Search

Magnetite nanoparticles (MNP)-caused enhancements for H2 yield (HY) are usually justified based on oxidation-reduction potential (ORP), iron ions (Fe+2/+3) concentration, and enzymatic activity, that is pH-dependent. However, the questions “If pH, ORP, and Fe+2/+3 impacts are excluded, will MNP-caused HY enhancement be still present? and how electrical energy input (EEI) affects HY?” are still unanswered. Herein, control, MNP-supplemented, EEI-applied, FeCl3-, and Na2S-supplemented batches, referred as G1, G2, G3, G5, and G6, respectively were conducted, under fixed pH (6.0 ± 0.1). G5 and G6 targeted quantifying sole impact of Fe+2/+3, and ORP, respectively on HY. G1, G2, G3, G5, and G6 achieved HY values of 1.10 ± 0.05, 1.66 ± 0.07, 1.38 ± 0.06, 1.18 ± 0.04, and 1.16 ± 0.05 mol H2/molhexose, respectively. Neither Fe+2/+3 release nor ORP reduction significantly affected HY. Further, Clostridium amylolyticum dominance was almost similar among G2, and G3. Metabolites flux analysis and functional genes’ prediction highlighted that G2 achieved highest hydrogenase expression and lowest homoacetogenic H2 consumption.

In this brief review mainly based on our own work, we summarised the electrochemical investigations into those iron carbonyl complexes relevant to the iron centres of [FeFe]-and [Fe]-hydrogenases in following aspects: (i) electron transfer (E) coupled with a chemical reaction (C), EC process; (ii) two-electron process with potential inversion (ECisoE); and (iii) Proton-coupled electron transfer (PCET) and the role of an internal base group in non-coordination sphere. Through individual examples, these processes involved in the electrochemistry of the iron carbonyl complexes are discussed. In probing the complexes involving two-electron process with potential inversion, the co-existence of one- and two-electron for a complex is demonstrated by incorporating intramolecularly a ferrocenyl group(s) into the complex. Our studies on proton reduction catalysed by three diiron complexes involving the PCET mechanism are also briefly summarised. Finally, the perspectives of the electrochemical study in iron carbonyl complexes inspired by the iron-containing enzymes are mentioned in the sense of developing mimics of low overpotentials for hydrogen evolution through exploiting the PCET effect.

As a paradigm for multiple-site concerted proton-electron transfer (MS-CPET) in the process of proton reduction or hydrogen oxidation, the active site of [FeFe]-Hydrogenase (H 2 ase) is preorganized with an amine ('azadithiolate') as a proton relay and a [4Fe4S] subunit as an electron reservoir. The synergy of two individual factors efficiently lowers the overpotential for these reactions. In this study, we report a mononuclear manganese complex fac- [Mn(CO) 3 (6-(2-hydroxyphenol)-2-pyridine-2-quinoline) Br] ( 1 ) as a rare model to fully mimic the functions of the H 2 ase. In 1 , a redox active bidentate ligand decorated with a pendent phenol replicates the roles of the electron reservoir and the proton relay in natural enzyme. Experimental and theoretical studies reveal two consecutive MS-CPET processes in the catalytic cycle. For each MS-CPET, electron prestored in the reductive ligand and proton at the proximal phenol synchronously transfer to the Mn center in a concerted way. By virtue of this mechanism, complex 1 exhibited a low overpotential comparable to that of natual enzyme in electrochemical hydrogen production using phenol as a proton source. .

Metabolic engineering 68, 199-209 (2021). doi:10.1016/j.ymben.2021.10.006, Published by Academic Press, Orlando, Fla.

In this work, Manganese ferrite nanoparticles (MnFe 2O4 NPs) were synthesized, and their potential to increase the biohydrogen (bio-H2) yield of glucose in mesophilic and thermophilic fermentations was evaluated. Material characterization showed that the MnFe 2O4 NPs were perfectly synthesized, with a specific surface area of 43.97 m2g−1. Both mesophilic and thermophilic fermentation obtained the highest bio-H2 yields of 272.7 and 183.4 mL H2/g glucose in the 400 mg/L MnFe 2O4 NPs group, which values were increased by 40.1% and 131.9% (37 °C and 55 °C), respectively, compared to their control groups. Soluble microbial products (SMPs) showed that MnFe 2O4 NPs enhanced the butyrate pathway. Three-dimensional fluorescence spectroscopy analysis showed that MnFe 2O4 NPs increased the extracellular polymer content of anaerobic sludge. The results showed that MnFe 2O4 NPs distinctly increased the abundance of Clostridium_sensu_stricto 1 (39.49%) in the microbial community during mesophilic fermentation and improved the abundance of the dominant bacteria Clostridium_sensu_stricto_7 (25.59%) and Longlinea (21.36%) during thermophilic fermentation. Real-time PCR analysis showed that hydrogenase gene (hycl) expression increased exponentially after the addition of MnFe 2O4 NPs, and increasing the fermentation temperature improved the hydrogenase activity. We found that MnFe 2O4 NPs promoted to bio-H2 production in both mesophilic and thermophilic fermentation, and it contributed more significantly to thermophilic fermentation than mesophilic fermentation.

