title: Dimer interface of bovine cytochrome c oxidase is influenced by local posttranslational modifications and lipid binding.
abstract: Bovine cytochrome c oxidase is an integral membrane protein complex comprising 13 protein subunits and associated lipids. Dimerization of the complex has been proposed; however, definitive evidence for the dimer is lacking. We used advanced mass spectrometry methods to investigate the oligomeric state of cytochrome c oxidase and the potential role of lipids and posttranslational modifications in its subunit interfaces. Mass spectrometry of the intact protein complex revealed that both the monomer and the dimer are stabilized by large lipid entities. We identified these lipid species from the purified protein complex, thus implying that they interact specifically with the enzyme. We further identified phosphorylation and acetylation sites of cytochrome c oxidase, located in the peripheral subunits and in the dimer interface, respectively. Comparing our phosphorylation and acetylation sites with those found in previous studies of bovine, mouse, rat, and human cytochrome c oxidase, we found that whereas some acetylation sites within the dimer interface are conserved, suggesting a role for regulation and stabilization of the dimer, phosphorylation sites were less conserved and more transient. Our results therefore provide insights into the locations and interactions of lipids with acetylated residues within the dimer interface of this enzyme, and thereby contribute to a better understanding of its structure in the natural membrane. Moreover dimeric cytochrome c oxidase, comprising 20 transmembrane, six extramembrane subunits, and associated lipids, represents the largest integral membrane protein complex that has been transferred via electrospray intact into the gas phase of a mass spectrometer, representing a significant technological advance.
authors.affiliation: Department of Chemistry, University of Oxford, Oxford OX1 2JD, United Kingdom;
authors.forename: Idlir
authors.initials: I
authors.lastname: Liko
authors.affiliation: Department of Chemistry, University of Oxford, Oxford OX1 2JD, United Kingdom;
authors.forename: Matteo T
authors.initials: MT
authors.lastname: Degiacomi
authors.affiliation: Department of Chemistry, University of Oxford, Oxford OX1 2JD, United Kingdom; Department of Biochemistry, University of Oxford, Oxford OX1 2JD, United Kingdom;
authors.forename: Shabaz
authors.initials: S
authors.lastname: Mohammed
authors.affiliation: Picobiology Institute, Graduate School of Life Science, University of Hyogo, Kamigohri Ako, Hyogo 678-1297, Japan.
authors.forename: Shinya
authors.initials: S
authors.lastname: Yoshikawa
authors.affiliation: Department of Chemistry, University of Oxford, Oxford OX1 2JD, United Kingdom; carla.schmidt@biochemtech.uni-halle.de carol.robinson@chem.ox.ac.uk.
authors.forename: Carla
authors.initials: C
authors.lastname: Schmidt
authors.affiliation: Department of Chemistry, University of Oxford, Oxford OX1 2JD, United Kingdom; carla.schmidt@biochemtech.uni-halle.de carol.robinson@chem.ox.ac.uk.
authors.forename: Carol V
authors.initials: CV
authors.lastname: Robinson
mesh_terms: D000107:Acetylation; D000818:Animals; D002417:Cattle; D003576:Electron Transport Complex IV / Q000737:chemistry / Q000378:metabolism*; D008055:Lipids / Q000737:chemistry; D009206:Myocardium / Q000201:enzymology; D055503:Protein Multimerization; D011499:Protein Processing, Post-Translational; D021122:Protein Subunits / Q000737:chemistry / Q000378:metabolism*
chemical_list: D008055:Lipids; D021122:Protein Subunits; D003576:Electron Transport Complex IV
keywords: cytochrome c oxidase; lipids; mass spectrometry