To gain insight into the rate of H/D exchange at the Cyr493 SH group we have conducted two types of isotope-labelled experiments. First, we followed what products were formed in the reaction of fumarate with d8-toluene in H2O, detecting d8-benzylsuccinate and d7benzylsuccinate by MS. If no H/D exchange occurred at the SH group, we would expect only the d8-product to be formed. The rate of formation of the d7-product (with one deuteron from toluene replaced by a proton from the reaction environment) would reveal the rate of SD/SH exchange during the reaction. We indeed detected the simultaneous appearance of both products i.e., d8-and d7-labelled benzylsuccinate. The specific activity of d8-benzylsuccinate formation turned out to be 2. We may assume that the enzyme changes its confirmation upon product release, so the relative energy change and the equilibrium shifts back to that of the glycyl radical state. This may help to safeguard the enzyme from accidental proteolysis, oxygen-dependent cleavage or other side-reactions.