The oxygen-resistant [FeFe]-hydrogenase CbA5H harbors an unknown radical signal

[FeFe]-hydrogenases catalyze the reversible conversion of molecular hydrogen into protons and electrons with remarkable efficiency. However, their industrial applications are limited by their oxygen sensitivity. Recently, it was shown that the [FeFe]-hydrogenase from Clostridium beijerinckii (CbA5H) is oxygen-resistant and can be reactivated after oxygen exposure. In this work, we used multifrequency continuous wave and pulsed electron paramagnetic resonance (EPR) spectroscopy to characterize the active center of CbA5H, the H-cluster. Under oxidizing conditions, the spectra were dominated by an additional and unprecedented radical species. The generation of this radical signal depends on the presence of an intact H-cluster and a complete proton transfer pathway including the bridging azadithiolate ligand. Selective ⁵⁷Fe enrichment combined with isotope-sensitive electron-nuclear double resonance (ENDOR) spectroscopy revealed a spin density distribution that resembles an H-cluster state. Overall, we uncovered a radical species in CbA5H that is potentially involved in the redox sensing of CbA5H.

Electron paramagnetic resonance spectroscopy revealed an unprecedented radical species in the oxygen-resistant [FeFe]-hydrogenase CbA5H. Analysis of the isotope-sensitive data suggests that it is related to the active site, the H-cluster.     

The electronic structure of the H-cluster in the [FeFe]-hydrogenase from Desulfovibrio desulfuricans: a Q-band 57Fe-ENDOR and HYSCORE study

The active site of the (57)Fe-enriched [FeFe]-hydrogenase (i.e., the "H-cluster") from Desulfovibrio desulfuricans has been examined using advanced pulse EPR methods at X- and Q-band frequencies. For both the active oxidized state (H(ox)) and the CO inhibited form (H(ox)-CO) all six (57)Fe hyperfine couplings were detected. The analysis shows that the apparent spin density extends over the whole H-cluster. The investigations revealed different hyperfine couplings of all six (57)Fe nuclei in the H-cluster of the H(ox)-CO state. Four large 57Fe hyperfine couplings in the range 20-40 MHz were found (using pulse ENDOR and TRIPLE methods) and were assigned to the [4Fe-4S](H) (cubane) subcluster. Two weak (57)Fe hyperfine couplings below 5 MHz were identified using Q-band HYSCORE spectroscopy and were assigned to the [2Fe](H) subcluster. For the H(ox) state only two different 57Fe hyperfine couplings in the range 10-13 MHz were detected using pulse ENDOR. An (57)Fe line broadening analysis of the X-band CW EPR spectrum indicated, however, that all six (57)Fe nuclei in the H-cluster are contributing to the hyperfine pattern. It is concluded that in both states the binuclear subcluster [2Fe](H) assumes a [Fe(I)Fe(II)] redox configuration where the paramagnetic Fe(I) atom is attached to the [4Fe-4S](H) subcluster. The (57)Fe hyperfine interactions of the formally diamagnetic [4Fe-4S](H) are due to an exchange interaction between the two subclusters as has been discussed earlier by Popescu and Münck [Popescu, C.V.; Münck, E., J. Am. Chem. Soc. 1999, 121, 7877-7884]. This exchange coupling is strongly enhanced by binding of the extrinsic CO ligand. Binding of the dihydrogen substrate may induce a similar effect, and it is therefore proposed that the observed modulation of the electronic structure by the changing ligand surrounding plays an important role in the catalytic mechanism of [FeFe]-hydrogenase.     

In silico evaluation of proposed biosynthetic pathways for the unique dithiolate ligand of the H-cluster of [FeFe]-hydrogenase

The biosynthesis of the active site of the [FeFe]-hydrogenases (H-cluster) remains a tantalizing puzzle due to its unprecedented and complex ligand environment. It contains a [2Fe] cluster ([2Fe](H)) bearing cyanide and carbon monoxide ligands attached to low-valence Fe ions and an abiological dithiolate ligand (SCH(2)XCH(2)S)(2-) that bridges the two iron centers. Various experimentally testable hypotheses have already been put forward regarding the precursor molecule and the biosynthetic mechanism that leads to the formation of the dithiolate ligand. In this work, we report a density functional theory-based theoretical evaluation of these hypotheses. We find preference for a mechanistically simple and energetically favorable pathway that includes known radical-SAM (S-adenosylmethionine) catalyzed reactions. We modeled this pathway using a long alkyl chain precursor molecule that leads to the formation of pronanadithiolate (X = CH(2)). However, the same pathway can be readily adopted for the biosynthesis of the dithiomethylamine (X = NH) or the dithiomethylether (X = O) analog, provided that the proper precursor molecule is available.     

