Molecular characterization and homologous overexpression of [FeFe]-hydrogenase in Clostridium tyrobutyricum JM1

The H₂-evoving [FeFe]-hydrogenase in Clostridium tyrobutyricum JM1 was isolated to elucidate molecular characterization and modular structure of the hydrogenase. Then, homologous overexpression of the hydrogenase gene was for the first time performed to enhance hydrogen production. The hydA open reading frame (ORF) was 1734-bp, encodes 577 amino acids with a predicted molecular mass of 63,970 Da, and presents 80% and 75% identity at the amino acid level with the [FeFe]-hydrogenase genes of Clostridium kluyveri DSM 555 and Clostridium acetobutylicum ATCC 824, respectively. One histidine residue and 19 cysteine residues, known to fasten one [2Fe–2S] cluster, three [4Fe–4S] clusters and one H-cluster, were conserved in hydA of C. tyrobutyricum.     

Maturation and processing of the recombinant [FeFe] hydrogenase from Desulfovibrio vulgaris Hildenborough (DvH) in Escherichia coli

The need of an efficient and well-characterized heterologous expression system of [FeFe]-hydrogenase for the production of O₂-resistant mutants prompted us to explore the use of Escherichia coli as a possible expression system. O₂-resistant hydrogenase mutants could be instrumental when coupling oxygenic photosynthesis with hydrogen bio-production. In general, expression of Desulfovibrio vulgaris Hildenborough active enzyme in E. coli was very modest indicating that the co-expression of the HydE, HydF and HydG maturases with hydrogenase structural genes in this bacterium is not optimal. A 28-fold increase in activity was obtained when these proteins were co-expressed with the Iron–Sulfur Cluster operon, indicating that one of the problems with over-expression is the correct insertion of FeS clusters. However, the measured activity is still about 4000-fold lower than the one measured in the native hydrogenase indicating that additional, so far unidentified factors may be necessary for optimal heterologous expression of [FeFe]-hydrogenase.     

Characterization and overexpression of a [FeFe]-hydrogenase gene of a novel hydrogen-producing bacterium Ethanoligenens harbinense

Ethanoligenens, a novel ethanologenic and hydrogen-producing genus, has capability of hydrogen production at low pH. A [FeFe]-hydrogenase gene with [4Fe-4S] and [2Fe-2S] clusters from Ethanoligenens harbinense YUAN-3 was cloned and overexpressed in a non-hydrogen-producing Escherichia coli BL-21. This hydA gene consisted of an open reading frame of 1743 bp encoding 580 amino acids with an estimated molecular weight of 63 188.1 Da. Six characteristic sequence signatures were present within the H-cluster domain of [FeFe]-H₂ases, and three of them were described previously. The overexpressed and purified hydrogenases from recombinant cells showed catalytic activity in vitro and in vivo.     

Arginine of Chlamydomonas reinhardtii [Fe–Fe] hydrogenase HydA1 plays a crucial role in electron transfer to its catalytic center

[Fe–Fe] hydrogenases, with hydrogen evolution activities outperforming [Ni–Fe] hydrogenases by 3–4 orders of magnitude, are still the most promising enzyme class for hydrogen production purposes. For Chlamydomonas reinhardtii [Fe–Fe] hydrogenase HydA1 the question of catalytic activity and electron transport is of main importance. Here we report the characterization of two mutant forms of C. reinhardtii HydA1. An aspartic acid in place of arginine¹⁷¹ leads to a six-fold increase of the catalytic activity in comparison to the wild type protein during methyl viologen-dependent hydrogen production. Tryptophan in position 171 does not result in any change in methyl viologen-induced activity. At the same time these mutations lead to a strong decrease in ferredoxin-dependent hydrogen production while the catalytic center of mutant forms stays intact. The localization of this amino acid (arginine¹⁷¹) in the environment of CrHydA1 H-cluster indicates that the limitation of the catalytic activity of this hydrogenase is due to the electron transfer step to the catalytic center where the reduction of protons takes place.     

