pH-sensitive vibrational probe reveals a cytoplasmic protonated cluster in bacteriorhodopsin

Significance The vectorial transport of protons across membranes by pumps is central to cellular bioenergetics. A persistent problem in their study is the technical unfeasibility to simultaneously resolve the dynamics of all the relevant proton transfer steps by the same method, that is, those within the protein as well as those involving protonation changes of the aqueous medium, currently relying on complementary methods to map both. Here, we solved this limitation and monitored both internal and external protonation changes during the proton-pump mechanism of bacteriorhodopsin by time-resolved infrared spectroscopy. Our findings reveal inconsistencies with the proton uptake mechanism accepted for the last 25 years, highlighting the need for simultaneous and comprehensive monitoring of protonation changes to resolve the molecular mechanism of ion pumps.