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.[NiFe]-hydrogenases are generally much less active in proton reduction, which is most likely based on a different architecture of the active site as compared to [FeFe]-hydrogenases. However,[NiFe]-hydrogenases perform H2 oxidation at turnover rates of up to 10,000 s− 1, which meets the activity of platinum-based catalysts [4, 5]. Moreover, several [NiFe]-hydrogenases have been described that are capable of sustaining catalysis in the presence of O2. Four representatives of this type of hydrogenases are present in the aerobic H2-oxidizing β-proteobacterium Ralstonia eutropha [3, 6, 7](Figure 4.1); they will be in the spotlight of this chapter. According to the classification of Vignais and Billoud [1],[NiFe]-hydrogenases are categorize d in four different groups, in reference to their physiological function, cellular localization, and quaternary structure. This classification can now be expanded to a fifth group that has been defined recently [8](see Section 4.5). It is interesting to note that the four individual hydrogenases in R. eutropha are all members of different groups, demonstrating that the ability to cycle H2 in the presence of O2 evolved as a phylogenetically widespread feature.

So far, O2-tolerant H2 cycling has been defined as the capability of sustaining H2 cycling in the presence of O2. However, we have to anticipate that “O2-tolerant hydrogenases” display diverse behaviors towards O2. Two of the R. eutropha hydrogenases (reviewed in Sections 4.1 and 4.2) show a decline in H2 oxidizing activity