O2-independent formation of the inactive states of NiFe hydrogenase

Biotechnological applications of hydrogenases are limited by their susceptibility to inactivation by oxygen, thought to proceed by trapping a reduced O 2 in the active site. Electrochemical and spectroscopic studies using various electron acceptors now show that oxygen inactivation is not linked to oxygen atom donation. We studied the mechanism of aerobic inactivation of Desulfovibrio fructosovorans nickel-iron (NiFe) hydrogenase by quantitatively examining the results of electrochemistry, EPR and FTIR experiments. They suggest that, contrary to the commonly accepted mechanism, the attacking O 2 is not incorporated as an active site ligand but, rather, acts as an electron acceptor. Our findings offer new ways toward the understanding of O 2 inactivation and O 2 tolerance in NiFe hydrogenases.