Role of nuclear-encoded subunit Vb in the assembly and stability of cytochrome c oxidase complex: implications in mitochondrial dysfunction and ROS production.
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.
Galati
Domenico
D
Department of Animal Biology, School of Veterinary Medicine, 3800 Spruce Street, University of Pennsylvania, Philadelphia, PA 19104, U.S.A.
Srinivasan
Satish
S
Raza
Haider
H
Prabu
Subbuswamy K
SK
Hardy
Michael
M
Chandran
Karunakaran
K
Lopez
Marcos
M
Kalyanaraman
Balaraman
B
Avadhani
Narayan G
NG