Isolation and Characterization of the Proton-translocating NADH:ubiquinone Oxidoreductase from Escherichia coli

Abstract

The proton-translocating NADH:ubiquinone oxidoreductase (complex I) was isolated from Escherichia coli by chromatographic steps performed in the presence of an alkylglucoside detergent at pH 6.0. The complex is obtained in a monodisperse state with a molecular mass of approximately 550,000 Da and is composed of 14 subunits. The subunits were assigned to the 14 genes of the nuo operon, partly based on their N-terminal sequences and partly on their apparent molecular masses. The preparation contains one noncovalently bound FMN/molecule. At least two binuclear (N1b and N1c) and three tetranuclear (N2, N3 and N4) iron-sulfur clusters were detected by EPR in the preparation when reduced with NADH. Their EPR characteristics remained mostly unaltered during the isolation process. After reconstitution in phospholipid membranes, the preparation catalyses piericidin-A-sensitive electron transfer from NADH to ubiquinone-2 with Km values similar to those of complex I in cytoplasmic membranes but with only 10% of the Vmax value. The isolated complex I was cleaved into three fragments when the pH was raised from 6.0 to 7.5 and the detergent exchanged to Triton X-100. One of these fragments is a water-soluble NADH dehydrogenase fragment which is composed of three subunits bearing at least four iron-sulfur clusters (N1b, N1c, N3 and N4) that can be reduced with NADH, one of them bearing FMN. The second, amphipathic, fragment, which is presumed to connect the NADH dehydrogenase fragment with the membrane, contains four subunits and at least one EPR-detectable iron-sulfur cluster whose spectral properties are reminiscent of the eucaryotic cluster N2. The third membrane fragment is composed of seven homologues of the mitochondrially encoded subunits of the eucaryotic complex I. This subunit arrangement coincidences to some extent with the order of the genes on the nuo operon. A topological model of the E. coli complex I is proposed.

Evidence for an association of ndh B, ndh J gene products and ferredoxin‐NADP‐reductase as components of a chloroplastic NAD(P)H dehydrogenase complex

“…Hypothetical scheme of the chloroplastic NAD(P)H dehydrogenase subunit organization. This scheme is based on the published organizations of the cyanobacterial [12] and bacterial [13] NADH dehydrogenase complexes as well as on the results presented in this paper. We suggest that the association of a FNR subunit allows the complex to use NADPH instead of NADH as a preferential substrate.…”

“…Alternatively, the putative NAD(P)H dehydrogenase complex might participate in cyclic electron flow around photosystem 1 (PS1) by allowing electrons to enter the plastoquinone (PQ) pool from a stromal NAD(P)H pool [9,10]. NADH dehydrogenase complexes from mitochondria [11], cyanobacteria [12] and more recently from Eseheriehia coli [13] have been *Corresponding author. Fax: (33) (1) 42 25 42 25.…”

Supramolecular organizations in the aerobic respiratory chain of Escherichia coli

“…coli K-12 (ATCC 23716) and selected respiratory chain mutants were grown manually in LuriaeBertani medium adjusted to pH 7, at 37 C, under vigorous agitation, the volume of cultures corresponding to one fifth of the total volume of the flasks, and harvested at early stationary phase. Upon suspension in MES 50 mM pH 6.0 [31] and disruption in a French press (6000 psi), cells were submitted to low speed centrifugation (14000 Â g, 15 min) to remove intact cells and cell debris and the supernatant was ultracentrifuged (138000 Â g, 2 h) to separate the soluble from the membrane fraction. The isolated membrane fraction was aliquoted, frozen in liquid nitrogen and stored at À80 C.…”

Genetic inactivation of the H+‐translocating NADH:ubiquinone oxidoreductase of Paracoccus denitrificans is facilitated by insertion of the ndh gene from Escherichia coli

“…Mitochondrial complex I is composed of more than 40 different protein subunits while NDH1 of Paracoccus denitrificans and Escherichia coli may contain 14 subunits [4,5]. Despite having about half the molecular mass of complex I and a third of its subunits, the P. denitrificans NDH1 contains the same set of redox centres [6,7].…”

“…Despite having about half the molecular mass of complex I and a third of its subunits, the P. denitrificans NDH1 contains the same set of redox centres [6,7]. Furthermore, the EPR spectra of all the detectable Fe-S clusters in P. denitrificans NDH1, unlike those of E. coli, are very similar to the respective clusters in complex I [5][6][7][8].…”