Maurizio Bruschi

Synthesis of the H-cluster framework of iron-only hydrogenase

The metal-sulphur active sites of hydrogenases catalyse hydrogen evolution or uptake at rapid rates. Understanding the structure and function of these active sites—through mechanistic studies of hydrogenases, synthetic assemblies and in silico models—will help guide the design of new materials for hydrogen production or uptake. Here we report the assembly of the iron-sulphur framework of the active site of iron-only hydrogenase (the H-cluster), and show that it functions as an electrocatalyst for proton reduction. Through linking of a di-iron subsite to a {4Fe4S} cluster, we achieve the first synthesis of a metallosulphur cluster core involved in small-molecule catalysis. In addition to advancing our understanding of the natural biological system, the availability of an active, free-standing analogue of the H-cluster may enable us to develop useful electrocatalytic materials for application in, for example, reversible hydrogen fuel cells. (Platinum is currently the preferred electrocatalyst for such applications, but is expensive, limited in availability and, in the long term, unsustainable.)

Molecular determinants of the physicochemical properties of a critical prion protein region comprising residues 106-126.

Prion diseases are marked by the cerebral accumulation of conformationally modified forms of the cellular prion protein (PrP(C)), known as PrP(res). The region comprising the residues 106-126 of human PrP seems to have a key role in this conformational conversion, because a synthetic peptide homologous with this sequence (PrP106-126) adopts different secondary structures in different environments. To investigate the molecular determinants of the physicochemical characteristics of PrP106-126, we synthesized a series of analogues including PrP106-126 H(D), PrP106-126 A and PrP106-126 K, with l-His-->d-His, His-->Ala and His-->Lys substitutions respectively at position 111, PrP106-126 NH(2) with amidation of the C-terminus, PrP106-126 V with an Ala-->Val substition at position 117, and PrP106-126 VNH(2) with an Ala-->Val substitution at position 117 and amidation of the C-terminus. The analysis of the secondary structure and aggregation properties of PrP106-126 and its analogues showed the following. (1) His(111) is central to the conformational changes of PrP peptides. (2) Amidation of the C-terminal Gly(126) yields a predominantly random coil structure, abolishes the molecular polymorphism and decreases the propensity of PrP106-126 to generate amyloid fibrils. (3) PrP106-126 V, carrying an Ala-->Val substitution at position 117, does not demonstrate a fibrillogenic ability superior to that of PrP106-126. However, the presence of Val at position 117 increases the aggregation properties of the amidated peptide. (4) Amyloid fibrils are not required for neurotoxicity because the effects of PrP106-126 NH(2) on primary neuronal cultures were similar to those of the wild-type sequence. Conversely, astroglial proliferation is related to the presence of amyloid fibrils, suggesting that astrogliosis in prion encephalopathies without amyloid deposits is a mediated effect rather than a direct effect of disease-specific PrP isoforms.

Influence of the [2Fe]H Subcluster Environment on the Properties of Key Intermediates in the Catalytic Cycle of [FeFe] Hydrogenases: Hints for the Rational Design of Synthetic Catalysts†

Natures recipe: A theoretical study analyzes how the environment of the [FeFe] hydrogenases catalytic cofactor affects its chemical properties, particularly the relative stability of complexes with bridging and terminal hydride ligands (see picture; Fe teal, S yellow, C green, N blue, O red, H gray). The results help to elucidate key rules for the design of bioinspired synthetic catalysts for H(2) production.

The oxidative inactivation of FeFe hydrogenase reveals the flexibility of the H-cluster

Nature is a valuable source of inspiration in the design of catalysts, and various approaches are used to elucidate the mechanism of hydrogenases, the enzymes that oxidize or produce H2. In FeFe hydrogenases, H2 oxidation occurs at the H-cluster, and catalysis involves H2 binding on the vacant coordination site of an iron centre. Here, we show that the reversible oxidative inactivation of this enzyme results from the binding of H2 to coordination positions that are normally blocked by intrinsic CO ligands. This flexibility of the coordination sphere around the reactive iron centre confers on the enzyme the ability to avoid harmful reactions under oxidizing conditions, including exposure to O2. The versatile chemistry of the diiron cluster in the natural system might inspire the design of novel synthetic catalysts for H2 oxidation.

Investigations on the Role of Proton-Coupled Electron Transfer in Hydrogen Activation by [FeFe]-Hydrogenase

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.

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]-hydrogenases 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.

