F1F0-ATP synthase is a multisubunit enzyme responsible for the synthesis of ATP. Among its multiple subunits (8 in E. coli,17 in yeast S. cerevisiae,16 in vertebrates), two subunits a and c are known to play a central role controlling the H+ flow through the inner mitochondrial membrane which allows the subsequent synthesis of ATP, but the pathway followed by H+ within the two proteins is still a matter of debate. In fact, even though the structure of ATP synthase is now well defined, the molecular mechanisms determining the function of both F-1 and F-o domains are still largely unknown. In this study, we propose a pathway for proton migration along the ATP synthase by hydrogen-bonded chain mechanism, with a key role of serine and threonine residues, by X-ray diffraction data on the subunit a of E. coli Fo. (C) 2016 Elsevier Inc. All rights reserved.