The catalytic properties of hydrogenases are nature's answer to the seemingly simple reaction H ⇌ 2H + 2e. Members of the phylogenetically diverse subgroup of [NiFe] hydrogenases generally consist of at least two subunits, where the large subunit harbors the H-activating [NiFe] site and the small subunit contains iron-sulfur clusters mediating e transfer. Typically, [NiFe] hydrogenases are susceptible to inhibition by O. Here, we conducted system minimization by isolating and analyzing the large subunit of one of the rare members of the group of O-tolerant [NiFe] hydrogenases, namely the preHoxG protein of the membrane-bound hydrogenase from Ralstonia eutropha. Unlike previous assumptions, preHoxG was able to activate H as it clearly performed catalytic hydrogen/deuterium exchange. However, it did not execute the entire catalytic cycle described for [NiFe] hydrogenases. Remarkably, H activation was performed by preHoxG even in the presence of O, although the unique [4Fe-3S] cluster located in the small subunit and described to be crucial for tolerance toward O was absent. These findings challenge the current understanding of O tolerance of [NiFe] hydrogenases. The applicability of this minimal hydrogenase in basic and applied research is discussed.

Background: Biosynthesis of complex metal cofactors in [NiFe] hydrogenase is sensitive toward molecular oxygen. Results: A rubredoxin-like protein is required for hydrogenase maturation under aerobic conditions. Conclusion: The rubredoxin-like protein prevents oxidative damage of metallocenters, including the recently discovered [4Fe3S] center. Significance: Dedicated protection mechanisms enable biosynthesis of sophisticated metal centers in the presence of dioxygen.