Structural role of phospholipids in ubiquinol-cytochrome c reductase.
The role of phospholipids in ubiquinol-cytochrome c reductase has been studied by the following methods: (1) removal and restoration of phospholipids, (2) circular dichroism measurements, and (3) phospholipase A2 treatment. Over 90% of the phospholipids in the cytochrome b--c1 III complex (a highly purified ubiquinol-cytochrome c reductase) can be removed by repeated precipitation with ammonium sulfate in the presence of 0.5% sodium cholate. The delipidated enzyme complex is inactive. Full restoration of enzymatic activity can only be achieved with a freshly prepared delipidated enzyme complex, made in the presence of 20% glycerol. As the age of the delipidated enzyme increased, the amount of activity restored decreased and the incubation time required to reach maximal activity increased. Removal of phospholipids from the cytochrome b--c1 III complex resulted in an immediate decrease of approximately 15% in molar ellipticities in both the far-UV and the Soret regions. A further decrease in ellipticities was observed upon incubation of the delipidated enzyme at 0 degrees C in 50 mM phosphate buffer, pH 7.4. Replenishing phospholipids to the delipidated enzyme complex restored enzymatic activity and the molar ellipticity in both regions. The absolute requirement for phospholipids in the cytochrome b--c1 III complex was also demonstrated by treatment of the enzyme with purified phospholipase A2. The inactivation of the cytochrome b--c1 III complex by phospholipase A2 was not prevented by the presence of excess exogenous ubiquinone but was prevented by the presence of ethylenediaminetetracetic acid (EDTA). The enzymatic activity of the phospholipase A2 treated complex is fully restorable upon the addition of EDTA and phospholipids.
D002942:Circular Dichroism; D003574:Cytochrome c Group / Q000378:metabolism; D003580:Cytochromes; D014450:Electron Transport Complex III; D004789:Enzyme Activation; D007700:Kinetics; D046911:Macromolecular Substances; D009097:Multienzyme Complexes / Q000378:metabolism*; D009247:NADH, NADPH Oxidoreductases / Q000378:metabolism*; D010741:Phospholipases A; D054467:Phospholipases A2; D010743:Phospholipids / Q000302:isolation & purification / Q000502:physiology*; D011485:Protein Binding; D011487:Protein Conformation; D011808:Quinone Reductases / Q000378:metabolism*
D003574:Cytochrome c Group; D003580:Cytochromes; D046911:Macromolecular Substances; D009097:Multienzyme Complexes; D010743:Phospholipids; D009247:NADH, NADPH Oxidoreductases; D011808:Quinone Reductases; D010741:Phospholipases A; D054467:Phospholipases A2; D014450:Electron Transport Complex III