neutralisation of specific surface carboxylates speeds up translocation of botulinum neurotoxin type b enzymatic domain botulinum neurotoxins translocate their enzymatic domain across vesicular membranes the molecular triggers of this process are unknown here we tested the possibility that this is elicited by protonation of conserved surface carboxylates glutamate48 glutamate653 and aspartate877 were identified as possible candidates and changed into amide this triple mutant showed increased neurotoxicity due to faster cytosolic delivery of the enzymatic domain membrane translocation could take place at less acidic ph thus neutralisation of specific negative surface charges facilitates membrane contact permitting a faster initiation of the toxin membrane insertion