Inhibition of [FeFe]-hydrogenases by formaldehyde and wider mechanistic implications for biohydrogen activation

Formaldehyde-a rapid and reversible inhibitor of hydrogen evolution by [FeFe]-hydrogenases-binds with a strong potential dependence that is almost complementary to that of CO. Whereas exogenous CO binds tightly to the oxidized state known as H(ox) but very weakly to a state two electrons more reduced, formaldehyde interacts most strongly with the latter. Formaldehyde thus intercepts increasingly reduced states of the catalytic cycle, and density functional theory calculations support the proposal that it reacts with the H-cluster directly, most likely targeting an otherwise elusive and highly reactive Fe-hydrido (Fe-H) intermediate.     

Time-resolved vibrational spectroscopy of [FeFe]-hydrogenase model compounds

Model compounds have been found to structurally mimic the catalytic hydrogen-producing active site of Fe-Fe hydrogenases and are being explored as functional models. The time-dependent behavior of Fe(2)(μ-S(2)C(3)H(6))(CO)(6) and Fe(2)(μ-S(2)C(2)H(4))(CO)(6) is reviewed and new ultrafast UV- and visible-excitation/IR-probe measurements of the carbonyl stretching region are presented. Ground-state and excited-state electronic and vibrational properties of Fe(2)(μ-S(2)C(3)H(6))(CO)(6) were studied with density functional theory (DFT) calculations. For Fe(2)(μ-S(2)C(3)H(6))(CO)(6) excited with 266 nm, long-lived signals (τ = 3.7 ± 0.26 μs) are assigned to loss of a CO ligand. For 355 and 532 nm excitation, short-lived (τ = 150 ± 17 ps) bands are observed in addition to CO-loss product. Short-lived transient absorption intensities are smaller for 355 nm and much larger for 532 nm excitation and are assigned to a short-lived photoproduct resulting from excited electronic state structural reorganization of the Fe-Fe bond. Because these molecules are tethered by bridging disulfur ligands, this extended di-iron bond relaxes during the excited state decay. Interestingly, and perhaps fortuitously, the time-dependent DFT-optimized exited-state geometry of Fe(2)(μ-S(2)C(3)H(6))(CO)(6) with a semibridging CO is reminiscent of the geometry of the Fe(2)S(2) subcluster of the active site observed in Fe-Fe hydrogenase X-ray crystal structures. We suggest these wavelength-dependent excitation dynamics could significantly alter potential mechanisms for light-driven catalysis.     

On the structure of a proposed mixed-valent analogue of the diiron subsite of [FeFe]-hydrogenase

We show that a dinuclear assembly apparently providing the first example of a synthetic molecule exhibiting key features of the diiron subsite of [FeFe] hydrogenase, viz. CO-bridging of a coordinatively unsaturated, dithiolate-bridged mixed-valence diiron centre, is in fact a diamagnetic tetranuclear complex.     

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.     

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.     

Mild redox complementation enables H2 activation by [FeFe]-hydrogenase models

Mild oxidants such as [Fe(C(5)Me(5))(2)](+) accelerate the activation of H(2) by [Fe(2)[(SCH(2))(2)NBn](CO)(3)(dppv)(PMe(3))](+) ([1](+)), despite the fact that the ferrocenium cation is incapable of oxidizing [1](+). The reaction is first-order in [1](+) and [H(2)] but independent of the E(1/2) and concentration of the oxidant. The analogous reaction occurs with D(2) and proceeds with an inverse kinetic isotope effect of 0.75(8). The activation of H(2) is further enhanced with the tetracarbonyl [Fe(2)[(SCH(2))(2)NBn](CO)(4)(dppn)](+) ([2](+)), the first crystallographically characterized model for the H(ox) state of the active site containing an amine cofactor. These studies point to rate-determining binding of H(2) followed by proton-coupled electron transfer. Relative to that by [1](+), the rate of H(2) activation by [2](+)/Fc(+) is enhanced by a factor of 10(4) at 25 °C.     

Azadithiolate-bridged [FeFe]-hydrogenase mimics with bridgehead N-derivation: structural and electrochemical investigations

Transition Metal Chemistry - To further develop the active site mimics of azadithiolate-bridged [FeFe]-hydrogenases, a series of new diiron azadithiolate complexes...     

