H₂ turnover at the [FeFe]-hydrogenase cofactor (H-cluster) is assumed to follow a reversible heterolytic mechanism, first yielding a proton and a hydrido-species which again is double-oxidized to release another proton. Three of the four presumed catalytic intermediates (H_ox, H_red/H_red and H_sred) were characterized, using various spectroscopic techniques. However, in catalytically active enzyme, the state containing the hydrido-species, which is eponymous for the proposed heterolytic mechanism, has yet only been speculated about. We use different strategies to trap and spectroscopically characterize this transient hydride state (H_hyd) for three wild-type [FeFe]-hydrogenases. Applying a novel set-up for real-time attenuated total-reflection Fourier-transform infrared spectroscopy, we monitor compositional changes in the state-specific infrared signatures of [FeFe]-hydrogenases, varying buffer pH and gas composition. We selectively enrich the equilibrium concentration of H_hyd, applying Le Chatelier’s principle by simultaneously increasing substrate and product concentrations (H₂/H⁺). Site-directed manipulation, targeting either the proton-transfer pathway or the adt ligand, significantly enhances H_hyd accumulation independent of pH.

The unmatched catalytic turnover rates of [FeFe]-hydrogenases require an exceptionally efficient proton-transfer (PT) pathway to shuttle protons as substrates or products between bulk water and catalytic center. For clostridial [FeFe]-hydrogenase CpI such a pathway has been proposed and analyzed, but mainly on a theoretical basis. Here, eleven enzyme variants of two different [FeFe]-hydrogenases (CpI and HydA1) with substitutions in the presumptive PT-pathway are examined kinetically, spectroscopically, and crystallographically to provide solid experimental proof for its role in hydrogen-turnover. Targeting key residues of the PT-pathway by site directed mutagenesis significantly alters the pH-activity profile of these variants and in presence of H₂ their cofactor is trapped in an intermediate state indicative of precluded proton-transfer. Furthermore, crystal structures coherently explain the individual levels of residual activity, demonstrating e.g. how trapped H₂O molecules rescue the interrupted PT-pathway. These features provide conclusive evidence that the targeted positions are indeed vital for catalytic proton-transfer.

[FeFe]-hydrogenases are efficient H₂-catalysts, yet upon contact with dioxygen their catalytic cofactor (H-cluster) is irreversibly inactivated. Here, we combine X-ray crystallography, rational protein design, direct electrochemistry, and Fourier-transform infrared spectroscopy to describe a protein morphing mechanism that controls the reversible transition between the catalytic H_ox-state and the inactive but oxygen-resistant H_inact-state in [FeFe]-hydrogenase CbA5H of Clostridium beijerinckii. The X-ray structure of air-exposed CbA5H reveals that a conserved cysteine residue in the local environment of the active site (H-cluster) directly coordinates the substrate-binding site, providing a safety cap that prevents O₂-binding and consequently, cofactor degradation. This protection mechanism depends on three non-conserved amino acids situated approximately 13 Å away from the H-cluster, demonstrating that the 1st coordination sphere chemistry of the H-cluster can be remote-controlled by distant residues.

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.

Photohydrogen generation in microalgae is catalysed by hydrogenases, which receive electrons from photosystem I via the ferredoxin PETF. The dominant acceptor of photosynthetic electrons is, however, ferredoxin-NADP⁺-oxidoreductase (FNR). By utilizing targeted ferredoxin and FNR variants in a light-dependent competition assay, electrons can be redirected to the hydrogenase yielding a five-fold enhanced hydrogen evolution activity.

Significance: Activation of hydrogen in biology is achieved by enzymes rivalling the platinum metals in catalytic activity. Attached to an electrode, hydrogenases display electrocatalytic reversibility—just the tiniest potential bias from the equilibrium value drives oxidation or production of H ₂ at significant rates. Hydrogenases activate H ₂ heterolytically; hence, reversibility, an extreme marker for evolved efficiency, is expected to depend on electron and proton transfer processes occurring in concert. Experiments in which the long-range proton-transfer pathway in two [FeFe]-hydrogenases has been mildly disrupted without affecting the electron transfer pathway show that electrocatalytic reversibility fades as electron and proton transfers become temporally decoupled. These subtlest of observations demonstrate how evolution responded to the need for concerted electron–proton transfer in optimizing catalytic efficiency.