H/D exchange reactions and mechanistic aspects of the hydrogenases

In the presence of D2 gas (in a D2/H2O system), hydrogenases catalyze the proton-deuterium (H/D) exchange reaction, which results in the formation of HD and H2, measurable by mass spectrometry at mass peaks m/e 3 and 2, respectively. This technique can be used to study the activity and function of hydrogenases both in vitro, with isolated enzymes, and in vivo, with whole cells. It allows to follow the reductive activation of oxidized [NiFe]-hydrogenases and to identify physiological activators such as NADH and NADPH, of bidirectional hydrogenases. Besides, since the H/D exchange reaction involves proton exchange with the solvent, it has been used to monitor the effects of site-directed mutagenesis of amino acid residues postulated to belong to proton channels.