Geometrical influence on the non-biomimetic heterolytic splitting of H2 by bio-inspired [FeFe]-hydrogenase complexes: a rare example of inverted frustrated Lewis pair based reactivity

Despite the high levels of interest in the synthesis of bio-inspired [FeFe]-hydrogenase complexes, H₂ oxidation, which is one specific aspect of hydrogenase enzymatic activity, is not observed for most reported complexes. To attempt H–H bond cleavage, two disubstituted diiron dithiolate complexes in the form of [Fe₂(μ-pdt)L₂(CO)₄] (L: PMe₃, dmpe) have been used to play the non-biomimetic role of a Lewis base, with frustrated Lewis pairs (FLPs) formed in the presence of B(C₆F₅)₃ Lewis acid. These unprecedented FLPs, based on the bimetallic Lewis base partner, allow the heterolytic splitting of the H₂ molecule, forming a protonated diiron cation and hydrido-borate anion. The substitution, symmetrical or asymmetrical, of two phosphine ligands at the diiron dithiolate core induces a strong difference in the H₂ bond cleavage abilities, with the FLP based on the first complex being more efficient than the second. DFT investigations examined the different mechanistic pathways involving each accessible isomer and rationalized the experimental findings. One of the main DFT results highlights that the iron site acting as a Lewis base for the asymmetrical complex is the {Fe(CO)₃} subunit, which is less electron-rich than the {FeL(CO)₂} site of the symmetrical complex, diminishing the reactivity towards H₂. Calculations relating to the different mechanistic pathways revealed the presence of a terminal hydride intermediate at the apical site of a rotated {Fe(CO)₃} site, which is experimentally observed, and a semi-bridging hydride intermediate from H₂ activation at the Fe–Fe site; these are responsible for a favourable back-reaction, reducing the conversion yield observed in the case of the asymmetrical complex. The use of two equivalents of Lewis acid allows for more complete and faster H₂ bond cleavage due to the encapsulation of the hydrido-borate species by a second borane, favouring the reactivity of each FLP, in agreement with DFT calculations.

Bio-inspired [FeFe]-hydrogenase complexes and B(C₆F₅)₃ form FLPs that are able to activate H₂, providing rare examples of inverted enzymatic reactivity. The influence of the symmetry/asymmetry of coordination is studied via DFT.     

A site-differentiated [4Fe–4S] cluster controls electron transfer reactivity of Clostridium acetobutylicum [FeFe]-hydrogenase I

One of the many functions of reduction–oxidation (redox) cofactors is to mediate electron transfer in biological enzymes catalyzing redox-based chemical transformation reactions. There are numerous examples of enzymes that utilize redox cofactors to form electron transfer relays to connect catalytic sites to external electron donors and acceptors. The compositions of relays are diverse and tune transfer thermodynamics and kinetics towards the chemical reactivity of the enzyme. Diversity in relay design is exemplified among different members of hydrogenases, enzymes which catalyze reversible H₂ activation, which also couple to diverse types of donor and acceptor molecules. The [FeFe]-hydrogenase I from Clostridium acetobutylicum (CaI) is a member of a large family of structurally related enzymes where interfacial electron transfer is mediated by a terminal, non-canonical, His-coordinated, [4Fe–4S] cluster. The function of His coordination was examined by comparing the biophysical properties and reactivity to a Cys substituted variant of CaI. This demonstrated that His coordination strongly affected the distal [4Fe–4S] cluster spin state, spin pairing, and spatial orientations of molecular orbitals, with a minor effect on reduction potential. The deviations in these properties by substituting His for Cys in CaI, correlated with pronounced changes in electron transfer and reactivity with the native electron donor–acceptor ferredoxin. The results demonstrate that differential coordination of the surface localized [4Fe–4S]His cluster in CaI is utilized to control intermolecular and intramolecular electron transfer where His coordination creates a physical and electronic environment that enables facile electron exchange between electron carrier molecules and the iron–sulfur cluster relay for coupling to reversible H₂ activation at the catalytic site.

