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Franziska Hoeser

Assembly of the respiratory
NADH:ubiquinone oxidoreductase in Escherichia coli

Principal Investigator: Prof. Dr. Thorsten Friedrich

Institut für Biochemie
Albertstr. 21
79104 Freiburg

Phone: +49 (761) 203-6056
nuber@bio.chemie.uni-freiburg.de

 

Abstract

The enzyme complexes of respiratory chains convert energy of reducing equivalents into an ion gradient across the membrane. This gradient is used in turn for energy-consuming processes such as ATP synthesis, active transport and motion. The NADH:ubiquinone oxidoreductase, respiratory complex I, is the main entry point for electrons from NADH into the respiratory chains of most mitochondria and many bacteria. A defect in the assembly or dysfunction of the mitochondrial complex I is associated with diseases such as Parkinson’s or Leigh syndrome. The Escherichia coli complex I represents a minimal structural model of the eukaryotic NADH:ubiquinone oxidoreductase. It consists of 13 subunits (NuoA-N) separated in a hydrophobic arm located in the membrane and a peripheral arm. The enzyme links the transfer of two electrons from NADH to ubiquinone with the translocation of four protons across the membrane. The peripheral arm which catalyses the electron transfer protrudes into the cytoplasm and harbours all cofactors, namely nine iron-sulfur-clusters and one flavin mononucleotide. Proton translocation takes place in the membrane arm that does not contain any cofactors. The assembly of the bacterial complex I is still unknown but it is supposed that it is closely connected to the insertion of the Fe/S-clusters. Recent studies showed that LdcI, the inducible lysine decarboxylase, specifically interacts with a variant of complex I lacking NuoL, which is a subunit of the membrane arm. The LdcI is a cytoplasmic protein and protects E. coli against mild acid stress. Due to its attachment to the ΔNuoL variant, it is suggested to be involved in the assembly or the repair of Fe/S-clusters of complex I. To contribute to the understanding of the complex I assembly machinery in E. coli, the state of complex I will be investigated in different deletion strains.

 

Methods

  • Manipulation of genes (AQUA cloning, λ- red mediated recombination)
  • Heterologous production and purification
  • Enzyme kinetics
  • UV/Vis and EPR spectroscopy
  • Native PAGE
  • Fluorescence microscopy