The effects of the dimethylether bridging moiety in the H-cluster of the Clostridium pasteurianum hydrogenase on the mechanism of H2 production: A quantum mechanics/molecular mechanics study

[Fe–Fe]-hydrogenases are naturally occurring metalloenzymes that catalyze the reversible production of H₂ from two protons and two electrons. [Fe–Fe]-hydrogenases found in two species – Clostridium pasteurianum (CpI) and Desulfovibrio desulfuricans (DdH) – were shown with x-ray crystallography to have active sites that are very similar, although several atoms that bridge the dithiolate ligand were unresolved. In earlier work, we employed density functional theory (DFT) within a QM/MM method to investigate two previously proposed mechanisms of hydrogen production by DdH and CpI hydrogenases. In one mechanism (I), a CO ligand bridging two Fe atoms in the active site rotates to a terminal position while in the other (II) the CO bridge remains intact throughout the catalytic cycle. We previously assumed that the active sites for the two hydrogenases were identical; each had a dimethylamine bridging moiety, whose basicity is important for Mechanism II. Our overall conclusion, taking into consideration an energy comparison for the two mechanisms and activation energies for the CO-unbridging step in Mechanism I, was that Mechanism II was favored for both hydrogenases. In this paper, we extend our previous work to show that Mechanism II is favored over Mechanism I even if the bridging moiety in CpI hydrogenase is dimethylether, a significantly weaker base than dimethylamine, providing further support for Mechanism II even though experimental verification of the bridging moiety for the CpI H-cluster is lacking.     

Towards biomimetic models of the reduced [FeFe]-hydrogenase that preserve the key structural features of the enzyme active site; a DFT investigation

[FeFe]-hydrogenases are the most efficient biological catalysts available for the H₂ evolution reaction. Their active site – the H-cluster – features a diiron subsite which has the peculiar characteristic of bearing cyanide groups hydrogen-bonded to the apoprotein as well as carbonyl ligands. Notably, one of the CO ligands is disposed in bridging position between the metal centers. This allows one of the Fe ions to retain a square pyramidal coordination – which determines the assumption of the so-called “rotated structure” – with a vacant coordination site in trans to the μ-CO group, ready to bind protons when the active site is in the Fe^IFe^I state. Many Fe^IFe^I biomimetic models have been synthesized and characterized so far, but most of them fail to reproduce the orientation of the diatomic ligands that is observed in the enzyme active site.     

Maturation of [FeFe]-hydrogenases: Structures and mechanisms

Maturation of [FeFe]-hydrogenases, consisting in the synthesis and assembly of a di-iron center with a dithiolate bridging ligand as well as CO and CN ligands, depends on the concerted action of three metalloproteins, HydE, HydF and HydG. HydE and HydG are “Radical-SAM” enzymes involved in the synthesis of the ligands. HydF is proposed to function as a scaffold protein in which the di-iron center is assembled before being transferred to the hydrogenase. Here we review the current knowledge regarding the structure of the three maturases and the mechanisms of synthesis and assembly of the di-iron center of [FeFe]-hydrogenases.     

Ferredoxin has a pivotal role in the biosynthesis of the hydrogen-oxidizing hydrogenases in Escherichia coli

Iron–sulphur clusters (FeS) are essential cofactors in the small, electron-transfer subunits of [NiFe]-hydrogenases (Hyd). In this study we analyzed the in vivo role of ferredoxin in the biosynthesis of three of the Hyd in Escherichia coli. Our results reveal that a fdx mutant, which is unable to synthesize ferredoxin, lacks the activity of both hydrogen-oxidizing enzymes Hyd-1 and Hyd-2. In the case of Hyd-2 this was due to the absence of the FeS cluster-containing small subunit. In the case of Hyd-1, stability of the catalytic subunit was also impaired. Partial activity of the hydrogen-evolving Hyd-3 enzyme, as well as that of both respiratory formate dehydrogenases was retained in the fdx mutant. Analysis of lacZ fusions demonstrated that the fdx mutation had a limited effect on expression of the operon encoding Hyd-1. Rather, these data suggest that ferredoxin has a role in the maturation or assembly of the hydrogen-oxidizing [NiFe]-hydrogenases.     

