The [FeFe]-hydrogenases of bacteria and algae are the most efficient hydrogen conversion catalysts in nature. Their active-site cofactor (H-cluster) comprises a [4Fe-4S] cluster linked to a unique diiron site that binds three carbon monoxide (CO) and two cyanide (CN) ligands. Understanding microbial hydrogen conversion requires elucidation of the interplay of proton and electron transfer events at the H-cluster. We performed real-time spectroscopy on [FeFe]-hydrogenase protein films under controlled variation of atmospheric gas composition, sample pH, and reductant concentration. Attenuated total reflection Fourier-transform infrared spectroscopy was used to monitor shifts of the CO/CN vibrational bands in response to redox and protonation changes. Three different [FeFe]-hydrogenases and several protein and cofactor variants were compared, including element and isotopic exchange studies. A protonated equivalent (HoxH) of the oxidized state (Hox) was found, which preferentially accumulated at acidic pH and under reducing conditions. We show that the one-electron reduced state Hred' represents an intrinsically protonated species. Interestingly, the formation of HoxH and Hred' was independent of the established proton pathway to the diiron site. Quantum chemical calculations of the respective CO/CN infrared band patterns favored a cysteine ligand of the [4Fe-4S] cluster as the protonation site in HoxH and Hred'. We propose that proton-coupled electron transfer facilitates reduction of the [4Fe-4S] cluster and prevents premature formation of a hydride at the catalytic diiron site. Our findings imply that protonation events both at the [4Fe-4S] cluster and at the diiron site of the H-cluster are important in the hydrogen conversion reaction of [FeFe]-hydrogenases.

[FeFe]-hydrogenases are superior hydrogen conversion catalysts. They bind a cofactor (H-cluster) comprising a four-iron and a diiron unit with three carbon monoxide (CO) and two cyanide (CN) ligands. Hydrogen (H) and oxygen (O) binding at the H-cluster was studied in the C169A variant of [FeFe]-hydrogenase HYDA1, in comparison to the active oxidized (Hox) and CO-inhibited (Hox-CO) species in wildtype enzyme. Fe labeling of the diiron site was achieved by in vitro maturation with a synthetic cofactor analogue. Site-selective X-ray absorption, emission, and nuclear inelastic/forward scattering methods and infrared spectroscopy were combined with quantum chemical calculations to determine the molecular and electronic structure and vibrational dynamics of detected cofactor species. Hox reveals an apical vacancy at Fe in a [4Fe4S-2Fe] complex with the net spin on Fe whereas Hox-CO shows an apical CN at Fe in a [4Fe4S-2Fe(CO)] complex with net spin sharing among Fe and Fe (proximal or distal iron ions in [2Fe]). At ambient O pressure, a novel H-cluster species (Hox-O) accumulated in C169A, assigned to a [4Fe4S-2Fe(O)] complex with an apical superoxide (O) carrying the net spin bound at Fe. H exposure populated the two-electron reduced Hhyd species in C169A, assigned as a [(H)4Fe4S-2Fe(H)] complex with the net spin on the reduced cubane, an apical hydride at Fe, and a proton at a cysteine ligand. Hox-O and Hhyd are stabilized by impaired O protonation or proton release after H cleavage due to interruption of the proton path towards and out of the active site.

[FeFe]-Hydrogenases efficiently catalyze the uptake and evolution of H due to the presence of an inorganic [6Fe-6S]-cofactor (H-cluster). This cofactor is comprised of a [4Fe-4S] cluster coupled to a unique [2Fe] cluster where the catalytic turnover of H/H takes place. We herein report on the synthesis of a selenium substituted [2Fe] cluster [Fe{μ(SeCH)NH}(CO)(CN)] (ADSe) and its successful in vitro integration into the native protein scaffold of [FeFe]-hydrogenases HydA1 from Chlamydomonas reinhardtii and CpI from Clostridium pasteurianum yielding fully active enzymes (HydA1-ADSe and CpI-ADSe). FT-IR spectroscopy and X-ray structure analysis confirmed the presence of structurally intact ADSe at the active site. Electrochemical assays reveal that the selenium containing enzymes are more biased towards hydrogen production than their native counterparts. In contrast to previous chalcogenide exchange studies, the S to Se exchange herein is not based on a simple reconstitution approach using ionic cluster constituents but on the in vitro maturation with a pre-synthesized selenium-containing [2Fe] mimic. The combination of biological and chemical methods allowed for the creation of a novel [FeFe]-hydrogenase with a [2Fe2Se]-active site which confers individual catalytic features.

[FeFe] hydrogenases interconvert H2 into protons and electrons reversibly and efficiently. The active site H-cluster is composed of two sites: a unique [2Fe] subcluster ([2Fe]H) covalently linked via cysteine to a canonical [4Fe–4S] cluster ([4Fe–4S]H). Both sites are redox active and electron transfer is proton-coupled, such that the potential of the H-cluster lies very close to the H2 thermodynamic potential, which confers the enzyme with the ability to operate quickly in both directions without energy losses. Here, one of the cysteines coordinating [4Fe–4S]H (Cys362) in the [FeFe] hydrogenase from the green algae Chlamydomonas reinhardtii (CrHydA1) was exchanged with histidine and the resulting C362H variant was shown to contain a [4Fe–4S] cluster with a more positive redox potential than the wild-type. The change in the [4Fe–4S] cluster potential resulted in a shift of the catalytic bias, diminishing the H2 production activity but giving significantly higher H2 oxidation activity, albeit with a 200 mV overpotential requirement. These results highlight the importance of the [4Fe–4S] cluster as an electron injection site, modulating the redox potential and the catalytic properties of the H-cluster.

Alteration of the [4Fe–4S] cluster coordinating cysteines reveals their individual importance for [4Fe–4S] cluster binding, [2Fe] insertion and catalytic turnover.