Radical S-adenosylmethionine (radical SAM or rSAM) enzymes use their S-adenosylmethionine cofactor bound to a unique Fe of a [4Fe-4S] cluster to generate the hot 5-deoxyadenosyl radical, which drives highly selective radical reactions via specific interactions with a given rSAM enzymes substrate. This Perspective focuses on the two rSAM enzymes involved in the biosynthesis of the organometallic H-cluster of [FeFe] hydrogenases. We present here a detailed sequential model initiated by HydG, which lyses a tyrosine substrate via a 5-deoxyadenosyl H atom abstraction from those amino acids amino group, initially producing dehydroglycine and an oxidobenzyl radical. In this model, two successive radical cascade reactions lead ultimately to the formation of HydGs product, a mononuclear Fe organometallic complex: [Fe(II)(CN)(CO)2(cysteinate)]-, with the iron originating from a unique dangler Fe coordinated by a cysteine ligand providing a sulfur bridge to another [4Fe-4S] auxiliary cluster in the enzyme. In turn, in this model, [Fe(II)(CN)(CO)2(cysteinate)]- is the substrate for HydE, the second rSAM enzyme in the biosynthetic pathway, which activates this mononuclear organometallic unit for dimerization, forming a [Fe2S2(CO)4(CN)2] precursor to the [2Fe] H component of the H-cluster, requiring only the completion of the bridging azadithiolate (SCH2NHCH2S) ligand. This model is built upon a foundation of data that incorporates cell-free synthesis, isotope sensitive spectroscopies, and the selective use of synthetic complexes substituting for intermediates in the enzymatic assembly line. We discuss controversies pertaining to this model and some remaining open issues to be addressed by future work.

Thermoanaerobacter kivuiis an acetogenic model organism that reduces CO2with electrons derived from H2or CO, or from organic substrates in the Wood–Ljugdahl pathway (WLP). For the calculation of ATP yields, it is necessary to know the electron carriers involved in coupling of the oxidative and reductive parts of metabolism. Analyses of key catabolic oxidoreductases in cell-free extract (CFE) or with purified enzymes revealed the physiological electron carriers involved. The glyceraldehyde-3-phosphate dehydrogenase (GA3P-DH) assayed in CFE was NAD+-specific, NADP+was used with less than 4% and ferredoxin (Fd) was not used. The methylene-THF dehydrogenase was NADP+-specific, NAD+or Fd were not used. A Nfn-type transhydrogenase that catalyzes reduced Fd-dependent reduction of NADP+with NADH as electron donor was also identified in CFE. The electron carriers used by the potential electron-bifurcating hydrogenase (HydABC) could not be unambiguously determined in CFE for technical reasons. Therefore, the enzyme was produced homologously inT. kivuiand purified by affinity chromatography. HydABC contained 33.9 ± 4.5 mol Fe/mol of protein and FMN; it reduced NADP+but not NAD+. The methylene-THF reductase (MetFV) was also produced homologously inT. kivuiand purified by affinity chromatography. MetFV contained 7.2 ± 0.4 mol Fe/mol of protein and FMN; the complex did neither use NADPH nor NADH as reductant but only reduced Fd. In sum, these analysis allowed us to propose a scheme for entire electron flow and bioenergetics inT. kivui.

A gene encoding an archaeal transcription regulator (TON_1525) homologous to the Tfx family DNA-binding protein has been identified to affect gene expression of carbon monoxide metabolism in the hyperthermophilic archaeon Thermococcus onnurineus NA1. To broadly understand gene regulation by the TON_1525 gene, a gene deletion mutant (Δ1525) was constructed and transcriptome changes were investigated. A total of 351 genes were found to be differentially expressed in the Δ1525 mutant. The expression levels of the hydrogenase genes were significantly changed; mbh (membrane-bound hydrogenase) and mch (membrane-bound carbon monoxide-dependent hydrogenase) genes were increased, and mfh2 (membrane-bound formate-dependent hydrogenase) and soluble hydrogenase genes were decreased. The Δ1525 mutant was cultured in a medium supplemented with maltodextrin or carbon monoxide to investigate whether changes in gene expression lead to physiological changes. The maximum H2 production rates of the Δ1525 mutant were significantly enhanced on both substrates compared to the wild-type strain. These results indicate that the TON_1525 gene can affect the expression of various genes including mbh and mch, consequently regulating the metabolism of T. onnurineus NA1. This study expands our understanding of the functional role of the Tfx family DNA-binding proteins.

