Crystalline cytochrome c oxidase of bovine heart mitochondrial membrane: composition and x-ray diffraction studies.
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.
Yoshikawa
S
S
Department of Biology, Konan University, Kobe, Japan.
Tera
T
T
Takahashi
Y
Y
Tsukihara
T
T
Caughey
W S
WS
D000596:Amino Acids / Q000032:analysis; D000818:Animals; D002417:Cattle; D003461:Crystallography; D003576:Electron Transport Complex IV; D008929:Mitochondria, Heart / Q000201:enzymology; D014961:X-Ray Diffraction
D000596:Amino Acids; D003576:Electron Transport Complex IV