Model compound of [FeFe]-hydrogenase active site employing [2Fe–2S] subunit as non-innocent [4Fe–4S] cluster and its proton reduction performance

Synthesis of model compound of [FeFe]-hydrogenase active site is a promising and effective method to produce hydrogen. [μ-(SCH(CH₂CH₃)CH₂S)–Fe₂(CO)₅]₂-(κ¹-DPPE) (compound 1) and [μ-(SCH(CH₂CH₃)CH₂S)] Fe₂(CO)₅ (κ¹-DPPM) (compound 2) were designed and synthesized. Compared 1 and 2 via IR, X-ray single crystal diffraction and CV, it was found that even through 1 and 2 shared similar electronic environment as well as crystal structure features, 1 with two symmetric [2Fe–2S] subsites had distinct reduction peaks and also showed higher electrocatalytic proton reduction efficiency in the presence of equivalent amount of HOAc, which indicated two symmetric [2Fe–2S] subsites played different roles in the process of proton reduction. Furthermore, -(κ¹-DPPE)-[2Fe–2S] moiety with variable valence state in 1 can mimic the [4Fe–4S] cluster and serve as an electron shuttle.     

Enhanced aerobic H2 production by engineering an [FeFe] hydrogenase from Clostridium pasteurianum

Biological H₂ production is one of the alternative technologies for producing H₂ in a renewable and sustainable fashion. The technology often relies on the [FeFe] hydrogenase enzyme for catalyzing the H₂ production reaction from protons and electrons as it is highly efficient. The high O₂ sensitivity of the enzyme have been one of the key obstacles for implementing the technology, but recent findings showed the feasibility of improving O₂ tolerance via protein engineering. In this study, we investigated the changes in the O₂ tolerance and aerobic H₂ production activity of an [FeFe] hydrogenase by replacing methionines on the surface with leucines. None of the mutations were detrimental to protein folding, and a few were able to exert either positive or negative influence on the O₂ tolerance of the enzyme. We combined the mutation exhibiting the highest improvement in O₂ tolerance with the rest of the Met – Leu replacements; however, the results indicated non-synergistic impact on the tolerance. Combinations with the other types of mutations that have been reported previously also failed to further improve the O₂ tolerance of the hydrogenase. These results provide new insights on how mutagenesis affects the enzyme's functional properties, as well as the molecular mechanism(s) behind the tolerance.     

New insights into the systems for heterologous synthesis and maturation of hydrogenases,the most efficient biohydrogen producers

Biohydrogen is considered as an important key to a sustainable world power supply and is currently being seen as the versatile fuel of the future, with the potential to replace fossil fuels. The most efficient biohydrogen producers are hydrogenases. Nevertheless, due to the complex maturation processes of these enzymes, their heterologous production leaves some intriguing points not elucidated up to now. The limit of our understanding in this field makes a barrier for hydrogenases application in a variety of biotechnological processes. This review focuses on recent progresses in the development of heterologous production systems and cell-free maturation systems for the biosynthesis of active [Fe–Fe] and [Ni–Fe] hydrogenases. It also highlights some up to now un-discussed questions on the probable existence of unknown machinery able to maturate [Fe–Fe] hydrogenases or a contribution of the [Ni–Fe] hydrogenases maturases to the formation of an active H-cluster for the [Fe–Fe] hydrogenases.     

The effect of additives on the electrochemical properties of Fe/C composite for Fe/air battery anode

K₂S and FeS were employed as additives for electrolyte and electrode, respectively, to suppress hydrogen evolution and improve the cycleability of the Fe/C composite air battery anode. The effects of these additives on the electrochemical properties of Fe/C composite electrodes were investigated using cyclic voltammetry (CV). The results showed that both K₂S and FeS significantly suppressed hydrogen evolution on the Fe/C electrode characteristics. Among the carbons used, nano-carbons such as tubular carbon nanofibers (CNF), platelet CNF, vapor-grown carbon fibers (VGCF) and acetylene black (AB) improved the discharge capacity of the Fe/C electrode with additive to electrolyte or electrode. The FeS additive showed a larger beneficial effect for the Fe/C composite electrode than K₂S in term of cycleability and capacity.     

