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] ⇔ [4Fe-4S] and [Fe(I)Fe(II)] ⇔ [Fe(I)Fe(I)] thereby enabling the storage of the two electrons needed for the catalyzed reaction 2H + 2e ⇄ H. 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 of the H-cluster actually consists of two species: H = [4Fe-4S] - [Fe(I)Fe(II)] and HH = [4Fe-4S] - [Fe(I)Fe(I)] (H) related by proton coupled electronic rearrangement. The two redox events in the catalytic cycle occur on the [4Fe-4S] subcluster at similar midpoint-potentials (-375 vs -418 mV); the protonation event (H/HH) has a pK ≈ 7.2.

[FeFe]-hydrogenases are metalloenzymes that reversibly reduce protons to molecular hydrogen at exceptionally high rates. We have characterized the catalytically competent hydride state (Hhyd) in the [FeFe]-hydrogenases from both Chlamydomonas reinhardtii and Desulfovibrio desulfuricans using 57Fe Nuclear Resonance Vibrational Spectroscopy (NRVS) and Density Functional Theory (DFT). H/D exchange identified two Fe-H bending modes originating from the binuclear iron cofactor. DFT calculations show that these spectral features result from an iron-bound terminal hydride, with the Fe-H vibrational frequencies being highly dependent on interactions between the amine base of the catalytic cofactor with both hydride and the conserved cysteine terminating the proton transfer chain to the active site. The results indicate that Hhyd is the catalytic state one step prior H2 formation. The observed vibrational spectrum, therefore, provides mechanistic insight into the reaction coordinate for H2 bond formation by [FeFe]-hydrogenases.

Sensory type [FeFe] hydrogenases are predicted to play a role in transcriptional regulation by detecting the H level of the cellular environment. These hydrogenases contain the hydrogenase domain with distinct modifications in the active site pocket, followed by a Per-Arnt-Sim (PAS) domain. As yet, neither the physiological function nor the biochemical or spectroscopic properties of these enzymes have been explored. Here, we present the characterization of an artificially maturated, putative sensory [FeFe] hydrogenase from Thermotoga maritima (HydS). This enzyme shows lower hydrogen conversion activity than prototypical [FeFe] hydrogenases and a reduced inhibition by CO. Using FTIR spectroelectrochemistry and EPR spectroscopy, three redox states of the active site were identified. The spectroscopic signatures of the most oxidized state closely resemble those of the H state from the prototypical [FeFe] hydrogenases, while the FTIR spectra of both singly and doubly reduced states show large differences. The FTIR bands of both the reduced states are strongly red-shifted relative to the H state, indicating reduction at the diiron site, but with retention of the bridging CO ligand. The unique functional and spectroscopic features of HydS are discussed with regard to the possible role of altered amino acid residues influencing the electronic properties of the H-cluster.

Hydride state intermediates are known to occur in various hydrogen conversion enzymes, including the highly efficient [FeFe] hydrogenases. The intermediate state involving a terminal iron-bound hydride has been recognized as crucial for the catalytic mechanism, but its occurrence has up to now eluded unequivocal proof under (near) physiological conditions. Here we show that the terminal hydride in the [FeFe] hydrogenase from Chlamydomonas reinhardtii can be directly detected using solution H NMR spectroscopy at room temperature, opening new avenues for detailed in situ investigations under catalytic conditions.

[FeFe]-hydrogenases, HydAs, are unique biocatalysts for proton reduction to H. However, they suffer from a number of drawbacks for biotechnological applications: size, number and diversity of metal cofactors, oxygen sensitivity. Here we show that HydA from Megasphaera elsdenii (MeHydA) displays significant resistance to O. Furthermore, we produced a shorter version of this enzyme (MeH-HydA), lacking the N-terminal domain harboring the accessory FeS clusters. As shown by detailed spectroscopic and biochemical characterization, MeH-HydA displays the following interesting properties. First, a functional active site can be assembled in MeH-HydA in vitro, providing the enzyme with excellent hydrogenase activity. Second, the resistance of MeHydA to O is conserved in MeH-HydA. Third, MeH-HydA is more biased toward proton reduction than MeHydA, as the result of the truncation changing the rate limiting steps in catalysis. This work shows that it is possible to engineer HydA to generate an active hydrogenase that combines the resistance of the most resistant HydAs and the simplicity of algal HydAs, containing only the H-cluster.