title: Iron-sulfur cluster N5 is coordinated by an HXXXCXXCXXXXXC motif in the NuoG subunit of Escherichia coli NADH:quinone oxidoreductase (complex I).
abstract: NADH:quinone oxidoreductase (complex I) plays a central role in cellular energy metabolism, and its dysfunction is found in numerous human mitochondrial diseases. Although the understanding of its structure and function has been limited, the x-ray crystal structure of the hydrophilic part of Thermus thermophilus complex I recently became available. It revealed the localization of all redox centers, including 9 iron-sulfur clusters and their coordinating ligands, and confirmed the predictions mostly made by Ohnishi et al. (Ohnishi, T., and Nakamaru-Ogiso, E. (2008) Biochim. Biophys. Acta 1777, 703-710) based on various EPR studies. Recently, Yakovlev et al. (Yakovlev, G., Reda, T., and Hirst, J. (2007) Proc. Natl. Acad. Sci. U. S. A. 104, 12720-12725) claimed that the EPR signals from clusters N4, N5, and N6b were misassigned. Here we identified and characterized cluster N5 in the Escherichia coli complex I whose EPR signals had never been detected by any group. Using homologous recombination, we constructed mutant strains of H101A, H101C, H101A/C114A, and cluster N5 knock-out. Although mutant NuoEFG subcomplexes were dissociated from complex I, we successfully recovered these mutant NuoCDEFG subcomplexes by expressing the His-tagged NuoCD subunit, which had a high affinity to NuoG. The W221A mutant was used as a control subcomplex carrying wild-type clusters. By lowering temperatures to around 3 K, we finally succeeded in detecting cluster N5 signals in the control for the first time. However, no cluster N5 signals were found in any of the N5 mutants, whereas EPR signals from all other clusters were detected. These data confirmed that, contrary to the misassignment claim, cluster N5 has a unique coordination with His(Cys)(3) ligands in NuoG.
authors.affiliation: Department of Biochemistry and Biophysics, School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania 19104, USA. eikoo@mail.med.upenn.edu
authors.forename: Eiko
authors.initials: E
authors.lastname: Nakamaru-Ogiso
authors.forename: Akemi
authors.initials: A
authors.lastname: Matsuno-Yagi
authors.forename: Shinya
authors.initials: S
authors.lastname: Yoshikawa
authors.forename: Takao
authors.initials: T
authors.lastname: Yagi
authors.forename: Tomoko
authors.initials: T
authors.lastname: Ohnishi
mesh_terms: D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000818:Animals; D002417:Cattle; D018360:Crystallography, X-Ray / Q000379:methods; D004578:Electron Spin Resonance Spectroscopy; D042967:Electron Transport Complex I / Q000737:chemistry* / Q000502:physiology*; D007506:Iron-Sulfur Proteins / Q000737:chemistry*; D008024:Ligands; D008969:Molecular Sequence Data; D009154:Mutation; D009245:NADH Dehydrogenase / Q000737:chemistry* / Q000502:physiology*; D009841:Oligonucleotides / Q000737:chemistry; D010084:Oxidation-Reduction; D017386:Sequence Homology, Amino Acid; D013696:Temperature
chemical_list: D007506:Iron-Sulfur Proteins; D008024:Ligands; D009841:Oligonucleotides; C088908:NADH dehydrogenase II; D009245:NADH Dehydrogenase; D042967:Electron Transport Complex I