Metal-rich heterostructure of Ag-doped FeS/Fe2P for robust hydrogen evolution

The transition metal phosphides (TMPs) with highly active and low-cost are imperative electrocatalysts for hydrogen evolution reaction (HER). In particular, metal-rich interface engineering of iron phosphide could effectively modify the active sites for HER and accelerate the charge transfer, thus achieving the promoted efficiency. Herein, we report metal-rich heterostructure of Ag-doped Fe₂P shell attached to FeS core on Fe foams (FeS/Fe₂P–Ag@IF) for HER, which are synthesized by a simple hydrothermal method with subsequent low-temperature phosphorization. Notably, the phosphorization process simultaneously achieves the partial conversion of FeS to Fe₂P, and complete reduction of Ag₂S to Ag. Furthermore, the metal-rich structure of Fe₂P increases the active sites for hydrogen adsorption, which consequently contributes to hydrogen evolution. Simultaneously, the successful doping of metallic Ag enhanced the electroconductivity and the stability of the electrocatalyst. Benefiting from the ternary synergistic effect at FeS/Fe₂P–Ag@IF and metallic Ag doping, the optimal Ag-doped FeS/Fe₂P electrocatalyst exhibits a low overpotential of 214.9 mV at 100 mA cm^?2, even surviving at this large current density with long-term stability. This promising strategy involving metallic Ag doping may be a suitable option for the development of iron-based metal-rich phosphides heterostructured for HER.     

How hydrophobic side chain design affects water cluster connectivity in model polymer electrolyte membranes: Linear versus Y-shaped side chains

Dissipative Particle Dynamics is used to model water clustering in ion exchange membranes. We consider polymers with hydrophobic backbone alternatingly grafted with short hydrophilic ([C]) and hydrophobic linear (A₆) or Y-shaped side chains (ie. Ap [As][As]; p(s) = 6(0), 4(1), 2(2), 0(3)). At 16% water volume fraction water cluster connectivity (D_MC(W), water diffusivity, and inter water domain distance increase with length p and average number of bonds (<N_bond^A−C>) separating A and nearest C beads. Exchange of the pendent A and C beads for (A [C]A [A₆])₁₀ to form (A [A]A [A₅C])₁₀ decreases water cluster connectivity. This is because the lower number of bonds between nearest C beads for (A [C]A [A₆])₁₀ forces them to join the same water domain. This cooperative mechanism also explains deviations from the increase of D_MC(W) with <N_bond^A−C> for similar ion exchange capacity (IEC). At high IEC (A [A_x+1C])₁₀ architectures favour good connected pores which is surpassed by the H-phobic side chain (A [C]A [Ax])₁₀ architectures at low IEC.     

Molecular modulation of NiFe hydrogenase activity

In NiFe hydrogenases, electrons are transferred from the active site to the redox partner via a chain of three Iron–Sulfur clusters. Intriguingly, the surface-exposed [4Fe4S] cluster has three cysteines and one histidine as a ligand, which is quite unusual. When this Histidine (H184 in Desulfovibrio fructosovorans) was changed into a glycine, the distal cubane was still assembled but the oxidative activity of the mutants was 3% of that of the WT. As glycine is not a cubane ligand, a water molecule is likely to stabilize the fourth iron atom, making this coordination position labile. It was then possible to exchange the water ligand with an exogenous ligand. Depending on the molecule tested, the enzyme exhibited various activity levels, making possible a modulation of the enzyme activity.     

Removal of mercury from flue gases over iron modified activated carbon made by in situ ion exchange method

Ion exchange method was applied to synthesize iron modified activated carbon in the present study. The carbon in volatile compounds of cation-exchange resin could be fixed by ferric ion, resulting in the higher carbon yields of iron contained adsorbents. The optimal temperatures for carbonization and activation of ferric ion-exchanged resin were determined to be 800 °C. The iron modified activated carbon with (Fe/AC-800) and without (Fe/C-800) activation at 800 °C showed different mercury removal mechanisms, and they could be employed to remove mercury from flue gases at reaction temperatures of 180 and 150 °C, respectively. HgO, HgS and HgSO₄ were the mainly mercury compounds generated over spent Fe/C-800, whereas only HgO and HgS were observed over spent Fe/AC-800. The formation of HgO over spent Fe/C-800 and Fe/AC-800 were mainly due to the oxidation of mercury by chemisorbed oxygen and lattice oxygen, respectively. The HgSO₄ was derived from FeS with the aid of oxygen, and the HgS was formed through the reaction between mercury and FeS and/or elemental sulfur.     

