Role of nuclear-encoded subunit Vb in the assembly and stability of cytochrome c oxidase complex: Implications in mitochondrial dysfunction and ROS production

Domenica Galati, Satish Srinivasan, Haider Raza, Subbuswamy K. Prabu, Michael Hardy, Karunakaran Chandran, Marcos Lopez, Balaraman Kalyanaraman, Narayan G. Avadhani

Research output: Contribution to journalArticlepeer-review

69 Citations (Scopus)

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, nontransmembrane subunit in the formation as well as function of the terminal CcO complex.

Original languageEnglish
Pages (from-to)439-449
Number of pages11
JournalBiochemical Journal
Volume420
Issue number3
DOIs
Publication statusPublished - Jun 15 2009
Externally publishedYes

Keywords

  • Cytochrome c oxidase assembly
  • Cytochrome c subunit Vb
  • Membrane potential
  • Mitochondrion
  • Reactive oxygen species production
  • Respirosome

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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