Energy-converting NADH:ubiquinone oxidoreductase, respiratory complex I, is essential for cellular energy metabolism coupling NADH oxidation to proton translocation. The mechanism of proton translocation by complex I is still under debate. Its membrane arm contains an unusual central axis of polar and charged amino acid residues connecting the quinone binding site with the antiporter-type subunits NuoL, NuoM, and NuoN, proposed to catalyze proton translocation. Quinone chemistry probably causes conformational changes and electrostatic interactions that are propagated through these subunits by a conserved pattern of predominantly lysine, histidine, and glutamate residues. These conserved residues are thought to transfer protons along and across the membrane arm. The distinct charge distribution in the membrane arm is a prerequisite for proton translocation. Remarkably, the central subunit NuoM contains a conserved glutamate residue in a position that is taken by a lysine residue in the two other antiporter-type subunits. It was proposed that this charge asymmetry is essential for proton translocation, as it should enable NuoM to operate asynchronously with NuoL and NuoN. Accordingly, we exchanged the conserved glutamate in NuoM for a lysine residue, introducing charge symmetry in the membrane arm. The stably assembled variant pumps protons across the membrane, but with a diminished H
+
/e
−
stoichiometry of 1.5. Thus, charge asymmetry is not essential for proton translocation by complex I, casting doubts on the suggestion of an asynchronous operation of NuoL, NuoM, and NuoN. Furthermore, our data emphasize the importance of a balanced charge distribution in the protein for directional proton transfer.
NADH:ubiquinone oxidoreductase (respiratory complex I) plays a major role in cellular energy metabolism. Complex I deficiencies are the most common cause of mitochondrial dysfunction. Patients suffering from a variety of neurodegenerative diseases carry numerous mutations in the mitochondrially encoded subunits of the complex. The biochemical consequences of these mutations are largely unknown because these genes are difficult to access experimentally. Here, we use Escherichia coli as a model system to characterize the effect of a 7 bps inversion in mtND1 (m.3902‐3908inv7) that results in a triple mutation. The triple mutant grew poorly but contained a normal amount of the stably assembled variant. The variant showed no enzymatic activity, which might contribute to the deleterious effect of the mutation in humans.
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