Integral cytochrome-c oxidase. Preparation and progress towards a three-dimensional crystallization.
A new rapid procedure for the preparation of monodispersed highly active cytochrome-c oxidase from bovine heart is described. The crucial step is the separation of cytochrome-c oxidase from cytochrome-c reductase by selective solubilization in the non-ionic detergents Triton X-100 or lauryl beta-D-maltoside. The enzyme is purified by subsequent anion-exchange chromatography. The preparation is finished within two days yielding approximately 60% of the oxidase present in mitochondria. The enzyme has a heme alpha/protein ratio of 9.7 +/- 0.5 nmol/mg, approximately equal to the theoretical value of 9.77 nmol/mg based on a molecular mass of 204.696 kDa for the protein monomer. SDS/PAGE of the preparation reveals the presence of the well-known thirteen protein components. Quantitative Edman degradation of the enzyme exclusively releases the known ten N-terminal residues; three of the thirteen protein components 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 observed in the EPR spectrum. The enzyme exhibits a fast monophasic reaction with cyanide. Determination of the metal contents of the enzyme indicates the stoichiometric presence of three copper ions besides two iron, one magnesium and one zinc ion in relation to the 94 sulfur atoms of the protein monomer. Gel-filtration experiments show a monodispersed dimeric association to form a complex of approximately 500 kDa. The phosphorus content 44 +/- 6.8 atoms/dimer, results from 59% cardiolipin, 23% phosphatidylethanolamine and 18% phosphatidylcholine, indicating a stable lipid shell, different from other previously described preparations. Crystals have been obtained from these preparations and are investigated for their suitability for X-ray work.
Soulimane
T
T
Institut für Biochemie, Rheinisch-Westfälische Technische Hochschule, Aachen, Germany.
Buse
G
G
D000818:Animals; D002417:Cattle; D002852:Chromatography, Ion Exchange; D003460:Crystallization; D003576:Electron Transport Complex IV / Q000737:chemistry* / Q000302:isolation & purification; D004591:Electrophoresis, Polyacrylamide Gel; D005561:Formates / Q000737:chemistry; D006863:Hydrogen-Ion Concentration; D008929:Mitochondria, Heart / Q000201:enzymology
D005561:Formates; C030544:formic acid; D003576:Electron Transport Complex IV