Cytochrome c Is Cross-Linked to Subunit II of Cytochrome c Oxidase by a Water-Soluble Carbodiimide

Academic Article

Abstract

  • Modification of beef heart cytochrome c oxidase with l-ethyl-3-[3-(dimethylamino)propyl]carbodiimide (EDC) or 1-ethyl-3-[3-(trimethylamino)propyl] carbodiimide (CH3EDC) has been found to significantly inhibit the highaffinity phase of the reaction of this enzyme with cytochrome c. Reaction conditions leading to a 70% inhibition of Fmax 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 subunitspecific antibodies. We propose that EDC or CH3EDC reacts with one or more partially buried carboxyl groups on subunit II to form a positively charged TV-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. © 1982, American Chemical Society. All rights reserved.
  • Published In

  • Biochemistry  Journal
  • Digital Object Identifier (doi)

    Pubmed Id

  • 26694930
  • Author List

  • Millett F; Darley-Usmar V; Capaldi RA
  • Start Page

  • 3857
  • End Page

  • 3862
  • Volume

  • 21
  • Issue

  • 16