Protein-protein interactions of cytochrome oxidase in inner mitochondrial membranes. The effect of liposome fusion on protein rotational mobility.
Rotational diffusion of cytochrome oxidase in the inner membrane of rat liver mitochondria was measured by detecting the decay of absorption anisotropy after photolysis of the heme a3.CO complex by a vertically polarized laser flash. As in previous experiments with beef heart mitochondria (Kawato, S., Sigel, E., Carafoli, E., and Cherry, R. J. (1980) J. Biol. Chem. 255, 5508-5510), co-existence of rotating cytochrome oxidase (mean rotational relaxation time, phi, of 700 to 1400 microseconds) and immobilized cytochrome oxidase (phi greater than 20 ms) was observed in mitochondria and mitoplasts. The effect of lipid/protein ratio by weight (L/P) on the relative proportions of mobile and immobile cytochrome oxidase was investigated following the fusion of soybean phospholipid vesicles with mitoplasts. The fusion procedure yielded four separate fractions upon sucrose density gradient centrifugation with L/P as follows: 0.3 in Pellet, 0.7 in Band 3, 1.5 in Band 2, and 3.0 in Band 1. The percentage of rotationally mobile cytochrome oxidase (phi = 700 to 1000 microseconds) in each of the different bands was found to be 16% in Pellet, 25% in Band 3, 47% in Band 2, and 76% in Band 1 at 37 degrees C. The dependence of the amount of mobile cytochrome oxidase on L/P indicates that the fraction of aggregated protein progressively decreases with decreasing concentration of proteins in the membrane. Thus, the large immobile fraction of cytochrome oxidase in mitochondrial inner membranes can be explained by nonspecific protein aggregation which is a consequence of the low L/P. The decrease in the mobile fraction in Pellet compared with mitoplasts was shown to be due to the pH 6.5 incubation used for fusion.
D000818:Animals; D003574:Cytochrome c Group / Q000378:metabolism; D003576:Electron Transport Complex IV / Q000378:metabolism*; D005614:Freeze Fracturing; D007425:Intracellular Membranes / Q000201:enzymology* / Q000648:ultrastructure; D007700:Kinetics; D008081:Liposomes; D008297:Male; D008854:Microscopy, Electron; D008930:Mitochondria, Liver / Q000201:enzymology* / Q000648:ultrastructure; D011485:Protein Binding; D051381:Rats; D011919:Rats, Inbred Strains
D003574:Cytochrome c Group; D008081:Liposomes; D003576:Electron Transport Complex IV