Biological hydrogen activation is cooperative: One H2H2 activates the dinuclear center and a second H2H2 is oxidized

Molecular hydrogen is activated by two classes of enzymes, Fe-hydrogenases and NiFe-hydrogenases, which have similar sulfur-bridged dinuclear metal active sites with biologically unusual ligands CO and CN-CN-. While detailed structural knowledge on the biocatalyst is available, the mode and the locus of binding of the substrate, H2H2, has not yet been established. A kinetic study of the hydrogen oxidation reaction by Fe-hydrogenase from Desulfovibrio vulgaris   (Hildenborough) reveals a biphasic activation mechanism from as isolated, resting enzyme, via an intermediate state of relatively low activity, to maximally active enzyme. H2H2 itself is the causative agent for the two subsequent enzyme activation processes. Kinetic model analysis suggests that the steady-state assumption of Michaelis–Menten kinetics does not apply to this hydrogenase. It appears that activation by hydrogen is cooperative: a catalytic H2H2 with high binding affinity is turned over at high rate only after a second, regulatory H2H2 with low affinity has been bound to the active site. The proposed novel paradigm of two hydrogen molecules binding simultaneously to the active site may hold for hydrogenases in general.