Iron-Sulfur Cluster N5 Is Coordinated by an HXXXCXXCXXXXXC Motif in the NuoG Subunit of Escherichia coli NADH:Quinone Oxidoreductase (Complex I)

Abstract

, 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.The proton-translocating NADH:ubiquinone oxidoreductase (EC 1.6.5.3) (complex I) is the largest energy-transducing complex in the aerobic respiratory chains of many prokaryotes and eukaryotes (1-3). Complex I is one of the most complicated and elaborate iron-sulfur (Fe/S)3 proteins yet known (4). Recently, the three-dimensional structure of the hydrophilic domain of Thermus thermophilus HB-8 complex I has been determined at 3.3 Å resolution (5). It revealed the spatial localization of all of the redox centers and their coordinating amino acid residues. For the sake of simplicity, we will use the Escherichia coli nomenclature for each subunit. The primary electron acceptor of complex I is a noncovalently bound flavin mononucleotide (FMN (Fig. 1A) (5, 6). These seven Fe/S clusters are lined up to form the main electron transfer pathway, whereas a [2Fe-2S] cluster N1a in NuoE and a [4Fe-4S] cluster N7 in NuoG stick out from the main pathway. It has been suggested that clusters N1a and N7 may have different roles (5, 7). The sequence analysis has confirmed that all of the Fe/S clusters except cluster N7 are conserved (8).EPR spectroscopy has been most informative for the analysis of Fe/S clusters. However, because the sensitivity and resolution of EPR spectroscopy are much lower than those of spectrophotometry, considerable spectral overlaps exist. Therefore, to make a definitive assignment of the spectra to each of the specific Fe/S clusters is sometimes very difficult. In addition, some Fe/S clusters may not be detectable by EPR when the spin relaxation time is too short, or when the Fe/S cluster is not paramagnetic under certain chemical or electronic conditions. In fact, E. coli complex I contains at least six EPR-detectable Fe/S clusters as follows: N1a, N1b, N2, N3, N4, and N7. However, N5 signals have not been detected so far. Cluster N6a and N6b signals have not been characterized sufficiently. Another problem is that EPR identification of an Fe/S cluster residing in the overexpressed single subunit could be misleading, because its EPR signals may be altered from tho...

Copper binding in IscA inhibits iron‐sulphur cluster assembly in Escherichia coli

“…Indeed, similar results were obtained when recombinant aconitase B [4Fe-4S] cluster (Varghese et al, 2003) or phosphogluconate dehydratase [4Fe-4S] cluster (Jang and Imlay, 2007) was expressed in the E. coli copA/cueO/cusA mutant cells grown in LB media supplemented with CuSO 4 (200 μM) (data not shown). We also examined the effect of CuSO4 (200 μM) on native NADH dehydrogenase I which requires multiple iron-sulphur clusters for its catalytic activity (Nakamaru-Ogiso et al, 2008). Using deamino-NADH as specific substrate for NADH dehydrogenase I (Matsushita et al, 1987), we found that addition of CuSO4 (200 μM) dramatically inhibited the enzyme activity of NADH dehydrogenase I but without significantly affecting the activity of pre-existed NADH dehydrogenase I in the E. coli copA/ cueO/cusA mutant cells (Fig.…”

Bacteria, yeast, worms, and flies: Exploiting simple model organisms to investigate human mitochondrial diseases

“…In fact, utilizing P. denitrificans, Thermus thermophilus, Rhodobacter capsulatus and E. coli , several groups succeeded in identifying 8–9 iron-sulfur clusters, N1a, N1b, N2, N3, N4, N5, N6a, N6b, and N7 in each of the peripheral subcomplex arms of complex I (Chevallet et al 1997; Dupuis et al 1998; Flemming et al 2003; Nakamaru-Ogiso et al 2008; Nakamaru-Ogiso et al 2002; Nakamaru-Ogiso et al 2005; Rasmussen et al 2001; Velazquez et al 2005; Yano et al 1999; Yano et al 2003; Yano et al 1994; Yano et al 1995). Those assignments were recently confirmed by demonstration of the X-ray crystal structure of the hydrophilic peripheral part of T. thermophilus complex I that was determined at 3.3 angstrom (Å) resolution (Sazanov and Hinchliffe 2006).…”

Determination of the intrinsic redox potentials of FeS centers of respiratory complex I from experimental titration curves

“…A number of groups have reported spectroscopic parameters ( g -values) and redox properties (midpoint reduction potentials, E mi ) for individual FeS centers [10–14]. The unambiguous assignment of spectra to individual FeS clusters, however, has been difficult due to the similarity of the g -values for certain clusters, the electrostatic interaction between the redox centers, and the relatively similar midpoint reduction potentials (most of the central FeS clusters have long been considered equipotential).…”