| Budget Amount *help |
¥5,700,000 (Direct Cost: ¥5,700,000)
Fiscal Year 1987: ¥2,000,000 (Direct Cost: ¥2,000,000)
Fiscal Year 1986: ¥3,700,000 (Direct Cost: ¥3,700,000)
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| Research Abstract |
The integral mitochondrial membrane protein cytochrome c oxidase (ferrocytochrome c:oxygen oxidoreductase. EC1.9.3.1) was crystallized from solutions of the protein from bovine heart. 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<plus-minus> 66 amino acids) and 50 kDa is detergent (Brij-35). Adjustments in buffer concentration and other conditions have yield much larger green crystals, hexagonal bipyramids; a crystal 0.3 x 0.5 x 0.7 mm gave x-ray diffractions as hith as 8 A resolution and a space group of P6_2 or P6_4 and cell dimensions of a =b=174.5 <Ang>, c=282.2 <Ang>,<alpha>=<beta>=90゜and <gamma>=120゜ were obtained. A reasonable value of 3.1 <Ang>^3/Da for Vm, the average space per dalton of protein in the crystal, was obtained for the asymmetric unit, which containes four irons and is a dimer of two minimal catalytic units. Cylindrical dimers (80 x 100 A) estimated from two-dimensional electron diffraction studies pack well in the crystal lattice with the symmetry of the space group of the crystal. The crystallization procedure developed is useful in purfication of the enzyme and shows promies for the production of crysals of sufficiently high order to gain improved structural information from x-ray diffraction.
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