Characterization of a putative sensory [FeFe]-hydrogenase provides new insight into the role of the active site architecture

[FeFe]-hydrogenases are known for their high rates of hydrogen turnover, and are intensively studied in the context of biotechnological applications. Evolution has generated a plethora of different subclasses with widely different characteristics. The M2e subclass is phylogenetically distinct from previously characterized members of this enzyme family and its biological role is unknown. It features significant differences in domain- and active site architecture, and is most closely related to the putative sensory [FeFe]-hydrogenases. Here we report the first comprehensive biochemical and spectroscopical characterization of an M2e enzyme, derived from Thermoanaerobacter mathranii. As compared to other [FeFe]-hydrogenases characterized to-date, this enzyme displays an increased H₂ affinity, higher activation enthalpies for H⁺/H₂ interconversion, and unusual reactivity towards known hydrogenase inhibitors. These properties are related to differences in active site architecture between the M2e [FeFe]-hydrogenase and “prototypical” [FeFe]-hydrogenases. Thus, this study provides new insight into the role of this subclass in hydrogen metabolism and the influence of the active site pocket on the chemistry of the H-cluster.

Characterization of a group D putative sensory [FeFe]-hydrogenase reveals how the active site can be tuned to decrease CO inhibition and increase stability of a reduced H-cluster while retaining the ability to catalyze H⁺/H₂ interconversion.     

Probing the effects of one-electron reduction and protonation on the electronic properties of the Fe-S clusters in the active-ready form of [FeFe]-hydrogenases. A QM/MM investigation

A QM/MM investigation of the active-ready (H(ox)) form of [FeFe]-hydrogenase from D. desulfuricans, in which the electronic properties of all Fe-S clusters (H, F and F') have been simultaneously described using DFT, was carried out with the aim of disclosing a possible interplay between the H-cluster and the accessory iron-sulfur clusters in the initial steps of the catalytic process leading to H(2) formation. It turned out that one-electron addition to the active-ready form leads to reduction of the F'-cluster and not of the H-cluster. Protonation of the H-cluster in H(ox) is unlikely, and in any case it would not trigger electron transfer from the accessory Fe(4)S(4) clusters to the active site. Instead, one-electron reduction and protonation of the active-ready form trigger electron transfer within the protein, a key event in the catalytic cycle. In particular, protonation of the H-cluster after one-electron reduction of the enzyme lowers the energy of the lowest unoccupied molecular orbitals localized on the H-cluster to such an extent that a long-range electron transfer from the F'-cluster towards the H-cluster itself is allowed.     

Approaches to efficient molecular catalyst systems for photochemical H2 production using [FeFe]-hydrogenase active site mimics

The research on structural and functional biomimics of the active site of [FeFe]-hydrogenases is in an attempt to elucidate the mechanisms of H(2)-evolution and uptake at the [FeFe]-hydrogenase active site, and to learn from Nature how to create highly efficient H(2)-production catalyst systems. Undoubtedly, it is a challenging, arduous, and long-term work. In this perspective, the progresses in approaches to photochemical H(2) production using mimics of the [FeFe]-hydrogenase active site as catalysts in the last three years are reviewed, with emphasis on adjustment of the redox potentials and hydrophilicity of the [FeFe]-hydrogenase active site mimics to make them efficient catalysts for H(2) production. With gradually increasing understanding of the chemistry of the [FeFe]-hydrogenases and their mimics, more bio-inspired proton reduction catalysts with significantly improved efficiency of H(2) production will be realized in the future.     

Applications of X-ray absorption spectroscopy to biologically relevant metal-based chemistry

Recent developments in the understanding of the biosynthesis of the active site of the nitrogenase enzyme, the structure of the iron centre of [Fe]-hydrogenase and the structure and biomimetic chemistry of the [FeFe] hydrogenase H-cluster as deduced by application of X-ray spectroscopy are reviewed. The techniques central to this work include X-ray absorption spectroscopy either in the form of extended X-ray absorption fine structure (EXAFS), X-ray absorption near-edge structure (XANES) and nuclear resonant vibrational spectroscopy (NRVS). Examples of the advances in the understanding of the chemistry of the system through integration of a range of spectroscopic and computational techniques with X-ray spectroscopy are highlighted. The critical role played by ab initio calculation of structural and spectroscopic properties of transition-metal compounds using density functional theory (DFT) is illustrated both by the calculation of nuclear resonance vibrational spectroscopy (NRVS) spectra and the structures and spectra of intermediates through the catalytic reactions of hydrogenase model compounds.     

