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-(dimethylamo)propyl]carbodiimide (EDC) or 1-ethyl-3-[3-(trimethylamino)propyl]carbodiimide (CH3EDC) significantly inhibits 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 (SDS) 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 SDS gel electrophoresis. This cross-linked species was shown to contain subunit II by using subunit-specific antibodies. EDC or CH3EDC probably reacts with 1 or more partially 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 fo amide cross-links between lysine amino groups on cytochrome c and their complementary carboxyl groups on cytochrome c oxidase.