PMID:12718520

Citation	Amarneh, B and Vik, SB (2003) Mutagenesis of subunit N of the Escherichia coli complex I. Identification of the initiation codon and the sensitivity of mutants to decylubiquinone. Biochemistry 42:4800-8
Abstract	The last gene in the nuo operon of Escherichia coli, nuoN, encodes a membrane-bound subunit of Complex I (NADH:ubiquinone oxidoreductase). In this report, the gene for subunit N was disrupted by a 163 bp deletion in the chromosome, resulting in the loss of Complex I function, as measured by deamino-NADH oxidase activity. This activity could be recovered after transformation of the mutant strain by a plasmid that contains the previously identified nuoN gene and the upstream intergenic region between nuoM and nuoN. Mutagenesis of the first ATG downstream of nuoM led to a loss of function, indicating that this is the likely initiation codon for nuoN, and predicting a protein of 485 amino acids and 52 044 Da. Thirty site-specific mutations in nuoN at 19 different positions were constructed in a vector that expresses the full-length subunit N with both an octahistidine tag and an HA epitope tag at the carboxyl terminus. Highly conserved charged and aromatic residues were selected for mutagenesis, as well as a substitution that occurs as a secondary mutation in Leber's hereditary optic neuropathy (LHON). Membranes from the mutant strains were tested for production of subunit N by immunoblots and for NADH-linked activities. Mutants with substitutions at six different positions (K158, K217, H224, K247, Y300, and K395) had rates of deamino-NADH oxidase activity that were no more than 50% of that of the wild type and had reduced rates of proton translocation. These mutants also showed enhanced inhibition by decylubiquinone, indicating that subunit N interacts with quinones. The mutation associated with LHON, G391S, had little effect on these functions.
Links	PubMed Online version:10.1021/bi0340346
Keywords	Amino Acid Sequence; Amino Acid Substitution; Animals; Base Sequence; Cell Membrane/enzymology; Codon, Initiator/genetics; Electron Transport Complex I; Escherichia coli/drug effects; Escherichia coli/enzymology; Escherichia coli/genetics; Humans; Models, Molecular; Molecular Sequence Data; Mutagenesis, Site-Directed; NADH, NADPH Oxidoreductases/chemistry; NADH, NADPH Oxidoreductases/genetics; Plasmids/genetics; Protein Structure, Secondary; Protein Subunits/chemistry; Protein Subunits/genetics; Recombinant Proteins/chemistry; Ubiquinone/analogs & derivatives; Ubiquinone/pharmacology
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