Demonstration of separate genetic loci encoding distinct membrane-bound respiratory NADH dehydrogenases in Escherichia coli

Abstract

The nature of the Escherichia coli membrane-bound NADH dehydrogenases and their role in the generation of the proton motive force has been controversial. One E. coli NADH:ubiquinone oxidoreductase has previously been purified to homogeneity, and its corresponding gene (ndh) has been isolated. However, two biochemically distinct E. coli NADH:ubiquinone oxidoreductase activities have been identified by others (K. Matsushita, T. Ohnishi, and H. R. Kaback, Biochemistry 26:7732-7737, 1987). An insertional mutation in the ndh gene has been introduced into the E. coli chromosome, and the resulting strain maintains membrane-bound NADH dehydrogenase activity, demonstrating that a second genetically distinct NADH dehydrogenase must be present. By standard genetic mapping techniques, the map position of a second locus (nuo) involved in the oxidation of NADH has been determined. The enzyme encoded by this locus probably translocates protons across the inner membrane, contributing to the proton motive force.The aerobic respiratory chain of Escherichia coli contains several dehydrogenases which catalyze the oxidation of various substrates. These dehydrogenases, including succinate dehydrogenase, D-lactate dehydrogenase, NADH dehydrogenase, and pyruvate oxidase, donate electrons to ubiquinone, which transfers the electrons to either of the two terminal oxidase complexes (1). The function of this electron transport chain is to generate a proton motive force across the membrane. This force, in turn, is responsible for a number of energy-dependent processes, such as ATP synthesis and active transport.The nature of the E. coli membrane-bound NADH dehydrogenases and their role in the generation of the proton motive force have been controversial. Young and Wallace isolated mutants deficient in membrane-bound NADH dehydrogenase activity by the inability of such mutants to utilize mannitol as the sole carbon source (28). These mutants were believed to contain single-locus mutations which were mapped to min 22 of the E. coli linkage map (2, 28). Subsequently, a gene encoding an E. coli NADH dehydrogenase (ndh) was cloned and sequenced (26,27). This membrane-associated NADH dehydrogenase was then purified from a strain harboring a recombinant plasmid (10). The purified enzyme (NDH) consists of a single polypeptide with a molecular mass of 47 kDa which contains flavin adenine dinucleotide but no iron (10, 11). It is highly active with ubiquinone-1 (Qi) as an electron acceptor (11). The data presented by Young and colleagues suggest that there are no iron-sulfur centers and no energy-coupling site associated with this NADH dehydrogenase.However, studies with inside-out membrane vesicles from E. coli strongly suggested that NADH oxidation is coupled to the generation of a proton motive force (14). In addition, Owen and Kaback (18)

Fermentation and alternative respiration compensate for NADH dehydrogenase deficiency in a prokaryotic model of DJ-1-associated Parkinsonism

“…NADH dehydrogenases 1 and 2 activities in membrane extracts (prepared from exponentialphase bacteria harvested at OD 600 0.4) were assayed by measuring the oxidation of 2,6-dichlorophenolindophenol at 600 nm, with NADH (NADH dehydrogenase 1+NADH dehydrogenase 2) or deamino-NADH (NADH dehydrogenase 1) as substrates (Calhoun & Gennis, 1993). The assay mixture contained the following in a final volume of 1 ml: 0.1 M Tris, pH 7.5, 100 mM NADH or deamino-NADH, 30 mM KCN and 10 mg membranes prepared by the French press procedure.…”

“…Eukaryotic D-amino acid oxidases (which are restricted to D-amino acid detoxification) are of limited metabolic interest. Eukaryotic acyl-CoA dehydrogenases are involved in the first step of fatty acyl-CoA degradation and transfer hydrogens to quinones; however, the next oxidoreduction step of b-oxidation and acetyl-CoA oxidation in the Krebs cycle produce NADH, so they would be of little use to overcome NADH dehydrogenase defects (Abdallah et al, 2007;Bindoff et al, 1989;Calhoun & Gennis, 1993;Koebmann et al, 2002;Le et al, 2012;Schapira & Gegg, 2011). Proline oxidase catalyses the conversion of proline to D 1 -pyrroline-5-carboxylate and transfers hydrogens to quinones via FAD, whereas the reverse reaction, catalysed by D 1 -pyrroline-5-carboxylate reductase, uses NAD(P)H as a cofactor; consequently, both reactions produce a cycle of proline synthesis and degradation that can transfer redox potential between cellular compartments, and might contribute to bypassing NADH dehydrogenase 1 deficiency.…”

FNR-dependent repression of ndh gene expression requires two upstream FNR-binding sites

“…The facultative anaerobe Escherichia coli possesses two NADH dehydrogenases, NdhI and NdhII, which serve as primary dehydrogenases in the aerobic respiratory chain (Calhoun & Gennis, 1993;Calhoun et al, 1993).…”

Mutations in NADH:ubiquinone oxidoreductase of Escherichia coli affect growth on mixed amino acids

“…This reaction does not lead to the formation of a proton motive force (25). Although the function of these two NADH dhs remains unclear, Calhoun and Gennis (6) propose that E. coli might regulate the amount of energy recovered from NADH oxidation by modulating the relative levels of these two NADH dhs.…”

“…Both loci were identified in related mutant strains, AN589 (52) and IY12 (6). Cells containing mutations in both nuo and ndh, e.g., those Strain CP762 was constructed by transducing the TnlO insertion (subsequently designated nuoG::TnlO-1) (Fig.…”