Histidine coordination of the distal [4Fe–4S] cluster in [FeFe]-hydrogenase was demonstrated to tune the cluster spin-states, spin-pairing and surrounding molecular orbitals to enable more facile electron transfer compared to cysteine coordination.     

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.     

Theoretical studies of [FeFe]-hydrogenase: structure and infrared spectra of synthetic models

First-principles density functional theory calculations of synthetic models of [FeFe]-hydrogenase are used to show that the theoretical methods reproduce observed structures and infrared spectra to high accuracy. The accuracy is demonstrated for synthetic Fe(I)Fe(I) models ([(mu-PDT)Fe2(CO)6] and [(CN)(CO)2(mu-PDT)Fe2(CO)2(CN)]2-), for which we show that their infrared spectra are sensitive to the geometric arrangement of their CO/CN ligands and can be used in conjunction with quantum-mechanical total energies to predict the correct ligand geometry. We then analyze and predict the structure of mixed-valence Fe(II)Fe(I) models ([(mu-MeSCH2C(Me)(CH2S)2)Fe2(CO)4(CN)2]x-). These capabilities promise to distinguish among the various structural isomers of the enzyme's active site which are consistent with the limited accuracy of the X-ray observations.     

Nanoscale ensembles using building blocks inspired by the [FeFe]-hydrogenase active site

The hydrogenase model [Fe₂(S₂C₃H₆)(CN)₂(CO)₄]²⁻ was employed as a molecular tecton for the construction of supramolecular aggregates. IR spectroscopy indicated that cyanide bridged aggregates are formed when [Fe₂(S₂C₃H₆)(CN)₂(CO)₄]²⁻ was treated with Lewis acids such as Zn(tetraphenylporphyrinate), [Cu(NCMe)(2,2′-bipyridine)]PF₆ and [Cu(NCMe)₄]PF₆. Condensation of [Fe₂(S₂C₃H₆)(CN)₂(CO)₄]²⁻ with the tritopic Lewis acid [Cp¹Rh]²⁺ afforded the novel expanded tetrahedron cage, {[Fe₂(S₂C₃H₆)(CN)₂(CO)₄]₆[Cp¹Rh]₄}⁴⁻. The tetrahedron cage was characterized crystallographically as the PPN salt.     

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%.     

Terahertz, infrared and Raman vibrational assignments of [FeFe]-hydrogenase model compounds

Using Raman, terahertz (THz), and mid-infrared (IR) spectroscopies, the vibrational spectra of two chromophore models of hydrogen-producing [FeFe]-hydrogenase, Fe₂(μ-S₂C₃H₆)(CO)₆ and Fe₂(μ-S₂C₂H₄)(CO)₆, have been assigned. The combination of absorption and scattering techniques, along with DFT calculations, allows for assignments to be made without traditional isotopic substitution methods.     

ADT-Type [FeFe]-hydrogenase biomimics featuring monodentate phosphines: formation,structures, and electrocatalysis

Transition Metal Chemistry - To further develop the diiron subsite biomimics of [FeFe]-hydrogenases, two new diiron azadithiolate (adt) complexes Fe2(μ-adtNPh)(CO)5(Ph2PX)...     

Photocatalytic hydrogen production from a simple water-soluble [FeFe]-hydrogenase model system

Combined with a simple water soluble [FeFe]-hydrogenase mimic 1, Ru(bpy)(3)(2+) and ascorbic acid enable hydrogen production photocatalytically. More than 88 equivalents of H(2) were achieved in water, which is much better than that obtained in an organic solvent or a mixture of organic solvent and water.     

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

Correction for ‘The oxygen-resistant [FeFe]-hydrogenase CbA5H harbors an unknown radical signal’ by Melanie Heghmanns et al., Chem. Sci., 2022, 13, 7289–7294, https://doi.org/10.1039/D2SC00385F.

The authors realized that incorrect references were cited following the sentence “In conjunction with the signal''s significant width, the frequency dependence clearly indicates spin–spin interaction between the F-clusters.” The correct references are shown below as ref. 1 and 2.Additionally ref. 36 and 37 were reversed in the reference list. The correct ref. 36 is shown below as ref. 3 and the correct ref. 37 is shown below as ref. 4.The Royal Society of Chemistry apologises for these errors and any consequent inconvenience to authors and readers.     

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.