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