Towards engineering O2-tolerance in [Ni–Fe] hydrogenases

Abstract

Hydrogenases catalyze the conversion between 2H⁺ + 2e⁻ and H₂. Most of these enzymes are inhibited by O₂, which represents a major drawback for their use in biotechnological applications. Improving hydrogenase O₂ tolerance is therefore a major contemporary challenge to allow the implementation of a sustainable hydrogen economy. A few bacteria, however, contain hydrogenases that activate H₂ even in the presence of O₂. Intriguingly, kinetic and spectroscopic studies lead to assuming that different mechanisms might be responsible for the resistance, depending on the enzyme type. The various hypotheses that emerged from these studies are still a matter of debate. In order to better understand the molecular bases of resistance to O₂ inhibition, we explored different methods to improve the O₂-tolerance of the O₂-sensitive [Ni–Fe] hydrogenase from Desulfovibrio fructosovorans. A whole bunch of mutants has been studied and fully characterized, which revealed that actually, different mechanisms can lead to O₂ tolerance. These mechanisms are described in this review and compared to the current hypothesis of O₂ tolerance.