Respiratory complex I from Escherichia coli does not transport Na+ in the absence of its NuoL subunit

•E. coli complex I devoid of the NuoL still transport H+ but does not transport Na+.•Reinforce the hypothesis for the presence of different coupling sites in complex I.•NuoL subunit is involved in both Na+ and H+ transport by complex I.•NuoL may contain Na+ and H+ translocating site(s) and influence NuoM/N subunits.

We investigated H+ and Na+ transport by complex I from Escherichia coli devoid of the NuoL subunit, which is probably part of the ion translocating machinery. We observed that complex I devoid of the NuoL subunit still translocates H+, although to a smaller extension than the complete version of complex I, but does not transport Na+. Our results unequivocally reinforce the observation that E. coli complex I transports Na+ in the opposite direction to that of the H+ and show that NuoL subunit is involved in the translocation of both ions by complex I.