Nitrosyl Derivatives of Diiron(I) Dithiolates Mimic the Structure and Lewis Acidity of the FeFe-Hydrogenase Active Site

This study probes the impact of electronic asymmetry of diiron(I) dithiolato carbonyls. Treatment of Fe2(S2C(n)H(2n))(CO)(6-x)(PMe3)x compounds (n = 2, 3; x = 1, 2, 3) with NOBF4 gave the derivatives [Fe2(S2C(n)H(2n))(CO)(5-x)(PMe3)x(NO)]BF4, which are electronically unsymmetrical because of the presence of a single NO(+) ligand. Whereas the monophosphine derivative is largely undistorted, the bis(PMe3) derivatives are distorted such that the CO ligand on the Fe(CO)(PMe3)(NO)(+) subunit is semibridging. Two isomers of [Fe2(S2C3H6)(CO)3(PMe3)2(NO)]BF4 were characterized spectroscopically and crystallographically. Each isomer features electron-rich Fe(CO)2PMe3 and electrophilic Fe(CO)(PMe3)(NO)(+) subunits. These species are in equilibrium with an unobserved isomer that reversibly binds CO (DeltaH = -35 kJ/mol, DeltaS = -139 J mol(-1) K(-1)) to give the symmetrical adduct [Fe2(S2C3H6)(mu-NO)(CO)4(PMe3)2]BF4. In contrast to Fe2(S2C3H6)(CO)4(PMe3)2, the bis(PMe3) nitrosyl complexes readily undergo CO substitution to give the (PMe3)3 derivatives. The nitrosyl complexes reduce at potentials that are approximately 1 V milder than their carbonyl counterparts. Results of density functional theory calculations, specifically natural bond orbital analysis, reinforce the electronic resemblance of the nitrosyl complexes to the corresponding mixed-valence diiron complexes. Unlike other diiron dithiolato carbonyls, these species undergo reversible reductions at mild potentials. The results show that the novel structural and chemical features associated with mixed-valence diiron dithiolates (the so-called H(ox) models) can be replicated in the absence of mixed-valency by the introduction of electronic asymmetry.

A theoretical study of spin states in Ni-S4 complexes and models of the [NiFe] hydrogenase active site

We have applied density functional theory, using both pure (BP86) and hybrid (B3LYP and B3LYP*) functionals, to investigate structural parameters and reaction energies for nickel(II)-sulfur coordination compounds, as well as for small cluster models of the Ni-SI and Ni-R redox state of [NiFe] hydrogenases. Results obtained investigating experimentally well-characterized complexes show that BP86 is well suited to describe the structural features of this class of compounds. However, the singlet–triplet energy splitting and even the computed ground state are strongly dependent on the applied functional. Results for the cluster models of [NiFe] hydrogenases lead to the conclusion that in the reduced protein structures characterized by X-ray diffraction a hydride bridges the two metal centres. The energy splitting of the singlet and triplet states in Ni-R and Ni-SI models is calculated to be very small and may be overcome at room temperature to allow a spin crossover. Moreover, the relative stability of the Ni-SI and Ni-R structures adopted in the present investigation is fully compatible with their involvement in the reversible heterolytic cleavage of H2.

Disclosure of key stereoelectronic factors for efficient H2 binding and cleavage in the active site of [NiFe]-hydrogenases.

A comparative analysis of a series of DFT models of [NiFe]-hydrogenases, ranging from minimal NiFe clusters to very large systems including both the first and second coordination sphere of the bimetallic cofactor, was carried out with the aim of unraveling which stereoelectronic properties of the active site of [NiFe]-hydrogenases are crucial for efficient H2 binding and cleavage. H2 binding to the Ni-SIa redox state is energetically favored (by 4.0 kcal mol(-1)) only when H2 binds to Ni, the NiFe metal cluster is in a low spin state, and the Ni cysteine ligands have a peculiar seesaw coordination geometry, which in the enzyme is stabilized by the protein environment. The influence of the Ni coordination geometry on the H2 binding affinity was then quantitatively evaluated and rationalized analyzing frontier molecular orbitals and populations. Several plausible reaction pathways leading to H2 cleavage were also studied. It turned out that a two-step pathway, where H2 cleavage takes place on the Ni-SIa redox state of the enzyme, is characterized by very low reaction barriers and favorable reaction energies. More importantly, the seesaw coordination geometry of Ni was found to be a key feature for facile H2 cleavage. The discovery of the crucial influence of the Ni coordination geometry on H2 binding and activation in the active site of [NiFe]-hydrogenases could be exploited in the design of novel biomimetic synthetic catalysts.

Functionally relevant interplay between the Fe(4)S(4) cluster and CN(-) ligands in the active site of [FeFe]-hydrogenases.

[FeFe]-hydrogenases are highly efficient H(2)-evolving metalloenzymes that include cyanides and carbonyls in the active site. The latter is an Fe(6)S(6) cluster (the so-called H-cluster) that can be subdivided into a binuclear portion carrying the CO and CN(-) groups and a tetranuclear subcluster. The fundamental role of cyanide ligands in increasing the basicity of the H-cluster has been highlighted previously. Here a more subtle but crucial role played by the two CN(-) ligands in the active site of [FeFe]-hydrogenases is disclosed. In fact, QM/MM calculations on all-atom models of the enzyme from Desulfovibrio desulfuricans show that the cyanide groups fine-tune the electronic and redox properties of the active site, affecting both the protonation regiochemistry and electron transfer between the two subclusters of the H-cluster. Despite the crucial role of cyanides in the protein active site, the currently available bioinspired electrocatalysts generally lack CN(-) groups in order to avoid competition between the latter and the catalytic metal centers for proton binding. In this respect, we show that a targeted inclusion of phosphine ligands in hexanuclear biomimetic clusters may restore the electronic and redox features of the wild-type H-cluster.