O 2 -Tolerant H 2 Activation by an Isolated Large Subunit of a [NiFe] Hydrogenase.

The catalytic properties of hydrogenases are nature's answer to the seemingly simple reaction H-2 reversible arrow 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.