Characterization of the Overproduced NADH Dehydrogenase Fragment of the NADH:Ubiquinone Oxidoreductase (Complex I) from Escherichia coli

Abstract

The proton-pumping NADH:ubiquinone oxidoreductase of Escherichia coli is composed of 14 different subunits and contains one FMN and up to nine iron-sulfur clusters as prosthetic groups. By use of salt treatment, the complex can be split into an NADH dehydrogenase fragment, a connecting fragment and a membrane fragment. The water-soluble NADH dehydrogenase fragment has a molecular mass of approximately 170,000 Da and consists of the subunits NuoE, F, and G. The fragment harbors the FMN and probably six iron-sulfur clusters, four of them being observable by EPR spectroscopy. Here, we report that the fully assembled fragment can be overproduced in E. coli when the genes nuoE, F, and G were simultaneously overexpressed with the genes nuoB, C, and D. Furthermore, riboflavin, sodium sulfide, and ferric ammonium citrate have to be added to the culture medium. The fragment was purified from the cytoplasm by means of ammonium sulfate fractionation and chromatographic steps. The preparation contains one noncovalently bound FMN per molecule. Two binuclear (N1b and N1c) and two tetranuclear (N3 and N4) iron-sulfur clusters were detected by EPR in the NADH reduced preparation with spectral characteristics identical with those of the corresponding clusters in complex I. The preparation fulfills all prerequisites for crystallization of the fragment.

Identification of Mitochondrial Complex I Assembly Intermediates by Tracing Tagged NDUFS3 Demonstrates the Entry Point of Mitochondrial Subunits

“…It is conceivable that assembly reflects the conserved evolutionary structural relationship of CI subunits via the formation of distinct modules (14,30). The evolutionary conserved relation between the NDUFS2 and NDUFS3 as fused proteins in several bacterial species and as part of the hydrogenase module of CI (31,32) corresponds well with their early association during the assembly process. The hydrogenase module is important early in assembly, as illustrated by the finding that the overexpressed dehydrogenase module of E. coli NDH-1 is only incorporated when the homologues of the NDUFS3, NDUFS2, and NDUFS7 subunits are also overexpressed (31).…”

Inactivation of genes coding for mitochondrial Nd7 and Nd9 complex I subunits in Chlamydomonas reinhardtii. Impact of complex I loss on respiration and energetic metabolism

“…In the fungus N. crassa, the disruption of the corresponding genes prevents the activity and assembly of the matricial arm while a membrane part of the complex is still present in mitochondrial membranes (Duarte et al, 1998;Schulte and Weiss, 1995). In E. coli, the nuoC gene encodes a fused version of the two subunits (Braun et al, 1998). Although no structurally intact complex is present in membranes of the nuoC-null mutant, a soluble 170 kD fragment corresponding to the NADH dehydrogenase module (NuoE/NDUFV2/24 kD, NuoF/NDUFV1/51 kD, NuoG/NDUFS1/75 kD) is still active (Erhardt et al, 2012).…”

Site-Specific S-Glutathiolation of Mitochondrial NADH Ubiquinone Reductase

“…al. has suggested that the 75 kDa subunit is associated structurally with the 51-and 24 kDa subunits of the Fp subcomplex based on the structural homology related to the α (51 and 24 kDa) and γ (75 kDa) subunits of the NAD-reducing hydrogenases and that the NuoE, F and G subunits of E. coli Complex I can be co-expressed to form a catalytically active recombinant subcomplex (1,34,35). The information of X-ray structure from T. thermophilius Complex I supports this prediction (32).…”