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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 (CrHydA1) 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 …