Overexpression of the Escherichia coli nuo-Operon and Isolation of the Overproduced NADH:Ubiquinone Oxidoreductase (Complex I)

Abstract

The proton-pumping NADH:ubiquinone oxidoreductase (complex I) of Escherichia coli is composed of 13 different subunits. The corresponding genes are organized in the nuo-operon (from NADH:ubiquinone oxidoreductase) at min 51 of the E. coli chromosome. To study the structure and function of this complex enzyme, a suitable purification protocol yielding sufficient amount of a stable protein is needed. Here, we report the overproduction of complex I in E. coli and a novel isolation procedure of the complex. Overexpression of the nuo-operon on the chromosome was achieved by replacing its 5'-promotor region with the phage-T7 RNA polymerase promotor and by expressing the genes with the T7 RNA polymerase coded on an inducible plasmid. It is shown by means of enzymatic activity and EPR spectroscopy of cytoplasmic membranes that complex I is overproduced 4-fold after induction. Complex I was isolated by chromatographic steps performed in the presence of dodecyl maltoside. The preparation comprises all subunits and known cofactors and exhibits a high enzymatic activity and inhibitor sensitivity. Due to its stability over a wide pH range and at very high salt concentrations, this preparation is well suited for structural investigations.

The Escherichia coli NADH:Ubiquinone Oxidoreductase (Complex I) Is a Primary Proton Pump but May Be Capable of Secondary Sodium Antiport

“…Purification of Complex I-Our standard purification method was modified to circumvent the washing of the membranes with NaBr, which led to an incubation of complex I with 1.4 M Na ϩ (26). Complex I was extracted from the membranes with 3% dodecyl maltoside and eluted by anion exchange chromatography on Source 15Q at 280 mM NaCl.…”

A Novel, Enzymatically Active Conformation of the Escherichia coli NADH:Ubiquinone Oxidoreductase (Complex I)

“…The v max of this reaction was about 40 s Ϫ1 , and the K M app values for NADH and decylubiquinone were 5 and 15 M, respectively. Thus, in a buffer of low ionic strength, complex I performed an enzymatic activity with kinetic parameters comparable with the enzyme reconstituted into phospholipids (11). The reaction was inhibitor-sensitive.…”

“…Isolation and Splitting of the E. coli Complex I-E. coli complex I was isolated from a strain overproducing complex I (11). Membrane proteins were extracted from plasma membranes using dodecyl maltoside as detergent.…”

“…(Amersham Biosciences) and size-exclusion chromatography on Ultrogel AcA 34 (Serva); both steps were performed in the presence of dodecyl maltoside (11). Aliquots of the same preparation were adjusted to different buffers by repeated concentration (Ultrafree 15, Millipore) and dilution with the desired buffer.…”

“…As a particularity of E. coli, nuoC and -D are fused and code for one subunit NuoCD (9). The E. coli complex I has been isolated in the presence of alkylglycoside detergents by means of chromatographic steps (10,11). The preparation is solely made up of the 13 subunits encoded by the nuo genes, which disintegrates into a so-called NADH dehydrogenase fragment, a connecting fragment, and a membrane fragment by changing the pH and the detergent (10).…”