The [FeFe] hydrogenase catalyzes the redox interconversion of protons and H2 with a Fe-S "H-cluster" employing CO, CN, and azadithiolate ligands to two Fe centers. The biosynthesis of the H-cluster is a highly interesting reaction carried out by a set of Fe-S maturase enzymes called HydE, HydF, and HydG. HydG, a member of the radical S-adenosylmethionine (rSAM) family, converts tyrosine, cysteine, and Fe(II) into an organometallic Fe(II)(CO)2(CN)cysteine "synthon", which serves as the substrate for HydE. Although key aspects of the HydG mechanism have been experimentally determined via isotope-sensitive spectroscopic methods, other important mechanistic questions have eluded experimental determination. Here, we use computational quantum chemistry to refine the mechanism of the HydG catalytic reaction. We utilize quantum mechanics/molecular mechanics simulations to investigate the reactions at the canonical Fe-S cluster, where a radical cleavage of the tyrosine substrate takes place and proceeds through a relay of radical intermediates to form HCN and a COO•- radical anion. We then carry out a broken-symmetry density functional theory study of the reactions at the unusual five-iron auxiliary Fe-S cluster, where two equivalents of CN- and COOH• coordinate to the fifth "dangler iron" in a series of substitution and redox reactions that yield the synthon as the final product for further processing by HydE.

One of the more active areas in bioorganometallic chemistry is the preparation and reactivity studies of active site mimics of the [NiFe]-hydrogenases. One area of particular recent progress involves reactions that interconvert Ni(μ-X)Fe centers for X = OH, H, CO, as described by Song et al. Such reactions illustrate new ways to access intermediates related to the Ni-R and Ni-SI states of the enzyme. Most models are derivatives of the type (diphosphine)Ni(SR)2Fe(CO)3-n(PR′3)n. In recent work, the methodology has been generalized to include FeII(diphosphine) derivatives of Ni(N2S2), where N2S22- is the tetradentate diamine-dithiolate (CH2N(CH3)CH2CH2S-)2. Indeed, models based on Ni(N2S2) have proven valuable, but these studies also highlight challenges in working with heterobimetallic complexes, specifically the tendency of some such Ni-Fe complexes to convert to homometalliic Ni-Ni derivatives. This kind of problem is not readily detected by X-ray crystallography. With this caution in mind, we argue that one series of complexes recently described in this journal are almost certainly misassigned.

The [4Fe-4S] cluster containing scaffold complex HypCD is the central construction site for the assembly of the [Fe](CN)2CO cofactor precursor of [NiFe]-hydrogenase. While the importance of the HypCD complex is well established, not much is known about the mechanism by which the CN– and CO ligands are transferred and attached to the iron ion. We developed an efficient protocol for the production and isolation of the functional HypCD complex that facilitated detailed spectroscopic investigations. The results obtained by UV/Vis-, electron paramagnetic Resonance (EPR)-, Resonance Raman-, Fourier-transform infrared (FTIR), and Mössbauer spectroscopy provide comprehensive evidence for an electron inventory fit to drive multi-electron redox reactions. We demonstrate the redox activity of the HypCD complex reporting the interconversion of the [4Fe-4S]2+/+ couple. Additionally, we observed a reversible redox conversion between the [4Fe-4S]2+ and a [3Fe-4S]+ cluster. MicroScale thermophoresis indicated preferable binding between the HypCD complex and its interaction partner HypEF under reducing conditions. Together, these results suggest a redox cascade involving the [4Fe-4S] cluster and a conserved disulfide bond of HypD that may facilitate the synthesis of the [Fe](CN)2CO cofactor precursor on the HypCD scaffold complex.

TTF- and exTTF-containing [(μ-S2)Fe2(CO)6] complexes have been prepared by the photochemical reaction of TTF or exTTF and [(μ-S2)Fe2(CO)6]. These complexes are able to interact with PAHs. In the absence of air and in acid media an electrocatalytic dihydrogen evolution reaction (HER) occurs, similarly to analogous [(μ-S2)Fe2(CO)6] complexes. However, in the presence of air, the TTF and exTTF organic moieties strongly influence the electrochemistry of these systems. The reported data may be valuable in the design of [FeFe] hydrogenase mimics able to combine the HER properties of the [FeFe] cores with the unique TTF properties

The review considers the role of purple sulfur bacteria in the global cycles of hydrogen and sulfur, as well as the ecology and physiology of these bacteria in relation to the metabolism of sulfur and hydrogen. Information is presented on five types of hydrogenases involved in consumption or production of hydrogen, as well as on various enzymes involved in the oxidation/reduction of sulfur compounds. Advances in the biochemistry and genetics of the enzymes from these microorganisms make it possible to analyze the interconnection of processes at a new level.

