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J. Biol. Chem., Vol. 283, Issue 38, 25979-25987, September 19, 2008
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From the
Department of Biochemistry and Biophysics, School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania 19104, the Division of Biochemistry, Department of Molecular and Experimental Medicine, The Scripps Research Institute, La Jolla, California 92037, and the ¶Department of Life Science, University of Hyogo, Kamigohri Akoh, Hyogo 678-1297, Japan
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.
Received for publication, May 26, 2008 , and in revised form, July 2, 2008.
* This work was supported, in whole or in part, by National Institutes of Health Grants R01GM30736 (to T. O.) and R01GM33712 (to T. Y.) from the United States Public Health Service. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
1 To whom correspondence may be addressed: 215 Anatomy/Chemistry Bldg., 36th and Hamilton Walk, Philadelphia, PA 19104-6059. Tel.: 215-898-2939; Fax: 215-573-3748; E-mail: eikoo{at}mail.med.upenn.edu. 2 To whom correspondence may be addressed: 214A Anatomy/Chemistry Bldg., 36th and Hamilton Walk, Philadelphia, PA 19104-6059. Tel.: 215-898-8024; Fax: 215-573-3748; E-mail: ohnishi{at}mail.med.upenn.edu.
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