Analysis of ND5 mutations in nuoL of E. coli. (A) The locations of human mutations I149S (red) and Y159H (blue) are shown in ND5 (green), using PDB file 5xtd (Gu et al., 2016). (B) ND4 (light yellow) is shown. (C) The locations of E. coli residues L148 (red) and Y158 are shown in nuoL (green), using PDB file 3rko (Efremov and Sazanov, 2011). (D) nuoM (light yellow) is shown. (E, F) dNADH-oxidase activities of membrane vesicles prepared from the E. coli mutants are shown compared to a wild type sample prepared the same day. (G, H) Proton translocation rates from the same samples shown in panels E and F are indicated by fluorescence quenching of the acridine dye ACMA. (I) Membrane vesicles from E. coli cells carrying the modeled human mutations, nuoL_L148S and nuoL_Y158H, were prepared using a two-plasmid, time-delayed expression of nuoL. dNADH-oxidase rates are shown compared to that from cells expressing all nuo genes from a single plasmid. (J) Proton translocation rates from the same samples shown in panel I are indicated by fluorescence quenching of ACMA. (E and F) ns, not significant; ****P < 0.0001. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)