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. 1988 Mar;85(5):1354–1358. doi: 10.1073/pnas.85.5.1354

Crystalline cytochrome c oxidase of bovine heart mitochondrial membrane: composition and x-ray diffraction studies.

S Yoshikawa 1, T Tera 1, Y Takahashi 1, T Tsukihara 1, W S Caughey 1
PMCID: PMC279769  PMID: 2830615

Abstract

The integral mitochondrial membrane protein cytochrome c oxidase (ferrocytochrome-c:oxygen oxidoreductase, EC 1.9.3.1) was crystallized from solutions of the protein from bovine heart isolated as described earlier [Yoshikawa, S. & Caughey, W. S. (1982) J. Biol. Chem. 257, 412-420]. Crystallinity was demonstrated by x-ray diffraction. Microcrystals (tetragonal prisms, 0.02 mm in the largest dimension) were obtained in high yield with retention of activity and contained Fe, Cu, Zn, and Mg in approximate atom ratios of 1.0:1.25:0.5:0.5, respectively. Analysis of the amino acid residues and the tightly bound detergent support an apparent molecular mass of about 200 kDa, of which 150 kDa is protein (1316 +/- 66 amino acids) and 50 kDa is detergent (Brij 35). Seven major polypeptides are evident by sodium dodecyl sulfate/polyacrylamide gel electrophoresis. Adjustments in buffer concentration and other conditions have yielded much larger green crystals, hexagonal bipyramids; a crystal 0.3 X 0.5 X 0.7 mm gave x-ray diffractions as high as 8-A resolution and a space group of P6(2) or P6(4), and cell dimensions of a = b = 174.5 A, c = 282.2 A, alpha = beta = 90 degrees, and gamma = 120 degrees were obtained. A reasonable value of 3.1 A3/Da for Vm, the average space per dalton of protein in the crystal, was obtained for the asymmetric unit, which contains four irons and is a dimer of two minimal catalytic units. Cylindrical dimers (80 X 100 A) estimated from two-dimensional electron diffraction studies [Fuller, S. D., Capaldi, R. A. & Henderson, R. (1979) J. Mol. Biol. 134, 305-327] pack well in the crystal lattice with the symmetry of the space group of the crystal. The crystallization procedure developed is useful in purification of the enzyme and shows promise for the production of crystals of sufficiently high order to gain improved structural information from x-ray diffraction.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Anderson S., de Bruijn M. H., Coulson A. R., Eperon I. C., Sanger F., Young I. G. Complete sequence of bovine mitochondrial DNA. Conserved features of the mammalian mitochondrial genome. J Mol Biol. 1982 Apr 25;156(4):683–717. doi: 10.1016/0022-2836(82)90137-1. [DOI] [PubMed] [Google Scholar]
  2. Bombelka E., Richter F. W., Stroh A., Kadenbach B. Analysis of the Cu, Fe, and Zn contents in cytochrome C oxidases from different species and tissues by proton-induced X-ray emission (PIXE). Biochem Biophys Res Commun. 1986 Nov 14;140(3):1007–1014. doi: 10.1016/0006-291x(86)90735-7. [DOI] [PubMed] [Google Scholar]
  3. Buse G., Meinecke L., Bruch B. The protein formula of beef heart cytochrome c oxidase. J Inorg Biochem. 1985 Mar-Apr;23(3-4):149–153. doi: 10.1016/0162-0134(85)85019-4. [DOI] [PubMed] [Google Scholar]
  4. Caughey W. S., Smythe G. A., O'Keeffe D. H., Maskasky J. E., Smith M. I. Heme A of cytochrome c oxicase. Structure and properties: comparisons with hemes B, C, and S and derivatives. J Biol Chem. 1975 Oct 10;250(19):7602–7622. [PubMed] [Google Scholar]
  5. Chang C. H., Schiffer M., Tiede D., Smith U., Norris J. Characterization of bacterial photosynthetic reaction center crystals from Rhodopseudomonas sphaeroides R-26 by X-ray diffraction. J Mol Biol. 1985 Nov 5;186(1):201–203. doi: 10.1016/0022-2836(85)90270-0. [DOI] [PubMed] [Google Scholar]
  6. Downer N. W., Robinson N. C. Characterization of a seventh different subunit of beef heart cytochrome c oxidase. Similarities between the beef heart enzyme and that from other species. Biochemistry. 1976 Jun 29;15(13):2930–2936. doi: 10.1021/bi00658a036. [DOI] [PubMed] [Google Scholar]
  7. Einarsdóttir O., Caughey W. S. Bovine heart cytochrome c oxidase preparations contain high affinity binding sites for magnesium as well as for zinc, copper, and heme iron. Biochem Biophys Res Commun. 1985 Jun 28;129(3):840–847. doi: 10.1016/0006-291x(85)91968-0. [DOI] [PubMed] [Google Scholar]
