title: Heterologous production, isolation, characterization and crystallization of a soluble fragment of the NADH:ubiquinone oxidoreductase (complex I) from Aquifex aeolicus.
abstract: 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.
authors.affiliation: Institut fur Organische Chemie and Biochemie, Albert-Ludwigs-Universitat, Albertstrasse 21, 79104 Freiburg, Germany, and Institut de Chimie UMR 7177, Laboratoire de spectroscopie vib. et electrochimie des biomolecules, CNRS, Universite Louis Pasteur, 4, rue Blaise Pascal, 67070 Strasbourg, France thorsten.friedrich@uni-freiburg.de.
authors.forename: Markus
authors.initials: M
authors.lastname: Kohlstädt
authors.forename: Katerina
authors.initials: K
authors.lastname: Dörner
authors.forename: Ramona
authors.initials: R
authors.lastname: Labatzke
authors.forename: Cengiz
authors.initials: C
authors.lastname: Koç
authors.forename: Ruth
authors.initials: R
authors.lastname: Heilscher
authors.forename: Emile
authors.initials: E
authors.lastname: Schiltz
authors.forename: Oliver
authors.initials: O
authors.lastname: Einsle
authors.forename: Petra
authors.initials: P
authors.lastname: Hellwig
authors.forename: Thorsten
authors.initials: T
authors.lastname: Friedrich
mesh_terms: D001419:Bacteria / Q000201:enzymology*; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D003001:Cloning, Molecular; D003460:Crystallization; D004578:Electron Spin Resonance Spectroscopy; D004579:Electron Transport; D042967:Electron Transport Complex I / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D005798:Genes, Bacterial; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D021122:Protein Subunits / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012995:Solubility; D013056:Spectrophotometry, Ultraviolet
chemical_list: D001426:Bacterial Proteins; D010446:Peptide Fragments; D021122:Protein Subunits; D011994:Recombinant Proteins; D042967:Electron Transport Complex I