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.
Kohlstädt
Markus
M
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.
Dörner
Katerina
K
Labatzke
Ramona
R
Koç
Cengiz
C
Heilscher
Ruth
R
Schiltz
Emile
E
Einsle
Oliver
O
Hellwig
Petra
P
Friedrich
Thorsten
T
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
D001426:Bacterial Proteins; D010446:Peptide Fragments; D021122:Protein Subunits; D011994:Recombinant Proteins; D042967:Electron Transport Complex I