Abstract
The active site of the [FeFe]-hydrogenases features a binuclear [2Fe](H) sub-cluster that contains a unique bridging amine moiety close to an exposed iron center. Heterolytic splitting of H(2) results in the formation of a transient terminal hydride at this iron site, which, however is difficult to stabilize. We show that the hydride intermediate forms immediately when [2Fe](H) is replaced with [2Ru](H) analogues through artificial maturation. Outside the protein, the [2Ru](H) analogues form bridging hydrides, which rearrange to terminal hydrides after insertion into the apo-protein. H/D exchange of the hydride only occurs for [2Ru](H) analogues containing the bridging amine moiety.