The functional and physical form of mammalian cytochrome c oxidase determined by gel filtration, radiation inactivation, and sedimentation equilibrium analysis.
When solubilized in laurylmaltoside, cytochrome oxidases from beef heart and rat liver mitochondria exist as monodisperse populations that are stable, highly active, and have apparent molecular weights of 300,000 to 350,000, as measured by gel filtration. To determine whether these are monomeric (2 heme A, 2 Cu) or dimeric forms of the enzyme, we performed radiation inactivation and sedimentation equilibrium analyses. From radiation inactivation experiments under two different sets of conditions, we obtained estimates for the functional molecular weight of beef heart cytochrome oxidase of 114,000 and 99,000, much less than a dimer and significantly smaller than a 200,000 molecular weight monomer containing one copy of each of the 12 subunits normally present in the complex. The same functional size is obtained for a rat liver oxidase preparation depleted of subunit III. The physical molecular weight of cytochrome oxidase was determined by sedimentation equilibrium measurements in solvents of different densities using mixtures of H2O and D218O. Estimates of Mr = 194,000 +/- 9,000 for the beef heart oxidase and Mr = 152,000 +/- 6,000 for the rat liver enzyme were obtained, consistent with the size predicted for monomers of their subunit composition. From these results we conclude that mammalian cytochrome oxidases from beef heart and rat liver exist in laurylmaltoside as monomers capable of high rates of electron transfer and normal substrate binding. Further, these functions appear to be associated with a subset of the peptides present in the monomer, mainly composed of subunits I and II.
D000818:Animals; D001921:Brain / Q000201:enzymology; D002417:Cattle; D002850:Chromatography, Gel; D003576:Electron Transport Complex IV / Q000032:analysis*; D005960:Glucosides; D006593:Hexokinase / Q000378:metabolism; D007700:Kinetics; D008099:Liver / Q000201:enzymology; D008433:Mathematics; D008823:Micelles; D008970:Molecular Weight; D009206:Myocardium / Q000201:enzymology; D011487:Protein Conformation / Q000528:radiation effects
D005960:Glucosides; D008823:Micelles; C040358:dodecyl maltoside; D003576:Electron Transport Complex IV; D006593:Hexokinase