Development of a versatile, sustainable and efficient photosynthesis system that integrates intricate catalytic networks and energy modules at the same location is of considerable future value to energy transformation. In the present study, we develop a coenzyme-mediated supramolecular host-guest semibiological system that combines artificial and enzymatic catalysis for photocatalytic hydrogen evolution from alcohol dehydrogenation. This approach involves modification of the microenvironment of a dithiolene-embedded metal-organic cage to trap an organic dye and NADH molecule simultaneously, serving as a hydrogenase analogue to induce effective proton reduction inside the artificial host. This abiotic photocatalytic system is further embedded into the pocket of the alcohol dehydrogenase to couple enzymatic alcohol dehydrogenation. This host-guest approach allows in situ regeneration of NAD+/NADH couple to transfer protons and electrons between the two catalytic cycles, thereby paving a unique avenue for a synergic combination of abiotic and biotic synthetic sequences for photocatalytic fuel and chemical transformation., Abiotic–biotic hybrid systems are promising to trap light for fuel and chemical transformation with high efficacy and selectivity. This study reports a coenzyme-mediated supramolecular host-guest semibiological system combining supramolecular catalyst and enzymes for solar alcohol splitting.

This study describes the synthesis and characterization of magnetite nanoparticles (NPs) from water hyacinth (WH) extract and its regulatory effect on fermentative hydrogen production from lignocellulosic hydrolysate by Klebsiella sp. Characterization of WH-magnetite-NP revealed that it was a pure magnetite NP in a spherical shape with an average particle size of 13.5 ± 3.7 nm. The maximum cumulative hydrogen production with an increment of 23.49% and an optimum Y(H2/S) of 83.20 ± 2.19 mL/gsubstrate was obtained with WH-magnetite-NP at 20 mg/L. Monitoring of key node metabolites further established the potential of WH-magnetite-NP to increase the flux distribution of the hydrogen synthesis pathway. The hydrogenase activity was enhanced via WH-magnetite-NP addition, with peak value 2.1 times of the control. The expression of functional genes in key pathways assessed via RT-PCR highlighted the effect of WH-magnetite-NP on the evident promotion of hydrogenase and formate-hydrogen lyase. This is the first attempt to detect the expression of multiple functional genes in key metabolic pathways to explain the regulatory mechanism upon NP addition.

The reconstitution of [Mn]‐hydrogenases using a series of MnI complexes is described. These complexes are designed to have an internal base or pro‐base that may participate in metal–ligand cooperative catalysis or have no internal base or pro‐base. Only MnI complexes with an internal base or pro‐base are active for H2 activation; only [Mn]‐hydrogenases incorporating such complexes are active for hydrogenase reactions. These results confirm the essential role of metal–ligand cooperation for H2 activation by the MnI complexes alone and by [Mn]‐hydrogenases. Owing to the nature and position of the internal base or pro‐base, the mode of metal–ligand cooperation in two active [Mn]‐hydrogenases is different from that of the native [Fe]‐hydrogenase. One [Mn]‐hydrogenase has the highest specific activity of semi‐synthetic [Mn]‐ and [Fe]‐hydrogenases. This work demonstrates reconstitution of active artificial hydrogenases using synthetic complexes differing greatly from the native active site., A series of [Mn]‐hydrogenases were reconstituted using specifically designed MnI complexes. In two catalytically active [Mn]‐hydrogenases, the mode of metal–ligand cooperation is different from that of the native [Fe]‐hydrogenase. One such [Mn]‐hydrogenase exhibits the highest specific activity among all known semi‐synthetic [Mn]‐ and [Fe]‐hydrogenases.

It is shown that the catalytic center of Thiocapsa roseopersicina remains active after prolonged treatment with cyanide. It was found that the incubation of cyanide-treated hydrogenase in the presence of beta-mercaptoethanol, ferric iron, and sodium sulfide restored the hydrogenase activity in the hydrogen-oxidation reaction in the presence of methylviologen. The process of activity reconstruction depended on time and reached its maximum value (~ 60%) within 30 min at room temperature. In this case, an absorption band at 420 nm appeared in the absorption spectrum of the hydrogenase, which was present in the native hydrogenase and disappeared after treatment with cyanide; this indicated the reconstruction of iron–sulfur clusters. Thus, instead of growing bacteria in the presence of an iron isotope, one can replace 56Fe with 57Fe in the isolated enzyme, which will allow the use of smaller amounts of 57Fe.