Influence of transition metals Fe,Ni, and Nb on dehydrogenation characteristics of Mg(BH4)2: Electronic structure mechanisms

First principles calculations on Fe, Ni, and Nb doped Mg(BH₄)₂ were carried out to study the influence of dopants on dehydrogenation properties of Mg(BH₄)₂. It was shown that all dopants considered prefer to substitute for Mg with relatively smaller occupation energies comparing to the B substitution and the interstitial occupation. However, the B substitution shows smaller hydrogen dissociation energy than the Mg substitution. Mechanisms that dopants used to improve dehydrogenation properties of Mg(BH₄)₂ are different. For Mg substitution, Fe strongly interacts with one H atoms of the [BH₄] group, distorts its structural stability and therefore lowers the hydrogen dissociation energy, Ni may attract one particular H atom of the [BH₄] group and weakens the interactions between the B and other H atoms reducing the hydrogen dissociation energy, and the Nb however may drive the formation of NbB₂ and improves the dehydrogenation properties as well. In the B substitution, Fe interacts with the one of H atoms and decreases its structure stability, the Ni will attract its neighbor atoms to form a regular group which is almost identical in structure to that of the NiH₄ group in Mg₂NiH₄, and the NbH₂ and MgH₂ are likely to be generated by Nb doping.     

Redox state-dependent changes in the crystal structure of [NiFeSe] hydrogenase from Desulfovibrio vulgaris Hildenborough

Hydrogenases are enzymes that can potentially be used in bioelectrical devices or for biological hydrogen production, the most studied of which are the [NiFe] type. Most [NiFe] hydrogenases are inactivated by oxygen and the few known O₂-tolerant enzymes are hydrogen-uptake enzymes, unsuitable for hydrogen production, due to strong product inhibition. In contrast, the [NiFeSe] hydrogenases, where a selenocysteine is bound to the nickel, are very attractive alternatives because of their high hydrogen production activity and fast reactivation after O₂ exposure. Here we report five high-resolution crystallographic 3D structures of the soluble form of the [NiFeSe] hydrogenase from Desulfovibrio vulgaris Hildenborough in three different redox states (oxidized as-isolated, H₂-reduced and air re-oxidized), which revealed the structural changes that take place at the active site during enzyme reduction and re-oxidation. The results provide new insights into the pathways of O₂ inactivation in [NiFe], and in particular [NiFeSe], hydrogenases. In addition, they suggest that different enzymes may display different oxidized states upon exposure to O₂, which are probably determined by the protein structure.     

Mechanism of hydrogen production in [Fe–Fe]-hydrogenases: A quantum mechanics/molecular mechanics study

[Fe–Fe]-hydrogenases are a class of metalloenzymes that catalyze the production of H₂ from two protons and two electrons. Crystal structures for [Fe–Fe]-hydrogenases found in two species – Clostridium pasteurianum (CpI) and Desulfovibrio desulfuricans (DdH) – show very similar active sites. However, the catalytic mechanism has not as yet been fully clarified. We employed density functional theory (DFT) within a QM/MM method to investigate proposed mechanisms of hydrogen production by DdH and CpI hydrogenases and their dependence on the protein environment of the active sites. For each mechanism investigated, we found only minor differences between the CpI and DdH hydrogenases in terms of the intermediate active site structures, although one mechanism follows a lower energy path for DdH hydrogenase, while the other mechanism follows a lower energy path for the CpI hydrogenase. We note, however, that the high activation energy we calculated for a step unique to one of the mechanisms might preclude it, making the energy-path comparison for the two mechanisms unnecessary.     

Ni–Fe/Mg(Al)O alloy catalyst for carbon dioxide reforming of methane: Influence of reduction temperature and Ni–Fe alloying on coking

Various Ni–Fe/Mg(Al)O alloy catalysts were obtained by calcination of Ni–Fe–Mg–Al hydrotalcite-like compounds, followed by reduction at different temperatures (973–1173 K). The characterizations of XRD and STEM-EDX suggest that the resulting Ni–Fe alloy particles are composition-uniform and size-controllable. The alloy composition is little affected by the reduction temperature, whereas the particle size (5.8–8.2 nm) increases with the increase of reduction temperature. This property is ascribed to the homogeneous distribution of nickel and iron species during the catalyst preparation. All of the Ni–Fe/Mg(Al)O alloy catalysts show relatively high and stable activity for CH₄–CO₂ reforming during 25 h of investigation at 773–1073 K. Particularly, the 973 K-reduced catalyst exhibits higher coke-resistance due to its smaller particle size. E_a-CH₄ and CH₄-TPSR measurements indicate that Ni–Fe alloying inhibits CH₄ dissociation. It is considered that during DRM CH₄ is dissociated at the Ni sites and CO₂ may be activated at the metal-support interface as well as the Fe sites. Ni–Fe alloying may inhibit CH₄ dissociation and/or promote CO₂ activation, thus contributing to the suppression of coke deposition.