INTEGRAL CYTOCHROME-C-OXIDASE - PREPARATION AND PROGRESS TOWARDS A 3-DIMENSIONAL CRYSTALLIZATION

A new rapid procedure for the preparation of monodispersed highly acti ve cytochrome-c oxidase from bovine heart is described. The crucial st ep is the separation of cytochrome-c oxidase from cytochrome-c reducta se by selective solubilization in the non-ionic detergents Triton X-10 0 or lauryl beta-D-maltoside. The enzyme is purified by subsequent ani on-exchange chromatography. The preparation is finished within two day s yielding approximately 60% of the oxidase present in mitochondria. T he enzyme has a heme a/protein ratio of 9.7 +/- 0.5 nmol/mg, approxima tely equal to the theoretical value of 9.77 nmol/mg based on a molecul ar mass of 204.696 kDa for the protein monomer. SDS/PAGE of the prepar ation reveals the presence of the well-known thirteen protein componen ts. Quantitative Edman degradation of the enzyme exclusively releases the known ten N-terminal residues; three of the thirteen protein compo nents are blocked at the N-terminus. The preparation is highly active with maximal turnover numbers of approximately 600 s(-1), identical to the maximal activity found in the mitochondrial membrane under these conditions. No g = 12 signal and no adventitious copper signal are obs erved in the EPR spectrum. The enzyme exhibits a fast monophasic react ion with cyanide. Determination of the metal contents of the enzyme in dicates the stoichiometric presence of three copper ions besides two i ron, one magnesium and one zinc ion in relation to the 94 sulfur atoms of the protein monomer. Gel-filtration experiments show a monodispers ed dimeric association to form a complex of approximately 500 kDa. The phosphorus content, 44 +/- 6.8 atoms/dimer, results from 59% cardioli pin, 23% phosphatidylethanolamine and 18% phosphatidylcholine, indicat ing a stable lipid shell, different from other previously described pr eparations. Crystals have been obtained from these preparations and ar e investigated for their suitability for X-ray work.