Hydrogenase enzymes produce H(2) gas, which can be a potential source of alternative energy. Inspired by the [NiFe] hydrogenases, we report the construction of a de novo designed artificial hydrogenase (ArH). The ArH is a dimeric coiled coil where two cysteine (Cys) residues are introduced at tandem a/d positions of a heptad to create a tetrathiolato Ni binding site. Spectroscopic studies show that Ni binding significantly stabilizes the peptide producing electronic transitions characteristic of Ni-thiolate proteins. The ArH produces H(2) photocatalytically, demonstrating a bell-shaped pH-dependence on activity. Fluorescence lifetimes and transient absorption spectroscopic studies are undertaken to elucidate the nature of pH-dependence, and to monitor the reaction kinetics of the photochemical processes. pH titrations are employed to determine the role of protonated Cys on reactivity. Combined, we find that a fine balance between solution acidity and the electron transfer steps need to be maintained such that the yield of reduced photosensitizer can be maximized to produce the Ni(I)-peptide and the Ni(II)-H(−) intermediate (Ni-R) is protonated by a Cys (pK(a)~6.4) to produce H(2).

As S-donor metalloligands, the dithiolato [NiN2S2] complexes are employed to synthesize the bimetallic model of the active site of [NiFe]-Hydrogenases ([NiFe]-H2ases). In this work, three [NiN2S2] complexes were used to react with [NiCl2(PNP)] (PNP = (Ph2PCH2)2NCH3), respectively, to find out the influences of [NiN2S2] ligands on the structure and property of model complexes. It’s interesting to find dianion [Ni(phma)]2− (H4phma = N,N'-1,2-phenylenebis(2-mercaptoacetamide)) and [Ni(ema)]2− (H4ema = N,N'-1,2-ethylenebis(2-mercaptoacetamide)) could afford the desired dinuclear models [Ni(phma)(μ-S,S′)Ni(PNP)] (1) and [Ni(ema)(μ-S,S′)Ni(PNP)] (2), while the reaction of neutral [Ni(bme-dach)] (bme-dach = N,N′-bis-2-methyl-mercaptopropyl-1,4-diazacycloheptane) only yielded a trinuclear complex [Ni{Ni(bme-dach)(μ-S,S′)}2][BF4]2 (3). The structures of these complexes have been carefully characterized by NMR, high resolution mass spectrometry, elemental analysis, and X-ray diffraction structure analysis. As the model of [NiFe]-H2ases, complex 1 could electrocatalyze H2 evolution with a rate constant of 9.35 × 103 M−1 s−1 in the presence of CH3COOH.

Molecular oxygen (O2) is a highly reactive oxidizing agent and is harmful to many biological and industrial systems. Although O2 often interacts via metals or reducing agents, a binding mechanism involving an organic supramolecular structure has not been described to date. In this work, the prominent dipeptide hydrogelator fluorenylmethyloxycarbonyl-diphenylalanine is shown to encage O2 and significantly limit its diffusion and penetration through the hydrogel. Molecular dynamics simulations suggested that the O2 binding mechanism is governed by pockets formed between the aromatic rings in the supramolecular structure of the gel, which bind O2 through hydrophobic interactions. This phenomenon is harnessed to maintain the activity of the O2-hypersensitive enzyme [FeFe]-hydrogenase, which holds promising potential for utilizing hydrogen gas for sustainable energy applications. Hydrogenase encapsulation within the gel allows hydrogen production following exposure to ambient O2. This phenomenon may lead to utilization of this low molecular weight gelator in a wide range of O2-sensitive applications.

The biomethane generation in microbial electrosynthesis systems (MESs) was affected by the addition of trace metals (TMs) during biocatalyst’s metabolic activity. The functional role of various TMs (Mg2+, Fe2+, Ni2+, Zn2+, Co2+, Mn2+, and Mo2+) in regulating the CH4 production potential of a biocatalyst was evaluated under three different ranges of TM concentrations, and their performances were compared with the control operation (no trace metals). The TM level in a relatively medium concentration range exhibited the best efficiency and could enhance the CH4 production and currents generation by 3.9 and 7.7 folds higher than the values from the control. Cyclic voltammogram profiles depicted increment in redox catalytic currents during MES operation with TMs and also supported the involvement of mediators towards CH4 generation. The optimum TM concentrations could enhance MES performance as a constituent of ferredoxin and hydrogenase linked to energy metabolism.

