Fig. 1: Generation and evolution of unbranched ETS variants.

a Schematic showing the respiratory enzymes involved in the flow of electrons from NADH (donor) to oxygen (acceptor). NDH-I and NDH-II are the proton pumping and non-pumping NADH: quinone oxidoreductase, respectively. Dashed arrows for CBDs represent the vectorial mode of PMF generation. b Scheme for generating ETS variants translocating 1, 2, 3, or 4 proton(s) per electron. uETS is the unevolved strain and eETS is the evolved strain. A–D are the four independently evolved lineages of each strain. c Growth rates of ETS variants before and after ALE. d, f Predictive mechanistic interpretation of the impact of mutations observed in the evolved strains of (d) ETS-3H (sdhA) and (f) ETS-4H (yjjX) mutations on the structure and function of the protein. Mutations displayed are those from this study and other ALE experiments in ALEdb seen to mutate these genes¹⁰. Horizontal tracks display the reported features associated with the region of the protein. The mutations collected from ALEdb refer to experiments from the following set, respectively^12,49,50. Protein structures showing the amino acid residues mutated in (e) SdhA and (g) YjjX. Source Data available in Supplementary Table 3.