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.

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 (Hox, Hred/Hred and Hsred) 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 (Hhyd) 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 Hhyd, 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 Hhyd 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 contain a H-converting cofactor (H-cluster) in which a canonical [4Fe-4S] cluster is linked to a unique diiron site with three carbon monoxide (CO) and two cyanide (CN) ligands (e.g., in the oxidized state, Hox). There has been much debate whether reduction and hydrogen binding may result in alternative rotamer structures of the diiron site in a single (Hred) or double (Hsred) reduced H-cluster species. We employed infrared spectro-electrochemistry and site-selective isotope editing to monitor the CO/CN stretching vibrations in [FeFe]-hydrogenase HYDA1 from Chlamydomonas reinhardtii. Density functional theory calculations yielded vibrational modes of the diatomic ligands for conceivable H-cluster structures. Correlation analysis of experimental and computational IR spectra has facilitated an assignment of Hred and Hsred to structures with a bridging hydride at the diiron site. Pronounced ligand rotation during μH binding seems to exclude Hred and Hsred as catalytic intermediates. Only states with a conservative H-cluster geometry featuring a μCO ligand are likely involved in rapid H turnover.

The six-iron cofactor of [FeFe]-hydrogenases (H-cluster) is the most efficient H 2 -forming catalyst in nature. It comprises a diiron active site with three carbon monoxide (CO) and two cyanide (CN − ) ligands in the active oxidized state (H ox ) and one additional CO ligand in the inhibited state (H ox -CO). The diatomic ligands are sensitive reporter groups for structural changes of the cofactor. Their vibrational dynamics were monitored by real-time attenuated total reflection Fouriertransform infrared spectroscopy. Combination of 13 CO gas exposure, blue or red light irradiation, and controlled hydration of three different [FeFe]-hydrogenase proteins produced 8 H ox and 16 H ox -CO species with all possible isotopic exchange patterns. Extensive density functional theory calculations revealed the vibrational mode couplings of the carbonyl ligands and uniquely assigned each infrared spectrum to a specific labeling pattern. For H ox -CO, agreement between experimental and calculated infrared frequencies improved by up to one order of magnitude for an apical CN − at the distal iron ion of the cofactor as opposed to an apical CO. For H ox , two equally probable isomers with partially rotated ligands were suggested. Interconversion between these structures implies dynamic ligand reorientation at the H-cluster. Our experimental protocol for site-selective 13 CO isotope editing combined with computational species assignment opens new perspectives for characterization of functional intermediates in the catalytic cycle.[FeFe]-hydrogenase | isotope editing | infrared spectroscopy | density functional theory | cofactor dynamics T he reduction of protons to form molecular hydrogen (H 2 ) is catalyzed by [FeFe]-hydrogenases (1, 2). With a turnover rate of up to 10,000 H 2 molecules per second in a thermodynamically reversible reaction (3-5), [FeFe]-hydrogenases inspired synthetic hydrogen catalysts (6-8) and renewable fuel technology applications (9, 10). The mechanism of catalysis at the active site cofactor (H-cluster) needs to be elucidated. Further information on functional intermediates is required (11-16) and expected to emerge from spectroscopic studies on H-cluster constructs carrying siteselective isotopic reporter groups (17)(18)(19)(20) Formation of H ox -CO does not affect the formal redox state of the H-cluster, but leads to increased spin delocalization over the diiron site (28). CO binding inhibits H 2 turnover and protects the enzyme against O 2 and light-induced degradation (24,29,30).The vibrational modes of the CO and CN − ligands at the diiron site are well accessible by infrared (IR) spectroscopy because they are separated from protein backbone and liquid water bands.Infrared spectroscopy therefore has pioneered elucidation of the molecular structure of the H-cluster and identification of several redox states (24, 31). In particular, the CO stretching frequencies are highly sensitive to structural isomerism, redox transitions, ligand binding, and isotope exchange (11,12,15,18,24,31,32). 13 CO editing ...