Crystallization and X-ray crystallographic analysis of Complexes III and I in mitochondrial respiratory system

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Based on the experience about crystallization of beef heart cytochrome c oxidase, purification procedure of beef heart Complex III, reported by Rieske, was improved to increase the yeald and the reproducibility. Complete replacement of the residual cholate in the purified sample with a nonionic detergent, dodecyl sucrose, increased the stability of the sample significantly. The preparation dissolved in Tris-HCl buffer was crystallizable in a monoclinic form with polyethyleneglycol as the precipitant, giving an x-ray diffraction pattern with 7.5 A resolution. However, the crystals were fragil and easily to be twinned. The preparation in K-phosphate buffer provided hexagonal crystals which diffracted x-rays upto 6.5 A resolution. An addition of An^<2+> increased the reproducibility of crystallization of the hexagonal crystals. On the other hand, the preparation purified with crystallization gave tetragonal crystals with a parfection similar to the hexagonal one.Purification procedure by Rieske for Complex I was also reexamined to establish the reproducibility and the yeald(0.9 grams from 1.2 kg of beef heart muscle). Survey of the crystallization conditions with "crystal screen"(Hampton Research Inc.) provided spherlite crystals which showed birefringe under polarized light.