Hydrogenases are nature’s catalysts designed to extract protons from molecular hydrogen (H2) thereby forming a reactive hydride species (H−). Those enzymes capable of catalyzing the complete reaction, H2 ↔ 2 e− + 2 H+, come in two different flavors, namely [NiFe]- and [FeFe]-hydrogenases [1, 2, 3]. They are assigned according to the metal content in their active sites. [FeFe]-hydrogenases, which are described in detail in Chapters 3 and 7, are usually highly active in the direction of proton reduction and display turnover rates of up to 20,000 hydrogen molecules per second [4]. However, [FeFe]-hydrogenases are exclusively synthesized under strictly anoxic conditions in their host organisms, and are immediately, and in most cases irreversibly, inactivated by traces of O2. The proposed mechanism of O2-mediated inactivation is described in Chapters 3 and 5. This property makes it extremely difficult to use these enzymes for applied purposes under aerobic conditions.