Mutagenesis of Subunit N of the Escherichia coli Complex I. Identification of the Initiation Codon and the Sensitivity of Mutants to Decylubiquinone

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.

Functional Roles of Four Conserved Charged Residues in the Membrane Domain Subunit NuoA of the Proton-translocating NADH-Quinone Oxidoreductase from Escherichia coli

“…Because this length does not allow incorporation of the whole operon into expression vectors, a site-specific mutation is traditionally carried out by complementation of a cassette-inserted gene with a mutated gene in the expression plasmid (designated in trans complementation) (37)(38)(39)(40)(41)(42)(43). However, the in trans complementation procedure presents some problems when applied to a gene cluster.…”

“…For example, the cassette inserted in chromosomal DNA might interrupt the expression of the downstream genes (44). The only case that does not suffer this polar effect is when the mutated gene is at the last position in the operon (38). Another problem is that the mutated gene is under the control of a promoter in the expression plasmid, which often leads to overexpression of the mutated subunit.…”

Energy Transducing Roles of Antiporter-like Subunits in Escherichia coli NDH-1 with Main Focus on Subunit NuoN (ND2)

“…The NDH-1 crystal structure showed that NuoN is located close to the NuoAJK bundle (7,17). Previously Amarneh and Vik (35) reported that conserved lysine residues in the middle of the transmembrane helices were required for the energytransducing NDH-1 activity and that mutation of conserved N Glu 133 in the membrane helix only moderately (30%) reduced the energy-transducing NDH-1 activities. On the other hand, we and others showed that mutations of corresponding glutamic acids in NuoM and NuoL lead to almost total elimination of energy-transducing NDH-1 activities (24,27,36).…”

“…Uncoupler FCCP was added at a final concentration of 2 M to dissipate the potential. The H ϩ pump activity was followed by ACMA fluorescence quenching (35). Fifty-g of protein/ml of membrane vesicles, 2 M ACMA, and 200 M dNADH were used for the assay.…”

Electron Transfer in Subunit NuoI (TYKY) of Escherichia coli NADH:Quinone Oxidoreductase (NDH-1)

“…The proton ionophore FCCP was added to a final concentration of 2 M to uncouple the reaction. Proton pumping activity of NDH-1 was estimated from ACMA fluorescence quenching as described by Amarneh and Vik (45). The reaction mixture was the same as that of the membrane potential assay except that the E. coli membrane samples were added at 150 g of protein/ml, whereas oxonol VI was replaced with 2 M ACMA.…”