Johannes Schimpf

Abstract

The energy-converting NADH:ubiquinone oxidoreductase, also known as respiratory complex I, is the first and largest enzyme of the respiratory chains of many prokaryotes and most eukaryotes. Complex I catalyzes the transfer of two electrons from NADH to ubiquinone. The redox-reaction is coupled with the translocation of four protons across the membrane. The enzyme has an L-shaped form and is made up of a peripheral arm catalyzing electron transfer and a membrane arm catalyzing proton translocation. E. coli complex I consists of 13 subunits and has numerous cofactors, namely one non-covalently bound FMN und nine iron sulfur (Fe/S)-clusters. The electrons are transferred through the FMN and a chain of seven iron sulfur clusters to ubiquinone. However, the mechanism of coupling electron transfer and proton translocation is still unknown. The most distal Fe/S-cluster N2 is in close proximity to the ubiquinone-binding site and it has a redox-dependent midpoint potential. It is coordinated by two adjacent cysteine residues, a binding motif rarely found in Fe/S‑proteins. It is suspected to play an essential role in coupling electron transfer and proton translocation. To investigate the importance of the cluster, the unusual binding motif of N2 will be replaced by a regular motif and the variants will be characterized in terms of electron transfer and proton translocation activity and by EPR-spectroscopy.

Methods

λ-Red mediated mutagenesis, protein production and purification, reconstitution of proteins in liposomes, fluorescence spectroscopy, EPR-spectroscopy, enzyme kinetics

Publications

• Dörner, K, Vranas, M, Schimpf, J, Straub,IR , Hoeser, J and Friedrich T (2017) Significance of the [2Fe-2S] Cluster N1a for Electron Transfer and Assembly of Escherichia coli Respiratory Complex I. Biochemistry, 56, 2770-2778. Doi: 10.1021/acs.biochem.6b01058
• Gnandt, E, Schimpf, J, Harter, C, Hoeser, J and Friedrich, T (2017) Reduction of the off- pathway iron-sulphur cluster N1a of Escherichia coli respiratory complex I restrains NAD+ dissociation. Sci. Rep., 7:8754. DOI:10.1038/s41598-017-09345-4.