Two anionic asymmetric Fe-Fe hydrogenase model compounds containing a single cyano (CN) and five carboxyl (CO) ligands, [Et4N][Fe2(μ-S2C3H6)(CO)5(CN)1] and [Et4N][Fe2(μ-S2C2H4)(CO)5(CN)1], dissolved in room-temperature acetonitrile, are examined. The molecular asymmetry affects the redox potentials of the central iron atoms, thus changing the photophysics and possible catalytic properties of the compounds. Femtosecond ultraviolet excitation with mid-infrared probe spectroscopy of the model compounds was employed to better understand the ultrafast dynamics of the enzyme-active site. Continuous ultraviolet lamp excitation with Fourier transform infrared (FTIR) spectroscopy was also used to explore stable product formation on the second timescale. For both model compounds, two timescales are observed; a 20-30 ps decay and the formation of a long-lived photoproduct. The picosecond decay is assigned to vibrational cooling and rotational dynamics, while the residual spectra remain for up to 300 ps, suggesting the formation of new photoproducts. Static FTIR spectroscopy yielded a different stable photoproduct than that observed on the ultrafast timescale. Density functional theory calculations simulated photoproducts for CO-loss and CN-loss isomers, and the resulting photoproduct spectra suggest that the picosecond transients arise from a complex mixture of isomerization after CO-loss, while dimerization and formation of a CN-containing Fe-CO-Fe bridged species are also considered.

Hydrogenases are versatile enzymatic catalysts with an unmet hydrogen evolution reactivity (HER) from synthetic bio-inspired systems. The binuclear active site only has one-site reactivity of the distal Fed atom. Here, binuclear complexes [Fe2(CO)5(μ-Mebdt)(P(4-C6H4OCH3)3)] 1 and [Fe2(CO)5(μ-Mebdt)(PPh2Py)] 2 are presented, which show electrocatalytic activity in the presence of weak acids as a proton source for the HER. Despite almost identical structural and spectroscopic properties (bond distances and angles from single-crystal X-ray; IR, UV/vis, and NMR), introduction of a nitrogen base atom in the phosphine ligand in 2 markedly changes site reactivity. The bridging benzenedithiolate ligand Mebdt interacts with the terminal ligand’s phenyl aromatic rings and stabilizes the reduced states of the catalysts. Although 1 with monodentate phosphine terminal ligands only shows a distal iron atom HER activity by a sequence of electrochemical and protonation steps, the lone pair of pyridine nitrogen in 2 acts as the primary site of protonation. This swaps the iron atom catalytic activity toward the proximal iron for complex 2. Density-functional theory (DFT) calculations reveal the role of terminal phosphines ligands without/with pendant amines by directing the proton transfer steps. The reactivity of 1 is a thiol-based protonation of a dangling bond in 1– and distal iron hydride mechanism, which may follow either an ECEC or EECC sequence, depending on the choice of acid. The pendant amine in 2 enables a terminal ligand protonation and an ECEC reactivity. The introduction of a terminal nitrogen atom enables the control of site reactivity in a binuclear system.

Gaining knowledge about the algal hydrogen metabolism is prerequisite for the biotechnological exploitation of photosynthetic H2 production. Model organism Chlamydomonas reinhardtii encodes two [FeFe]-hydrogenases, which are individually capable of catalysing the reversible reduction of protons to molecular hydrogen. While physiological results indicated that HYDA1, connected to the photosynthetic electron transfer pathway via plant-type ferredoxin PETF, is accountable for a major part of algal H2 evolution, the role of HYDA2 in the algal metabolism is not understood yet. Herein, a comprehensive screening of enzymatic attributes was conducted, revealing that the two enzymes differ in their affinity to oxidised PETF. Notably, utilising protein film voltammetry, a higher catalytic preference of HYDA2 to consume H2 was observed. Site directed exchange mutagenesis revealed the contribution of a hydroxyl group in place of threonine 226, present in HYDA1, but not in HYDA2, potentially influencing the electronic properties of the active site, thereby fine-tuning catalytic function.

Renewable energy production using microorganisms is one of the challenging issues for environmental sustainability. Algal hydrogen (H2) production has often been achieved by sulfur (S) and chloride ion (Cl−) deprivation in a growth medium; however, it may not be realistic to control S or Cl− concentrations in natural sources (e.g., wastewater). In this study, two different green algal species, Chlamydomonas reinhardtii and Chlorella sorokiniana were selected and their photosynthetic activities were compared with different acetate/Cl− ratios both in batch and continuous modes. At 150 of acetate/Cl− ratio, the H2 production rates were 0.25–0.33 μmol L−1 min−1 for C. sorokiniana and 0.20–0.38 μmol L−1 min−1 for C. reinhardtii, respectively. The hydrogenase (HydA) reactivation and photosystem II (PSII) inhibitor test revealed that biohydrogen production by algae is due to photosynthetic activity. It was found that maintaining acetate/Cl− ratios greater than 60–100 leads to continuous O2 depletion and thus renewable H2 production for both algal species. Molecular dynamics (MD) simulations of hydrogen bonding between Yz and His190 in PSII supported O2 inhibition using acetate. Using fermenter effluents, C. sorokiniana and C. reinhardtii showed a successful continuous H2 production of ~80 μmol L−1 and ~95 μmol L−1, respectively, for 15 days.

