The gene order in the nuo-operon is not essential for the assembly of E. coli complex I.

Energy-converting NADH: ubiquinone oxidoreductase, respiratory complex I, plays an important role in cellular energy metabolism. Bacterial complex I is generally composed of 14 different subunits, seven of which are membranous and the other seven are globular proteins. They are encoded by the nuo-operon, whose gene order is strictly conserved in bacteria. The operon starts with nuoA encoding a membranous subunit followed by genes encoding globular subunits. To test the idea that NuoA acts as a seed to initiate the assembly of the complex in the membrane, we generated mutants that either lacked nuoA or contain nuoA at a different position within the operon. To enable the detection of putative assembly intermediates, the globular subunit NuoF and the membranous subunit NuoM were individually decorated with the fluorescent protein mCherry. Deletion of nuoA led to the assembly of an inactive complex in the membrane containing NuoF and NuoM. Re-arrangement of nuoA within the nuo-operon led to a slightly diminished amount of complex I in the membrane that was fully active. Thus, nuoA but not its distinct position in the operon is required for the assembly of E. coli complex I. Furthermore, we detected a previously unknown assembly intermediate in the membrane containing NuoM that is present in greater amounts than complex I.