Cytochrome oxidase subunit composition

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). 

These are similar to the mitochondrial enzyme, apart from the simpler subunit composition. EXAFS studies on the oxidases from Paracoccus denitrijicans and Thermus thermophilus have confirmed the metal centres to be similar to those in the mitochondrial enzyme. In the latter case, the ESR silent cytochrome Us in the reduced enzyme has been shown by the use of Mossbauer spectroscopy to involve five-coordinate, high-spin On addition of cyanide to the oxidized... 

Fig. 3. Subunit composition of two types of cytochrome oxidase. Enzymes isolated and purified as described in the text, with the properties shown in Table II. Gel electrophoresis of dissociated proteins was as described by Fairbanks et al. on 10% gels at 6 mA per gel for 4 h staining was with Coomassie blue and scanning at 630 nm. (A) and (B) enzymes from beef heart (C) and (D) enzymes from yeast (A) and (C) type I (B) and (D) type II.

In order to define the translation site of the cytochrome b subunit(s) we proceeded as described for cytochrome oxidase (Table I) N. crassa was first labeled with [ C]leucine in the absence of inhibitors, and then with PHJleucine, either (1) in the absence of inhibitors, (2) in the presence of cycloheximide, (3) in the presence of chloramphenicol, or (4) during transitory presence of chloramphenicol. Purified cytochrome b was analyzed for and after gel filtration in bile acid-KCl solution and after gel electrophoresis in dodecylsulfate solution. It should be remembered that in the bile acid solution, the polypeptide and heme composition of cytochrome b remains intact, whereas in dodecylsulfate, the heme is dissociated from polypeptides. The results of these labeling experiments are shown in Table I and in Figs. 16 and 17. 

Analysis of the amino acid composition of the mitochondrially synthesized subunits of cytochrome oxidase of the rutamycin-sensitive ATP-ase (another membrane component which requires mitochondrial protein synthesis for some of its component polypeptides) and cytochrome b displayed a high proportion of nonpolar amino acid residues. The resulting unusually hydrophobic composition explains their insolubility in aqueous solutions. This hydrophobic character of the mitochondrially translated polypeptides may have relevance for speculations on the existence of mitochondrial protein synthesis. It has been suggested that their hydro-phobic properties make it necessary that these polypeptides be delivered to the inner mitochondrial membrane from the matrix side, since they cannot be transported through the cytoplasm and the intercristae space. 

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