Heterologous production, isolation, characterization and crystallization of a soluble fragment of the NADH:ubiquinone oxidoreductase (complex I) from Aquifex aeolicus
| Autor: | Thorsten Friedrich, Katerina Dörner, Emile Schiltz, Cengiz Koç, Petra Hellwig, Ruth Hielscher, Markus Kohlstädt, Ramona Labatzke, Oliver Einsle |
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| Rok vydání: | 2008 |
| Předmět: |
Enzyme complex
NADH binding Protein subunit Flavin mononucleotide medicine.disease_cause Biochemistry Cofactor Electron Transport chemistry.chemical_compound Bacterial Proteins Oxidoreductase medicine Cloning Molecular Escherichia coli chemistry.chemical_classification Aquifex aeolicus Electron Transport Complex I biology Bacteria Electron Spin Resonance Spectroscopy biology.organism_classification Peptide Fragments Recombinant Proteins Protein Subunits chemistry Solubility Genes Bacterial biology.protein Spectrophotometry Ultraviolet Crystallization |
| Zdroj: | Biochemistry. 47(49) |
| ISSN: | 1520-4995 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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