  8. Einarsdóttir O., Caughey W. S. Zinc is a constituent of bovine heart cytochrome c oxidase preparations. Biochem Biophys Res Commun. 1984 Nov 14;124(3):836–842. doi: 10.1016/0006-291x(84)91033-7. [DOI] [PubMed] [Google Scholar]
  9. Fuller S. D., Capaldi R. A., Henderson R. Structure of cytochrome c oxidase in deoxycholate-drived two-dimensional crystals. J Mol Biol. 1979 Oct 25;134(2):305–327. doi: 10.1016/0022-2836(79)90037-8. [DOI] [PubMed] [Google Scholar]
  10. Garavito R. M., Hinz U., Neuhaus J. M. The crystallization of outer membrane proteins from Escherichia coli. Studies on lamB and ompA gene products. J Biol Chem. 1984 Apr 10;259(7):4254–4257. [PubMed] [Google Scholar]
  11. Garavito R. M., Jenkins J., Jansonius J. N., Karlsson R., Rosenbusch J. P. X-ray diffraction analysis of matrix porin, an integral membrane protein from Escherichia coli outer membranes. J Mol Biol. 1983 Feb 25;164(2):313–327. doi: 10.1016/0022-2836(83)90079-7. [DOI] [PubMed] [Google Scholar]
  12. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  13. Li P. M., Gelles J., Chan S. I., Sullivan R. J., Scott R. A. Extended X-ray absorption fine structure of copper in CuA-depleted, p-(hydroxymercuri)benzoate-modified, and native cytochrome c oxidase. Biochemistry. 1987 Apr 21;26(8):2091–2095. doi: 10.1021/bi00382a005. [DOI] [PubMed] [Google Scholar]
  14. Matthews B. W. Solvent content of protein crystals. J Mol Biol. 1968 Apr 28;33(2):491–497. doi: 10.1016/0022-2836(68)90205-2. [DOI] [PubMed] [Google Scholar]
  15. Michel H., Oesterhelt D. Three-dimensional crystals of membrane proteins: bacteriorhodopsin. Proc Natl Acad Sci U S A. 1980 Mar;77(3):1283–1285. doi: 10.1073/pnas.77.3.1283. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Penke B., Ferenczi R., Kovács K. A new acid hydrolysis method for determining tryptophan in peptides and proteins. Anal Biochem. 1974 Jul;60(1):45–50. doi: 10.1016/0003-2697(74)90129-8. [DOI] [PubMed] [Google Scholar]
  17. Poynter D., Landon M. Polypeptide components of bovine heart cytochrome c oxidase. Cross-identifications in two high-resolution polyacrylamide-gel electrophoresis systems. Int J Biochem. 1985;17(12):1349–1356. doi: 10.1016/0020-711x(85)90059-x. [DOI] [PubMed] [Google Scholar]
  18. SMITH L. Spectrophotometric assay of cytochrome c oxidase. Methods Biochem Anal. 1955;2:427–434. doi: 10.1002/9780470110188.ch13. [DOI] [PubMed] [Google Scholar]
  19. Steffens G. C., Biewald R., Buse G. Cytochrome c oxidase is a three-copper, two-heme-A protein. Eur J Biochem. 1987 Apr 15;164(2):295–300. doi: 10.1111/j.1432-1033.1987.tb11057.x. [DOI] [PubMed] [Google Scholar]
  20. Steffens G., Buse G. Studien an Cytochrom-c-Oxidase, I. Reinigung und Charakterisierung des Enzyms aus Rinderherzen und Identifizierung der im Komplex enthaltenen Peptidketten. Hoppe Seylers Z Physiol Chem. 1976 Aug;357(8):1125–1137. [PubMed] [Google Scholar]
  21. Vanneste W. H. The stoichiometry and absorption spectra of components a and a-3 in cytochrome c oxidase. Biochemistry. 1966 Mar;5(3):838–848. doi: 10.1021/bi00867a005. [DOI] [PubMed] [Google Scholar]
  22. YONETANI T. Studies on cytochrome oxidase. III. Improved preparation and some properties. J Biol Chem. 1961 Jun;236:1680–1688. [PubMed] [Google Scholar]
  23. Yewey G. L., Caughey W. S. Metals and activity of bovine heart cytochrome c oxidase are independent of polypeptide subunits III, VII, a, and b. Biochem Biophys Res Commun. 1987 Nov 13;148(3):1520–1526. doi: 10.1016/s0006-291x(87)80304-2. [DOI] [PubMed] [Google Scholar]
  24. Yoshida Y., Furuya E., Tagawa K. A direct colorimetric method for the determination of phospholipids with dithiocyanatoiron reagent. J Biochem. 1980 Aug;88(2):463–468. doi: 10.1093/oxfordjournals.jbchem.a132993. [DOI] [PubMed] [Google Scholar]
  25. Yoshikawa S., Choc M. G., O'Toole M. C., Caughey W. S. An infrared study of CO binding to heart cytochrome c oxidase and hemoglobin A. Implications re O2 reactions. J Biol Chem. 1977 Aug 10;252(15):5498–5508. [PubMed] [Google Scholar]

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