Preparation, Structure and Electrochemical Property of a Pyrazole-substituted Diiron Dithiolate Complex

A pyrazole-substituted diiron dithiolate complex [Fe2(μ-pdt)(CO)5(3,5-Me2Pz)](1,3,5-Me 2Pz=3,5-dimethylpyrazole) was prepared as a biomimetic model for the active site of [FeFe]-hydrogenase by CO-substitution of all-carbonyl complex [Fe2(μ-pdt)(CO)6] with 3,5-Me2Pz. The molecular structure was confirmed by MS, IR, 1H NMR, elemental analysis and single-crystal X-ray analysis. Complex 1 crystallizes in the triclinic system, space group P1 with a=9.108(7), b=9.743(8), c=11.192(9), α=109.235(5), β=101.914(9), γ=96.605(6)o. In CH3CN solution, reversible transformation between 1 and the acetonitrile-substituted species [Fe2(μ-pdt)-(CO)5(NCCH3)] was detected by both IR and cyclic voltammetry (CV). The electrochemical proton reduction catalyzed by 1 in the presence of acetic acid was also studied in CH2Cl2.     

Maturation of the [FeFe]-Hydrogenase: Direct Transfer of the (κ3-cysteinate)FeII(CN)(CO)2 Complex B from HydG to HydE

[FeFe]-hydrogenases efficiently catalyze the reversible oxidation of molecular hydrogen. Their prowess stems from the intricate H-cluster, combining a [Fe₄S₄] center with a binuclear iron center ([2Fe]_H). In the latter, each iron atom is coordinated by a CO and CN ligand, connected by a CO and an azadithiolate ligand. The synthesis of this active site involves a unique multiprotein assembly, featuring radical SAM proteins HydG and HydE. HydG initiates the transformation of L-tyrosine into cyanide and carbon monoxide to generate complex B, which is subsequently transferred to HydE to continue the biosynthesis of the [2Fe]_H-subcluster. Due to its instability, complex B isolation for structural or spectroscopic characterization has been elusive thus far. Nevertheless, the use of a biomimetic analogue of complex B allowed circumvention of the need for the HydG protein during in vitro functional investigations, implying a similar structure for complex B. Herein, we used the HydE protein as a nanocage to encapsulate and stabilize the complex B product generated by HydG. Using X-ray crystallography, we successfully determined its structure at 1.3 Å resolution. Furthermore, we demonstrated that complex B is directly transferred from HydG to HydE, thus not being released into the solution post-synthesis, highlighting a transient interaction between the two proteins.     

Silicon–Heteroaromatic [FeFe] Hydrogenase Model Complexes: Insight into Protonation,Electrochemical Properties,and Molecular Structures

To learn from Nature how to create an efficient hydrogen‐producing catalyst, much attention has been paid to the investigation of structural and functional biomimics of the active site of [FeFe]‐hydrogenase. To understand their catalytic activities, the μ‐S atoms of the dithiolate bridge have been considered as possible basic sites during the catalytic processes. For this reason, a series of [FeFe]‐H₂ase mimics have been synthesized and characterized. Different [FeFe]‐hydrogenase model complexes containing bulky Si–heteroaromatic systems or fluorene directly attached to the dithiolate moiety as well as their mono‐PPh₃‐substituted derivatives have been prepared and investigated in detail by spectroscopic, electrochemical, X‐ray diffraction, and computational methods. The assembly of the herein reported series of complexes shows that the μ‐S atoms can be a favored basic site in the catalytic process. Small changes in the (hetero)‐aromatic system of the dithiolate moiety are responsible for large differences in their structures. This was elucidated in detail by DFT calculations, which were consistent with the experimental results.     

Catalytic turnover of [FeFe]-hydrogenase based on single-molecule imaging

Hydrogenases catalyze the interconversion of protons and hydrogen according to the reversible reaction: 2H(+) + 2e(-) ? H(2) while using only the earth-abundant metals nickel and/or iron for catalysis. Due to their high activity for proton reduction and the technological significance of the H(+)/H(2) half reaction, it is important to characterize the catalytic activity of [FeFe]-hydrogenases using both biochemical and electrochemical techniques. Following a detailed electrochemical and photoelectrochemical study of an [FeFe]-hydrogenase from Clostridium acetobutylicum (CaHydA), we now report electrochemical and single-molecule imaging studies carried out on a catalytically active hydrogenase preparation. The enzyme CaHydA, a homologue (70% identity) of the [FeFe]-hydrogenase from Clostridium pasteurianum , CpI, was adsorbed to a negatively charged, self-assembled monolayer (SAM) for investigation by electrochemical scanning tunneling microscopy (EC-STM) techniques and macroscopic electrochemical measurements. The EC-STM imaging revealed uniform surface coverage with sufficient stability to undergo repeated scanning with a STM tip as well as other electrochemical investigations. Cyclic voltammetry yielded a characteristic cathodic hydrogen production signal when the potential was scanned sufficiently negative. The direct observation of the single enzyme distribution on the Au-SAM surface coupled with macroscopic electrochemical measurements obtained from the same electrode allowed the evaluation of a turnover frequency (TOF) as a function of potential for single [FeFe]-hydrogenase molecules.     

