Proton-coupled electron transfer (PCET) is a fundamental process at the core of oxidation-reduction reactions for energy conversion. The [FeFe]-hydrogenases catalyze the reversible activation of molecular H2 through a unique metallocofactor, the H-cluster, which is finely tuned by the surrounding protein environment to undergo fast PCET transitions. The correlation of electronic and structural transitions at the H-cluster with proton-transfer (PT) steps has not been well-resolved experimentally. Here, we explore how modification of the conserved PT network via a Cys → Ser substitution at position 169 proximal to the H-cluster of Chlamydomonas reinhardtii [FeFe]-hydrogenase (CrHydA1) affects the H-cluster using electron paramagnetic resonance (EPR) and Fourier transform infrared (FTIR) spectroscopy. Despite a substantial decrease in catalytic activity, the EPR and FTIR spectra reveal different H-cluster catalytic states under reducing and oxidizing conditions. Under H2 or sodium dithionite reductive treatments, the EPR spectra show signals that are consistent with a reduced [4Fe-4S]H(+) subcluster. The FTIR spectra showed upshifts of νCO modes to energies that are consistent with an increase in oxidation state of the [2Fe]H subcluster, which was corroborated by DFT analysis. In contrast to the case for wild-type CrHydA1, spectra associated with Hred and Hsred states are less populated in the Cys → Ser variant, demonstrating that the exchange of -SH with -OH alters how the H-cluster equilibrates among different reduced states of the catalytic cycle under steady-state conditions.

Hydrogenases couple electrochemical potential to the reversible chemical transformation of H and protons, yet the reaction mechanism and composition of intermediates are not fully understood. In this Communication we describe the biophysical properties of a hydride-bound state (H) of the [FeFe]-hydrogenase from Chlamydomonas reinhardtii. The catalytic H-cluster of [FeFe]-hydrogenase consists of a [4Fe-4S] subcluster ([4Fe-4S]) linked by a cysteine thiol to an azadithiolate-bridged 2Fe subcluster ([2Fe]) with CO and CN ligands. Mössbauer analysis and density functional theory (DFT) calculations show that H consists of a reduced [4Fe-4S] coupled to a diferrous [2Fe] with a terminally bound Fe-hydride. The existence of the Fe-hydride in H was demonstrated by an unusually low Mössbauer isomer shift of the distal Fe of the [2Fe] subcluster. A DFT model of H shows that the Fe-hydride is part of a H-bonding network with the nearby bridging azadithiolate to facilitate fast proton exchange and catalytic turnover.

The [FeFe]-hydrogenase catalytic site H cluster is a complex iron sulfur cofactor that is sensitive to oxygen (O2). The O2 sensitivity is a significant barrier for production of hydrogen as an energy source in water-splitting, oxygenic systems. Oxygen reacts directly with the H cluster, which results in rapid enzyme inactivation and eventual degradation. To investigate the progression of O2-dependent [FeFe]-hydrogenase inactivation and the process of H cluster degradation, the highly O2-sensitive [FeFe]-hydrogenase HydA1 from the green algae Chlamydomonas reinhardtii was exposed to defined concentrations of O2 while monitoring the loss of activity and accompanying changes in H cluster spectroscopic properties. The results indicate that H cluster degradation proceeds through a series of reactions, the extent of which depend on the initial enzyme reduction/oxidation state. The degradation process begins with O2 interacting and reacting with the 2Fe subcluster, leading to degradation of the 2Fe subcluster and leaving an inactive [4Fe-4S] subcluster state. This final inactive degradation product could be reactivated in vitro by incubation with 2Fe subcluster maturation machinery, specifically HydF(EG), which was observed by recovery of enzyme activity.

While a general model of H2 activation has been proposed for [FeFe]-hydrogenases, the structural and biophysical properties of the intermediates of the H-cluster catalytic site have not yet been discretely defined. Electron paramagnetic resonance (EPR) spectroscopy and Fourier transform infrared (FTIR) spectroscopy were used to characterize the H-cluster catalytic site, a [4Fe-4S]H subcluster linked by a cysteine thiolate to an organometallic diiron subsite with CO, CN, and dithiolate ligands, in [FeFe]-hydrogenase HydA1 from Chlamydomonas reinhardtii (CrHydA1). Oxidized CrHydA1 displayed a rhombic 2.1 EPR signal (g = 2.100, 2.039, 1.997) and an FTIR spectrum previously assigned to the oxidized H-cluster (Hox). Reduction of the Hox sample with 100% H2 or sodium dithionite (NaDT) nearly eliminated the 2.1 signal, which coincided with appearance of a broad 2.3-2.07 signal (g = 2.3-2.07, 1.863) and/or a rhombic 2.08 signal (g = 2.077, 1.935, 1.880). Both signals displayed relaxation properties similar to those of [4Fe-4S] clusters and are consistent with an S = 1/2 H-cluster containing a [4Fe-4S]H(+) subcluster. These EPR signals were correlated with differences in the CO and CN ligand modes in the FTIR spectra of H2- and NaDT-reduced samples compared with Hox. The results indicate that reduction of [4Fe-4S]H from the 2+ state to the 1+ state occurs during both catalytic H2 activation and proton reduction and is accompanied by structural rearrangements of the diiron subsite CO/CN ligand field. Changes in the [4Fe-4S]H oxidation state occur in electron exchange with the diiron subsite during catalysis and mediate electron transfer with either external carriers or accessory FeS clusters.

