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

Abstract

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] H 2+ ⇔ [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 – ⇄ H 2 . Using FTIR spectro-electrochemistry on the [FeFe] hydrogenase from Chlamydomonas reinhardtii ( Cr 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 red H + = [4Fe–4S] H 2+ – [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 red H + ) has a p K a ≈ 7.2.