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 was recently obtained. 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 and her collaborators based on various EPR studies. Recently, Yakovlev et al. claimed that the EPR signals from clusters N4, N5, and N6b were misassigned (Proc Natl Acad Sci USA (2007) 104, 12720-12725). Here, we identified and characterized cluster N5 in E. 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 3K, 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, while 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.