The [FeFe]-hydrogenases ([FeFe] Hases) catalyze reversible H activation at the H-cluster, which is composed of a [4Fe-4S] subsite linked by a cysteine thiolate to a bridged, organometallic [2Fe-2S] ([2Fe]) subsite. Profoundly different geometric models of the H-cluster redox states that orchestrate the electron/proton transfer steps of H bond activation have been proposed. We have examined this question in the [FeFe] Hase I from Clostridium acetobutylicum (CaI) by Fourier-transform infrared (FTIR) spectroscopy with temperature annealing and H/D isotope exchange to identify the relevant redox states and define catalytic transitions. One-electron reduction of H led to formation of HH ([4Fe-4S]-Fe-Fe) and H' ([4Fe-4S]-Fe-Fe), with both states characterized by low frequency μ-CO IR modes consistent with a fully bridged [2Fe]. Similar μ-CO IR modes were also identified for HH of the [FeFe] Hase from Chlamydomonas reinhardtii (CrHydA1). The CaI proton-transfer variant C298S showed enrichment of an H/D isotope-sensitive μ-CO mode, a component of the hydride bound H-cluster IR signal, H. Equilibrating CaI with increasing amounts of NaDT, and probed at cryogenic temperatures, showed HH was converted to H. Over an increasing temperature range from 10 to 260 K catalytic turnover led to loss of H and appearance of H, consistent with enzymatic turnover and H formation. The results show for CaI that the μ-CO of [2Fe] remains bridging for all of the "H" states and that HH is on pathway to H and H evolution in the catalytic mechanism. These results provide a blueprint for designing small molecule catalytic analogs.

Hydrogenases display a wide range of catalytic rates and biases in reversible hydrogen gas oxidation catalysis. The interactions of the iron−sulfur-containing catalytic site with the local protein environment are thought to contribute to differences in catalytic reactivity, but this has not been demonstrated. The microbe Clostridium pasteurianum produces three [FeFe]-hydrogenases that differ in "catalytic bias" by exerting a disproportionate rate acceleration in one direction or the other that spans a remarkable 6 orders of magnitude. The combination of high-resolution structural work, biochemical analyses, and computational modeling indicates that protein secondary interactions directly influence the relative stabilization/destabilization of different oxidation states of the active site metal cluster. This selective stabilization or destabilization of oxidation states can preferentially promote hydrogen oxidation or proton reduction and represents a simple yet elegant model by which a protein catalytic site can confer catalytic bias.

An [FeFe]-hydrogenase from Clostridium pasteurianum, CpI, is a model system for biological H activation. In addition to the catalytic H-cluster, CpI contains four accessory iron-sulfur [FeS] clusters in a branched series that transfer electrons to and from the active site. In this work, potentiometric titrations have been employed in combination with electron paramagnetic resonance (EPR) spectroscopy at defined electrochemical potentials to gain insights into the role of the accessory clusters in catalysis. EPR spectra collected over a range of potentials were deconvoluted into individual components attributable to the accessory [FeS] clusters and the active site H-cluster, and reduction potentials for each cluster were determined. The data suggest a large degree of magnetic coupling between the clusters. The distal [4Fe-4S] cluster is shown to have a lower reduction potential (∼ < -450 mV) than the other clusters, and molecular docking experiments indicate that the physiological electron donor, ferredoxin (Fd), most favorably interacts with this cluster. The low reduction potential of the distal [4Fe-4S] cluster thermodynamically restricts the Fd/Fd ratio at which CpI can operate, consistent with the role of CpI in recycling Fd that accumulates during fermentation. Subsequent electron transfer through the additional accessory [FeS] clusters to the H-cluster is thermodynamically favorable.

A combination of nuclear resonance vibrational spectroscopy (NRVS), FTIR spectroscopy, and DFT calculations was used to observe and characterize Fe-H/D bending modes in CrHydA1 [FeFe]-hydrogenase Cys-to-Ser variant C169S. Mutagenesis of cysteine to serine at position 169 changes the functional group adjacent to the H-cluster from a -SH to -OH, thus altering the proton transfer pathway. The catalytic activity of C169S is significantly reduced compared to that of native CrHydA1, presumably owing to less efficient proton transfer to the H-cluster. This mutation enabled effective capture of a hydride/deuteride intermediate and facilitated direct detection of the Fe-H/D normal modes. We observed a significant shift to higher frequency in an Fe-H bending mode of the C169S variant, as compared to previous findings with reconstituted native and oxadithiolate (ODT)-substituted CrHydA1. On the basis of DFT calculations, we propose that this shift is caused by the stronger interaction of the -OH group of C169S with the bridgehead -NH- moiety of the active site, as compared to that of the -SH group of C169 in the native enzyme.