Sabrina Burschel

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. In human its incomplete assembly or dysfunction leads to diseases such as Parkinson´s syndrome. The Escherichia coli complex I t represents a minimal structural model of the eukaryotic version consisting of 13 subunits (NuoA-N). It links the transfer of two electrons from NADH to ubiquinone with the translocation of four protons across the membrane. A hydrophilic arm catalysing electron transfer protrudes in the cytoplasm and harbours all cofactors, namely nine Fe/S-clusters and one FMN. Proton translocation takes place in the membrane arm. The assembly of complex I from its subunits and Fe/S-cluster is not well understood. Phylogenetic analyses have shown that the bacterial complex evolved from preexisting modules for electron transfer and proton translocation. The NADH dehydrogenase module delivers the electrons from NADH to the amphipathic hydrogenase module, which is also present in family of multi-subunit membranous [NiFe] hydrogenases. The third module, the transporter module, transports protons across the membrane and contains homologous to subunits of multi-subunit monovalent cation/proton antiporters. To contribute to the understanding of the complex I assembly machinery in E. coli different deletion strains will be investigated for a possible loss in fully assembled and active complex I.

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