The aggregation state of bovine heart cytochrome c oxidase and its kinetics in monomeric and dimeric form.
The monomeric and dimeric forms of bovine cytochrome c oxidase (EC 1.9.3.1) were obtained from gel filtration chromatography on Ultrogel AcA 34 and analyzed. Both species contained all 12-13 subunits described for this enzyme. In the dimer 320 molecules [3H]dodecyl-beta-D-maltoside were bound per heme aa3 and in the monomer 360 molecules per heme aa3. The monomers contained 10 mol of tightly bound phospholipid/mol heme aa3 and the dimers 14. Sedimentation coefficients of 15.5-18 S for the dimer and 9.6 S for the monomer were calculated from sucrose density centrifugation analysis and analytical centrifugation. By the laser beam light-scattering technique a Stokes radius of 70 A for the dimeric detergent-lipid-protein complex was measured. From those parameters and the densitometric determined partial specific volumes of the detergent and the enzyme, the molecular weights of 400,000 for the protein moiety of the dimer and 170,000-200,000 for the monomer were calculated. Under very low ionic strength conditions the monomer/dimer equilibrium was found to be dependent on the protein concentration. At low enzyme concentrations (10(-9) M) monomers were predominant, whereas at concentrations above 5 X 10(-6) M the amounts of dimers and higher aggregates were more represented. The cytochrome c oxidase activity, measured spectrophotometrically and analyzed by Eadie-Hofstee plot, was biphasic as a function of cytochrome c concentration for the dimeric enzyme. Pure monomers gave monophasic kinetics. The data, fitting with a homotropic negative cooperative mechanism for the dimer of cytochrome c oxidase, are discussed and compared with other described mechanisms.
Bolli
R
R
Nałecz
K A
KA
Azzi
A
A
D000818:Animals; D001665:Binding Sites; D002417:Cattle; D002499:Centrifugation, Density Gradient; D002850:Chromatography, Gel; D003574:Cytochrome c Group / Q000378:metabolism; D003902:Detergents; D004579:Electron Transport; D003576:Electron Transport Complex IV / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D005960:Glucosides / Q000378:metabolism; D007700:Kinetics; D008970:Molecular Weight; D009206:Myocardium / Q000201:enzymology*; D010743:Phospholipids / Q000378:metabolism; D011485:Protein Binding; D012492:Salts / Q000494:pharmacology; D013696:Temperature
D003574:Cytochrome c Group; D003902:Detergents; D005960:Glucosides; D010743:Phospholipids; D012492:Salts; C040358:dodecyl maltoside; D003576:Electron Transport Complex IV