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... 

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)... 

Figure 14.10 (Left) The CuA site in bacterial cytochrome oxidase. (From Messerschmidt et al., 2001. Reproduced with permission from John Wiley Sons., Inc.) (Right) The haem-a3/CuB site in the resting form of oxidized bovine heart cytochrome c oxidase showing peroxide bound between the haem Fe and CuB. (From Bento et al., 2006. With kind permission of Springer Science and Business Media.)...

The active site of bovine heart cytochrome c oxidase is constituted by a multimetallic assembly (Cu, Mg, Fe, Zn),4 but it is thought that... 

Steady state kinetics and protein-protein binding measurements have also been reported for the interaction of these mutant cytochromes with bovine heart cytochrome c oxidase [120]. The binding of cytochrome c variants to the oxidase occurred with increasing values of Kj in the order He (3 x 10 Mol L ) < Leu = Gly < wild-type < Tyr < Ser (3 x 10 molL ). Steady-state kinetic analysis indicated that the rate of electron transfer with cytochrome c oxidase increased in the order Ser < He < Gly < Leu < Tyr < wild-type, an order notably different from that observed for a related analysis of the oxidation of these mutants by cytochrome c peroxidase [85]. This difference in order of mutant turnover by the oxidase and peroxidase may arise from differences in the mode of interaction of the cytochrome with these two enzymes. 

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). 

The reaction with Oj of the fully reduced bovine heart cytochrome c oxidase (o Cua -CuboJ ) was followed at low temperature by optical and EPR spectroscopy With the mixed-valence-state cytochrome c oxidase (< Cu cytochrome and Cua remained unaffected and the presence of three intermediates could be deduced ... 

Robinson, N. C., Zborowski, J., and Talbert, L. H. (1990) Cardiolipin-depleted bovine heart cytochrome c oxidase binding stoichiometry and affinity for cardiolipin derivatives, Biochemistry 29, 8962-8969. 

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

Yoshikawa, S. et al. (1998). Redox-Coupled Crystal Structural Changes in Bovine Heart Cytochrome c Oxidase. Science 280 1723. 

It is well known that the O2 reduction site of bovine heart cytochrome c oxidase in the fuUy oxidized state exhibits variable reactivity to cyanide and ferrocytochrome c, which is dependent on the method of purihcation (Moody, 1996). Some preparations react with cyanide extremely slowly at an almost immeasurable rate and are known as the slow form. Other preparations, which react at a half-Ufe of about 30 s, are known as the fast form (Brandt et al., 1989). Electronic absorption spectra of the slow-and fast-form preparations exhibit Soret bands at 418 and 424 nm, respectively. The two forms often coexist in a single preparation (Baker et al., 1987). Both forms exhibit an identical visible-Soret spectrum in the fully reduced state. The slow-form preparation can be converted to the fast form by dithionite reduction followed by reoxidation with O2. The fast form thus obtained returns to the slow form spontaneously at a rate much slower than the enzymatic turnover rate. Thus, the slow form is unlikely to be involved in the enzymatic turnover (Antoniniei a/., 1977). It should be noted that no clear experimental evidence has been reported for direct involvement of the fast form in the enzyme turnover, although its direct involvement has been widely accepted. The third species of the fully oxidized O2 reduction site, which appears in the partially reduced enzyme, reacts with cyanide 10 —10 times more rapidly than the fast form (Jones et al., 1984). In the absence of a reducing system, no interconversion is detectable between the slow and the fast forms (Brandt et al., 1989). Thus, the heterogeneity is expected to inhibit the crystallization of this enzyme. In fact, the enzyme preparations providing crystals showing X-ray diffraction at atomic resolution are the fast form preparation. 

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.. 

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. 

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. 

Fig. 9. A reductive titration of the crystalline bovine heart cytochrome c oxidase with dithionite. Absolute spectra for each oxidation state are shown for the Soret (A) and visible (B) regions. The difference spectra against the spectrum in the fully reduced state are given for the near-infrared region (C). The insets show titration curves against the electron equivalent per enzyme. The reaction mixture contained 7.5 jlM bovine heart cytochrome c oxidase in 0.1 M sodium phosphate buffer, pH 7.4. The enzyme preparation was stabilized with a synthetic non-ionic detergent, CH3(CH2)ii(0CH2CH2)80H. The light path was 1 cm.

Fig. 10. Improvement of the infrared spectrophotometer. Infrared spectra of fully oxidized bovine heart cytochrome c oxidase cyanide derivatives measured with (A) a dispersive infrared spectrophotometer (Perkin-Elmer Model 180) and (B) a FTIR spectrometer equipped with a mercury/cadmium/tellurium detector (Perkin-Elmer Model 1800). Concentrations of the enzyme (O.VmM) and cyanide (19.4 M) were identical in both measurements.

Fig. 15. Resonance Raman spectra of the Fe +-02 stretching frequency region of bovine heart cytochrome c oxidase 0.1 ms after initiation of the reaction of the fully reduced enzyme with O2. Spectra on the left- and right-hand sides are the observed spectra and the calculated spectra with the differences of the observed versus calculated spectra, respectively. Spectrum (d) is obtained using the following calculation [Spectrum ( )—Spectrum (c)]/2. (e) Simulated bands for Fe— 02 (1), Fe—(2), Fe—(3), and Fe— 02 (4). The peak intensity ratio is 6 6 5 5. All bands have the Gaussian band shape with a half-maximal band width of 12.9cm h...

As stated above, for proton transfers inside cytochrome c oxidase, redox-coupled conformational changes are required. As shown in Fig. 18 (see color insert for A), a fairly large conformational change, including even a movement of the peptide backbone in a loop region between helices I and II of subunit I of bovine heart cytochrome c oxidase, was... 

The key residue of the H-channel of bovine heart cytochrome c oxidase, Asp-51, is conserved only in the animal kingdom. The enzyme of plants... 

---