The kinetics of the activation and anaerobic inactivation processes of Desulfovibrio gigas hydrogenase have been measured in D2O by FTIR spectroelectrochemistry. A primary kinetic solvent isotope effect was observed for the inactivation process but not for the activation step. The kinetics of these processes have been also measured after replacement of a glutamic residue placed near the active site of an analogous [NiFe] hydrogenase from Desulfovibrio fructosovorans. Its replacement by a glutamine affected greatly the kinetics of the inactivation process but only slightly the activation process. The interpretation of the experimental results is that the rate-limiting step for anaerobic inactivation is the formation from water of a μ-OH- bridge at the hydrogenase active site, and that Glu25 has a role in this step.