title: Role of nuclear-encoded subunit Vb in the assembly and stability of cytochrome c oxidase complex: implications in mitochondrial dysfunction and ROS production.
abstract: CcO (cytochrome c oxidase) is a multisubunit bigenomic protein complex which catalyses the last step of the mitochondrial electron transport chain. The nuclear-encoded subunits are thought to have roles either in regulation or in the structural stability of the enzyme. Subunit Vb is a peripheral nuclear-encoded subunit of mammalian CcO that is dramatically reduced under hypoxia. Although it has been shown to contain different ligand-binding sites and undergo modifications, its precise function is not known. In the present study we generated a cell line from RAW 264.7 murine macrophages that has a more than 80% reduced level of Vb. Functional analysis of these cells showed a loss of CcO activity, membrane potential and less ability to generate ATP. Resolution of complexes on blue native gel and two-dimensional electrophoretic analysis showed an accumulation of subcomplexes of CcO and also reduced association with supercomplexes of the electron transfer chain. Furthermore, the mitochondria from CcO Vb knock-down cells generated increased ROS (reactive oxygen species), and the cells were unable to grow on galactose-containing medium. Pulse-chase experiments suggest the role of the CcO Vb subunit in the assembly of the complex. We show for the first time the role of a peripheral, non-transmembrane subunit in the formation as well as function of the terminal CcO complex.
authors.affiliation: Department of Animal Biology, School of Veterinary Medicine, 3800 Spruce Street, University of Pennsylvania, Philadelphia, PA 19104, U.S.A.
authors.forename: Domenico
authors.initials: D
authors.lastname: Galati
authors.forename: Satish
authors.initials: S
authors.lastname: Srinivasan
authors.forename: Haider
authors.initials: H
authors.lastname: Raza
authors.forename: Subbuswamy K
authors.initials: SK
authors.lastname: Prabu
authors.forename: Michael
authors.initials: M
authors.lastname: Hardy
authors.forename: Karunakaran
authors.initials: K
authors.lastname: Chandran
authors.forename: Marcos
authors.initials: M
authors.lastname: Lopez
authors.forename: Balaraman
authors.initials: B
authors.lastname: Kalyanaraman
authors.forename: Narayan G
authors.initials: NG
authors.lastname: Avadhani