Identification of a Catalytic Iron-Hydride at the H-Cluster of [FeFe]-Hydrogenase

Hydrogenases couple electrochemical potential to the reversible chemical transformation of H₂ and protons, yet the reaction mechanism and composition of intermediates are not fully understood. In this Communication we describe the biophysical properties of a hydride-bound state (H_hyd) of the [FeFe]-hydrogenase from Chlamydomonas reinhardtii. The catalytic H-cluster of [FeFe]-hydrogenase consists of a [4Fe-4S] subcluster ([4Fe-4S]_H) linked by a cysteine thiol to an azadithiolate-bridged 2Fe subcluster ([2Fe]_H) with CO and CN-ligands. Mossbauer analysis and density functional theory (DFT) calculations show that H_hyd consists of a reduced [4Fe-4S]_H⁺ coupled to a diferrous [2Fe]_H with a terminally bound Fe-hydride. The existence of the Fe-hydride in H_hyd was demonstrated by an unusually low Mossbauer isomer shift of the distal Fe of the [2Fe]_H subcluster. A DFT model of H_hyd shows that the Fe-hydride is part of a H-bonding network with the nearby bridging azadithiolate to facilitate fast proton exchange and catalytic turnover.