Proton Coupled Electronic Rearrangement within the H‑Cluster as an Essential Step in the Catalytic Cycle of [FeFe] Hydrogenases

The active site of [FeFe] hydrogenases, the H-cluster, consists of a [4Fe–4S] cluster connected via a bridging cysteine to a [2Fe] complex carrying CO and CN^– ligands as well as a bridging aza-dithiolate ligand (ADT) of which the amine moiety serves as a proton shuttle between the protein and the H-cluster. During the catalytic cycle, the two subclusters change oxidation states: [4Fe–4S]_H²⁺ ⇔ [4Fe–4S]_H⁺ and [Fe­(I)­Fe­(II)]_H ⇔ [Fe­(I)­Fe­(I)]_H thereby enabling the storage of the two electrons needed for the catalyzed reaction 2H⁺ + 2e^– ⇄ H₂. Using FTIR spectro-electrochemistry on the [FeFe] hydrogenase from Chlamydomonas reinhardtii (<i>Cr</i>HydA1) at different pH values, we resolve the redox and protonation events in the catalytic cycle and determine their intrinsic thermodynamic parameters. We show that the singly reduced state H_red of the H-cluster actually consists of two species: H_red = [4Fe–4S]_H⁺ − [Fe­(I)­Fe­(II)]_H and H_redH⁺ = [4Fe–4S]_H²⁺ – [Fe­(I)­Fe­(I)]_H (H⁺) related by proton coupled electronic rearrangement. The two redox events in the catalytic cycle occur on the [4Fe–4S]_H subcluster at similar midpoint-potentials (−375 vs −418 mV); the protonation event (H_red/H_redH⁺) has a p<i>K</i>_a ≈ 7.2