The impact of the four membrane-bound [NiFe]-hydrogenases (Hyd) of Escherichia coli on total H2-oxidizing activity during fermentation of a mixture of glucose, glycerol and formate at different pHs was examined. It was shown that Hyd-2 had a major contribution to total Hyd activity at pH 7.5 in early-stationary phase (24 h) cells, while the main contribution was made by Hyd-3 in late-stationary phase (72 h). Hyd-4-dependent Hyd activity could be demonstrated at pH 6.5 in cells lacking Hyd-1, Hyd-2 and Hyd-3. at pH 7.5 Hyd-4-dependent formate dehydrogenase (FDH-H) activity was demonstrated. Growth properties and fermentation end product patterns during 72 h demonstrated that the cells retained viability deep into stationary phase. Our findings emphasize the importance of formate in modulating H2 metabolism, presumably by contributing to maintain redox, pH and pmf balance. This is important for regulating and enhancing H2 production when a mixture of carbon sources is applied.

International audience; A series of triiron clusters [Fe3(CO)5(2-dppe)(µ-pdtR2)(µ-pdt)] featuring different combination of dithiolate bridges have been prepared by using dinuclear and mononuclear precursors, [Fe2(CO)6(µ-xdt)] (xdt = pdt, pdtEt2, pdtBn2, adtBn) and [Fe(CO)2(2-dppe)(2-pdt)]. Novel triiron compounds, featuring sterically crowded dithiolate bridges (pdtEt2, pdtBn2), were spectroscopically and structurally characterized. Their protonation and redox behaviours are discussed. The effect of the dithiolate bridges on the electrochemical properties (oxidation and reduction) of the complexes [Fe3(CO)5(2-dppe)(µ-xdt)(µ-pdt)] has been examined by cyclic voltammetry and rationalized by DFT calculations. Studies of the protonation of these species and their proton reduction behaviour were considered.

The organometallic H-cluster of the [FeFe]-hydrogenase consists of a [4Fe-4S] cubane bridged via a cysteinyl thiolate to a 2Fe subcluster ([2Fe]H) containing CO, CN-, and dithiomethylamine (DTMA) ligands. The H-cluster is synthesized by three dedicated maturation proteins: the radical SAM enzymes HydE and HydG synthesize the non-protein ligands, while the GTPase HydF serves as a scaffold for assembly of [2Fe]H prior to its delivery to the [FeFe]-hydrogenase containing the [4Fe-4S] cubane. HydG uses l-tyrosine as a substrate, cleaving it to produce p-cresol as well as the CO and CN- ligands to the H-cluster, although there is some question as to whether these are formed as free diatomics or as part of a [Fe(CO)2(CN)] synthon. Here we show that Clostridium acetobutylicum (C.a.) HydG catalyzes formation of multiple equivalents of free CO at rates comparable to those for CN- formation. Free CN- is also formed in excess molar equivalents over protein. A g = 8.9 EPR signal is observed for C.a. HydG reconstituted to load the 5th "dangler" iron of the auxiliary [4Fe-4S][FeCys] cluster and is assigned to this "dangler-loaded" cluster state. Free CO and CN- formation and the degree of activation of [FeFe]-hydrogenase all occur regardless of dangler loading, but are increased 10-35% in the dangler-loaded HydG; this indicates the dangler iron is not essential to this process but may affect relevant catalysis. During HydG turnover in the presence of myoglobin, the g = 8.9 signal remains unchanged, indicating that a [Fe(CO)2(CN)(Cys)] synthon is not formed at the dangler iron. Mutation of the only protein ligand to the dangler iron, H272, to alanine nearly completely abolishes both free CO formation and hydrogenase activation, however results show this is not due solely to the loss of the dangler iron. In experiments with wild type and H272A HydG, and with different degrees of dangler loading, we observe a consistent correlation between free CO/CN- formation and hydrogenase activation. Taken in full, our results point to free CO/CN-, but not an [Fe(CO)2(CN)(Cys)] synthon, as essential species in hydrogenase maturation.