[FeFe]-Hydrogenase and its organic molecule mimics—Artificial and bioengineering application for hydrogenproduction

This study focuses on [FeFe]-hydrogenase and its metallorganic mimics in terms of electronic and photophysical properties, which can be applied to the electrochemical and/or photochemical production of molecular hydrogen. Natural [FeFe]-hydrogenase, synthetic mimics of its active site and recent progresses in hybrid-type hydrogen production, for example, inorganic-combination photoelectrochemical and photochemical hydrogen production, are reviewed.     

Direct electrochemistry of an [FeFe]-hydrogenase on a TiO2 electrode

[FeFe]-hydrogenases are efficient natural catalysts that can be exploited for hydrogen production. Immobilization of the recombinant [FeFe]-hydrogenase CaHydA was achieved for the first time on an anatase TiO(2) electrode. The enzyme is able to interact and exchange electrons with the electrode and to catalyze hydrogen production with an efficiency of 70%.     

Targeting intermediates of [FeFe]-hydrogenase by CO and CN vibrational signatures

In this work, we employ density functional theory to assign vibrational signatures of [FeFe]-hydrogenase intermediates to molecular structures. For this purpose, we perform an exhaustive analysis of structures and harmonic vibrations of a series of CN and CO containing model clusters of the [FeFe]-hydrogenase enzyme active site considering also different charges, counterions, and solvents. The pure density functional BP86 in combination with a triple-ζ polarized basis set produce reliable molecular structures as well as harmonic vibrations. Calculated CN and CO stretching vibrations are analyzed separately. Scaled vibrational frequencies are then applied to assign intermediates in [FeFe]-hydrogenase's reaction cycle. The results nicely complement the previous studies of Darensbourg and Hall, and Zilberman et al. The infrared spectrum of the H(ox) form is in very good agreement with the calculated spectrum of the Fe(I)Fe(II) model complex featuring a free coordination site at the distal Fe atom, as well as, with the calculated spectra of the complexes in which H(2) or H(2)O are coordinated at this site. The spectrum of H(red) measured from Desulfovibrio desulfuricans is compatible with a mixture of a Fe(I)Fe(I) species with all terminal COs, and a Fe(I)Fe(I) species with protonated dtma ligand, while the spectrum of H(red) recently measured from Chlamydomonas reinhardtii is compatible with a mixture of a Fe(I)Fe(I) species with a bridged CO, and a Fe(II)Fe(II) species with a terminal hydride bound to the Fe atom.     

A theoretical study on the enhancement of functionally relevant electron transfers in biomimetic models of [FeFe]-hydrogenases

Recent advances aimed at modeling the chemistry of the active site of [FeFe]-hydrogenases (the H-cluster, composed by a catalytic Fe(2)S(2) subcluster and an Fe(4)S(4) portion) have led to the synthesis of binuclear coordination compounds containing a noninnocent organophosphine ligand [2,3-bis(diphenylphosphino)maleic anhydride, bma] that is able to undergo monoelectron reduction, analogously to the tetranuclear Fe(4)S(4) subcluster portion of the H-cluster. However, such a synthetic model was shown to feature negligible electronic communication between the noninnocent ligand and the remaining portion of the cluster, at variance with the enzyme active site. Here, we report a theoretical investigation that shows why the electron transfer observed in the enzyme upon protonation of the catalytic Fe(2)S(2) subsite cannot take place in the bma-containing cluster. In addition, we show that targeted modifications of the bma ligand are sufficient to restore the electronic communication within the model, such that electron density can be more easily withdrawn from the noninnocent ligand, as a result of protonation of the iron centers. Similar results were also obtained with a ligand derived from cobaltocene. The relevance of our findings is discussed from the perspective of biomimetic reproduction of proton reduction to yield molecular hydrogen.     

