The preferential binding of dicyclohexylcarbodiimide to cytochrome b and phospholipids in soluble complex III from yeast mitochondria.
The binding of [14C]dicyclohexylcarbodiimide (DCCD) to soluble complex III from yeast mitochondria was examined under conditions which resulted in the inhibition of proton ejection but had a minimal effect on cytochrome c reductase activity. Incubation of the complex with 50-100 nmol of [14C]DCCD/nmol of cytochrome b at 12 degrees C did not result in any changes in the appearance of the high-molecular-weight subunits (I-V) after sodium dodecyl sulfate-gel electrophoresis, although a slight broadening of the three lowest molecular-weight subunits (VI-VIII) was observed. The [14C]DCCD was bound preferentially to subunit III (cytochrome b) and a wide band with an apparent low-molecular weight ranging from 8000 to 9000 to less than 2000 depending on the gel system used. Extraction of the [14C]DCCD-treated complex III with chloroform:methanol had no effect on subunit III but completely removed the low-molecular-weight radioactive band. Thin-layer chromatography of the chloroform:methanol extract revealed that the radioactive material extracted from the [14C]DCCD-treated complex III migrated with the same apparent RF as either free [14C]DCCD or cardiolipin. Amino acids were not detectable in an acid hydrolysate of the chloroform:methanol extract, suggesting the absence of protein. Digestion of the [14C]DCCD-treated complex III with either chymotrypsin or Staphylococcus aureus V8 protease resulted in the decrease of both staining intensity and labeling in subunit III but had no effect on the radioactivity in the low-molecular-weight material. These results confirm that DCCD binds preferentially to cytochrome b in complex III from yeast mitochondria and suggest that cytochrome b may play an important role in proton translocation at this site of the respiratory chain.
D001665:Binding Sites; D002234:Carbodiimides / Q000378:metabolism*; D002250:Carbon Radioisotopes; D003573:Cytochrome b Group / Q000378:metabolism*; D004024:Dicyclohexylcarbodiimide / Q000378:metabolism*; D014450:Electron Transport Complex III; D007700:Kinetics; D008928:Mitochondria / Q000201:enzymology*; D009097:Multienzyme Complexes / Q000378:metabolism*; D009247:NADH, NADPH Oxidoreductases / Q000378:metabolism*; D010743:Phospholipids / Q000378:metabolism*; D011485:Protein Binding; D011808:Quinone Reductases / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D013816:Thermodynamics
D002234:Carbodiimides; D002250:Carbon Radioisotopes; D003573:Cytochrome b Group; D009097:Multienzyme Complexes; D010743:Phospholipids; D004024:Dicyclohexylcarbodiimide; D009247:NADH, NADPH Oxidoreductases; D011808:Quinone Reductases; D014450:Electron Transport Complex III