A mechanism to prevent production of reactive oxygen species by Escherichia coli respiratory complex I.

Respiratory complex I plays a central role in cellular energy metabolism coupling NADH oxidation to proton translocation. In humans its dysfunction is associated with degenerative diseases. Here we report the structure of the electron input part of Aquifex aeolicus complex I at up to 1.8 Å resolution with bound substrates in the reduced and oxidized states. The redox states differ by the flip of a peptide bond close to the NADH binding site. The orientation of this peptide bond is determined by the reduction state of the nearby [Fe-S] cluster N1a. Fixation of the peptide bond by site-directed mutagenesis led to an inactivation of electron transfer and a decreased reactive oxygen species (ROS) production. We suggest the redox-gated peptide flip to represent a previously unrecognized molecular switch synchronizing NADH oxidation in response to the redox state of the complex as part of an intramolecular feed-back mechanism to prevent ROS production.

Heterologous production, isolation, characterization and crystallization of a soluble fragment of the NADH:ubiquinone oxidoreductase (complex I) from Aquifex aeolicus.

The proton-pumping NADH:ubiquinone oxidoreductase (complex I) is the first enzyme complex of the respiratory chains in many bacteria and most eukaryotes. It is the least understood of all, due to its enormous size and unique energy conversion mechanism. The bacterial complex is in general made up of 14 different subunits named NuoA-N. Subunits NuoE, -F, and -G comprise the electron input part of the complex. We have cloned these genes from the hyperthermophilic bacterium Aquifex aeolicus and expressed them heterologously in Escherichia coli. A soluble subcomplex made up of NuoE and NuoF and containing the NADH binding site, the primary electron acceptor flavin mononucleotide (FMN), the binuclear iron-sulfur cluster N1a, and the tetranuclear iron-sulfur cluster N3 was isolated by chromatographic methods. The proteins were identified by N-terminal sequencing and mass spectrometry; the cofactors were characterized by UV/vis and EPR spectroscopy. Subunit NuoG was not produced in this strain. The preparation was thermostable and exhibited maximum NADH/ferricyanide oxidoreductase activity at 85 degrees C. Analytical size-exclusion chromatography and dynamic light scattering revealed the homogeneity of the preparation. First attempts to crystallize the preparation led to crystals diffracting more than 2 A.