Cytochrome c is cross-linked to subunit II of cytochrome c oxidase by a water-soluble carbodiimide.
Modification of beef heart cytochrome c oxidase with 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide (EDC) or 1-ethyl-3-[3-(trimethylamino)propyl]carbodiimide (CH3EDC) has been found to significantly inhibit the high-affinity phase of the reaction of this enzyme with cytochrome c. Reaction conditions leading to a 70% inhibition of Vmax resulted in a 16-fold increase in the Km for cytochrome c. The loss in activity was accompanied by modification of subunit II to form a new species, II', which migrated somewhat more rapidly than the unmodified subunit during sodium dodecyl sulfate (NaDodSO4) gel electrophoresis. This new species was the major site of radiolabeling when cytochrome c oxidase was treated with [14C]CH3EDC, indicating covalent incorporation of the carbodiimide. Equimolar concentrations of cytochrome c dramatically protected cytochrome c oxidase from inhibition by the carbodiimide and in approximately the same proportion shielded subunit II from modification to the labeled II' species. In addition, cytochrome c was cross-linked to subunit II to form a new species migrating somewhat faster than subunit I during NaDodSO4 gel electrophoresis. This cross-linked species was shown to contain subunit II by using subunit-specific antibodies. We propose that EDC or CH3EDC reacts with one or more partially buried carboxyl groups on subunit II to form a positively charged N-acylurea which inhibits cytochrome c binding. In the presence of cytochrome c, EDC promotes formation of amide cross-links between lysine amino groups on cytochrome c and their complementary carboxyl groups on cytochrome c oxidase.
D000818:Animals; D002417:Cattle; D003432:Cross-Linking Reagents / Q000378:metabolism*; D003574:Cytochrome c Group / Q000378:metabolism*; D004024:Dicyclohexylcarbodiimide / Q000378:metabolism; D003576:Electron Transport Complex IV / Q000378:metabolism*; D005022:Ethyldimethylaminopropyl Carbodiimide / Q000378:metabolism*; D007700:Kinetics; D046911:Macromolecular Substances; D009206:Myocardium / Q000201:enzymology
D003432:Cross-Linking Reagents; D003574:Cytochrome c Group; D046911:Macromolecular Substances; D004024:Dicyclohexylcarbodiimide; D003576:Electron Transport Complex IV; D005022:Ethyldimethylaminopropyl Carbodiimide