Spectroscopic investigations of a semi-synthetic [FeFe] hydrogenase with propane di-selenol as bridging ligand in the binuclear subsite: comparison to the wild type and propane di-thiol variants

[FeFe] Hydrogenases catalyze the reversible conversion of H2 into electrons and protons. Their catalytic site, the H-cluster, contains a generic [4Fe–4S]H cluster coupled to a [2Fe]H subsite [Fe2(ADT)(CO)3(CN)2]2−, ADT = µ(SCH2)2NH. Heterologously expressed [FeFe] hydrogenases (apo-hydrogenase) lack the [2Fe]H unit, but this can be incorporated through artificial maturation with a synthetic precursor [Fe2(ADT)(CO)4(CN)2]2−. Maturation with a [2Fe] complex in which the essential ADT amine moiety has been replaced by CH2 (PDT = propane-dithiolate) results in a low activity enzyme with structural and spectroscopic properties similar to those of the native enzyme, but with simplified redox behavior. Here, we study the effect of sulfur-to-selenium (S-to-Se) substitution in the bridging PDT ligand incorporated in the [FeFe] hydrogenase HydA1 from Chlamydomonas reinhardtii using magnetic resonance (EPR, NMR), FTIR and spectroelectrochemistry. The resulting HydA1-PDSe enzyme shows the same redox behavior as the parent HydA1-PDT. In addition, a state is observed in which extraneous CO is bound to the open coordination site of the [2Fe]H unit. This state was previously observed only in the native enzyme HydA1-ADT and not in HydA1-PDT. The spectroscopic features and redox behavior of HydA1-PDSe, resulting from maturation with [Fe2(PDSe)(CO)4(CN)2]2−, are discussed in terms of spin and charge density shifts and provide interesting insight into the electronic structure of the H-cluster. We also studied the effect of S-to-Se substitution in the [4Fe–4S] subcluster. The reduced form of HydA1 containing only the [4Fe–4Se]H cluster shows a characteristic S = 7/2 spin state which converts back into the S = 1/2 spin state upon maturation with a [2Fe]–PDT/ADT complex

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

Spontaneous activation of [FeFe]-hydrogenases by an inorganic [2Fe] active site mimic

International audienceHydrogenases catalyze the formation of hydrogen. The cofactor ('H-cluster') of [FeFe]-hydrogenases consists of a [4Fe-4S] cluster bridged to a unique [2Fe] subcluster whose biosynthesis in vivo requires hydrogenase-specific maturases. Here we show that a chemical mimic of the [2Fe] subcluster can reconstitute apo-hydrogenase to full activity, independent of helper proteins. The assembled H-cluster is virtually indistinguishable from the native cofactor. This procedure will be a powerful tool for developing new artificial H₂-producing catalysts