Sulfide Protects [FeFe] Hydrogenases From O2

[FeFe] hydrogenases catalyze proton reduction and hydrogen oxidation with high rates and efficiency under physiological conditions, but are highly oxygen sensitive. The [FeFe] hydrogenase from Desulfovibrio desulfuricans (DdHydAB) can be purified under air in an oxygen stable inactive state Hox air. The formation of the Hox air state in vitro allows the handling of hydrogenases in air, making their implementation in biotechnological applications more feasible. Here, we report a simple and robust protocol for the formation of the Hox air state in DdHydAB and the [FeFe] hydrogenase from Chlamydomonas reinhardtii, which is based on high potential inactivation in the presence of sulfide.