Detection of bovine heart mitochondrial cytochrome c oxidase dimers in Triton X-100 and phospholipid vesicles by chemical cross-linking.
Bovine heart cytochrome c oxidase is a multisubunit enzyme whose oligomeric state is dependent on its detergent or phospholipid environment. We have utilized the cleavable, heterobifunctional cross-linking reagent N-succinimidyl 3-[(4-azidophenyl)dithio]propionate (SADP) to detect cytochrome c oxidase dimers. Monomeric or dimeric enzyme dispersed in Triton X-100 (as assessed by sedimentation velocity measurements) was reacted with SADP. A unique intersubunit cross-link having an apparent molecular mass of 136 kDa was identified in the dimeric enzyme; this product was insensitive to limited proteolysis by trypsin and contained a cross-link between two adjacent monomers. Two-dimensional NaDodSO4-PAGE (the second dimension containing beta-mercaptoethanol to cleave the cross-linking reagent) indicated that subunit I was the major component of the dimer-specific cross-link. The dimer-specific cross-link created by SADP was observed in phospholipid vesicles [cardiolipin/phosphatidylcholine (1:20, w/w)] containing dimeric (2 microM heme aa3) enzyme; a low yield of dimer-specific cross-link was observed in liposomes containing 6 microM (heme aa3) monomeric enzyme. The 136-kDa cross-link was not observed in liposomes containing 2 microM (heme aa3) monomeric enzyme. These results indicate that subunit I from each monomer may provide one site of interaction between monomers in the dimeric form of the enzyme and that cytochrome c oxidase monomers may reassociate to form dimeric complexes in phospholipid vesicles.
Estey
L A
LA
Department of Biochemistry and Molecular Biology, School of Medicine, College of Science and Mathematics, Wright State University, Dayton, Ohio 45435.
Prochaska
L J
LJ
D000818:Animals; D002417:Cattle; D003432:Cross-Linking Reagents; D003576:Electron Transport Complex IV / Q000737:chemistry*; D066298:In Vitro Techniques; D046911:Macromolecular Substances; D008563:Membrane Lipids / Q000737:chemistry; D008565:Membrane Proteins / Q000737:chemistry; D008929:Mitochondria, Heart / Q000201:enzymology*; D017830:Octoxynol; D010743:Phospholipids / Q000737:chemistry; D011485:Protein Binding
D003432:Cross-Linking Reagents; D046911:Macromolecular Substances; D008563:Membrane Lipids; D008565:Membrane Proteins; D010743:Phospholipids; D017830:Octoxynol; D003576:Electron Transport Complex IV