Formaldehyde--a rapid and reversible inhibitor of hydrogen production by [FeFe]-hydrogenases

Dihydrogen (H(2)) production by [FeFe]-hydrogenases is strongly inhibited by formaldehyde (methanal) in a reaction that is rapid, reversible, and specific to this type of hydrogenase. This discovery, using three [FeFe]-hydrogenases that are homologous about the active site but otherwise structurally distinct, was made by protein film electrochemistry, which measures the activity (as electrical current) of enzymes immobilized on an electrode; importantly, the inhibitor can be removed after addition. Formaldehyde causes rapid loss of proton reduction activity which is restored when the solution is exchanged. Inhibition is confirmed by conventional solution assays. The effect depends strongly on the direction of catalysis: inhibition of H(2) oxidation is much weaker than for H(2) production, and formaldehyde also protects against CO and O(2) inactivation. By contrast, inhibition of [NiFe]-hydrogenases is weak. The results strongly suggest that formaldehyde binds at, or close to, the active site of [FeFe]-hydrogenases at a site unique to this class of enzyme--highly conserved lysine and cysteine residues, the bridgehead atom of the dithiolate ligand, or the reduced Fe(d) that is the focal center of catalysis.     

Series of mixed valent Fe(II)Fe(I) complexes that model the Hox state of [FeFe]hydrogenase: redox properties, density-functional theory investigation, and reactivities with extrinsic CO

A series of asymmetrically disubstituted models of the active site of [FeFe]-hydrogenase, (mu-pdt)[Fe(CO) 2PMe 3][Fe(CO) 2NHC] (pdt = 1,3-propanedithiolate, NHC = IMes, 1,3-bis(2,4,6-trimethylphenyl)imidazol-2-ylidene IMes ( 1), IMesMe, 1-methyl,3-(2,4,6-trimethylphenyl)imidazol-2-ylidene ( 2) or IMe, 1,3-bis(methyl)imidazol-2-ylidene ( 3)), have been synthesized and characterized. The one-electron oxidation of these complexes to generate mixed valent models of the H ox state of [FeFe]-hydrogenase, such as the previously reported (mu-pdt)(mu-CO)[Fe(CO) 2PMe 3][Fe(CO)IMes] (+) ( 1 ox ) (Liu, T.; Darensbourg, M. Y. J. Am. Chem. Soc. 2007, 129, 7008-7009) has been examined to explore the steric and electronic effects of different N-atom substituents on the stability and structure of the mixed valent cations. The differences in spectroscopic properties, structures, and relative stabilities of 1 ox , (mu-pdt)[Fe(CO) 2PMe 3][Fe(CO) 2IMesMe] (+) ( 2 ox ), and (mu-pdt)[Fe(CO) 2PMe 3]-[Fe(CO) 2IMe] (+) ( 3 ox ) are discussed in the context of both experimental and theoretical data. Of the three derivatives, only that with greatest steric bulk on the NHC ligand, 1 ox , shows a clear indication of a mu-CO by solution nu(CO) IR and yields to crystallization as a rotated form, commensurate with the two-Fe subsite of H ox. In addition, the reactivity of the complexes with extrinsic CO to form CO adducts and/or exchange with (13)CO is explored by experiment and by using density-functional theory calculations.     

Structural and electronic properties of the [FeFe] hydrogenase H-cluster in different redox and protonation states. A DFT investigation

The molecular and electronic structure of the Fe 6S 6 H-cluster of [FeFe] hydrogenase in relevant redox and protonation states have been investigated by DFT. The calculations have been carried out according to the broken symmetry approach and considering different environmental conditions. The large negative charge of the H-cluster leads, in a vacuum, to structures different from those observed experimentally in the protein. A better agreement with experimental data is observed for solvated complexes, suggesting that the protein environment could buffer the large negative charge of the H-cluster. The comparison of Fe 6S 6 and Fe 2S 2 DFT models shows that the presence of the Fe 4S 4 moiety does not affect appreciably the geometry of the [2Fe] H cluster. In particular, the Fe 4S 4 cluster alone cannot be invoked to explain the stabilization of the mu-CO forms observed in the enzyme (relative to all-terminal CO species). As for protonation of the hydrogen cluster, it turned out that mu-H species are always more stable than terminal hydride isomers, leading to the conclusion that specific interactions of the H-cluster with the environment, not considered in our calculations, would be necessary to reverse the stability order of mu-H and terminal hydrides. Otherwise, protonation of the metal center and H 2 evolution in the enzyme are predicted to be kinetically controlled processes. Finally, subtle modifications in the H-cluster environment can change the relative stability of key frontier orbitals, triggering electron transfer between the Fe 4S 4 and the Fe 2S 2 moieties forming the H-cluster.