Pseudomonas chlororaphis B23 yields nitrile hydratase (NHase) used for the production of 5-cyanovaleramide at the industrial level. Although the nhpC gene (known as P47K) located just downstream of the NHase structural genes (nhpAB) has been important for efficient NHase expression, the key role of nhpC remains poorly studied. Here, we purified two NHases expressed in the presence and absence of nhpC, respectively, and characterized them. The purified NHase expressed with nhpC proved to be an iron-containing holo-NHase, while the purified one expressed without nhpC was identified as an apo-NHase, which was iron-deficient. These findings indicated that nhpC would play a crucial role in the post-translational incorporation of iron into the NHase active site as a metal chaperone. In the overall amino acid sequence of NhpC, only the N-terminus exhibited similarities to the CobW protein involved in cobalamin biosynthesis, the UreG and HypB proteins essential for the metallocenter biosynthesis of urease and hydrogenase, respectively. NhpC contains a P-loop motif known as a nucleotide-binding site, and Lys23 and Thr24 are conserved in the P-loop motif in NhpC. Expression analysis of NHase formed in the presence of each mutant NhpC (i.e., K23A and T24A) resulted in immunodetectable production of a mutant NhpC and remarkable expression of NHase lacking the enzyme activity. These findings suggested that an intact P-loop containing Lys23 and Thr24 would be essential for the NhpC function in vivo for the post-translational metallocenter assembly of NHase.

Bioelectrochemical systems (BESs) are engineered systems that utilize electrochemical interactions between electrochemically active bacteria (EAB) and electrodes. BESs have attracted considerable attention for their utility in biotechnological processes. In a BES, hydrogen is generated by the reduction of water on low-potential cathode electrodes. However, limited information is available on the effect of hydrogen on the metabolism and growth of EAB and current generation in BESs. Here, we investigated the effect of hydrogen on current generation by a model EAB, Shewanella oneidensis MR-1. We found that this strain utilizes hydrogen as an electron donor for electrode respiration, thereby facilitating current generation and cell growth in the presence of organic substrates. Inner membrane (IM) quinones (i.e., ubiquinone and menaquinone), IM quinone-reactive hydrogenase Hya, and IM-bound quinone reductase CymA are involved in hydrogen-dependent current generation, suggesting that the redox cycling of IM quinones catalyzed by Hya and CymA contributes to the generation of the proton motive force and the synthesis of ATP via F0F1-ATPase. These findings indicate that the evolution of hydrogen on the cathode facilitates energy metabolism and growth of hydrogen-utilizing EAB associated with anodes. The results also suggest that hydrogen cycling between cathodes and anodes can hinder quantitative evaluation of organic substrate-dependent current generation in BESs.

The H-cluster of [Fe–Fe] hydrogenase consists of a [4Fe]H subcluster linked by the sulfur of a cysteine residue to an organometallic [2Fe]H subcluster that utilizes terminal CO and CN ligands to each Fe along with a bridging CO and a bridging SCH2NHCH2S azadithiolate (adt) to catalyze proton reduction or hydrogen oxidation. Three Fe–S “maturase” proteins, HydE, HydF, and HydG, are responsible for the biosynthesis of the [2Fe]H subcluster and its incorporation into the hydrogenase enzyme to form this catalytically active H-cluster. We have proposed that HydG is a bifunctional enzyme that uses S-adenosylmethione (SAM) bound to a [4Fe–4S] cluster to lyse tyrosine via a transient 5′-deoxyadenosyl radical to produce CO and CN ligands to a unique cysteine-chelated Fe(II) that is linked to a second [4Fe–4S] cluster via the cysteine sulfur. In this “synthon model”, after two cycles of tyrosine lysis, the product of HydG is completed: a [Fe(CN)(CO)2(cysteinate)]− organometallic unit that is vectored directly into the synthesis of the [2Fe]H sub-cluster. However our HydG-centric synthon model is not universally accepted, so further validation is important. In this Frontiers article, we discuss recent results using a synthetic “Syn-B” complex that donates [Fe(CN)(CO)2(cysteinate)]− units that match our proposed HydG product. Can Syn-B activate hydrogenase in the absence of HydG and its tyrosine substrate? If so, since Syn-B can be synthesized with specific magnetic nuclear isotopes and with chemical substitutions, its use could allow its enzymatic conversions on the route to the H-cluster to be monitored and modeled in fresh detail.

Hydrogen production is key to carbon-free energy production and the use of hydrogenases is an excellent approach for this achievement since they produce hydrogen with extraordinary efficiencies. However, these enzymes have several shortcomings, which may be resolved using hydrogenase mimics. The design and synthesis of these mimics is a very active area of research, and the understanding of the mode of the action of mimics is particularly relevant to the future design of robust and productive catalysts. In this review, we discuss the role of isotopes (mainly deuterium) in disentangling key steps of the mechanisms of H2 activation by mimics of hydrogenases. Through selected examples, we focus on the key aspects of processes that, in many cases, have allowed a better understanding of the mode of action of the natural enzymes, making a valuable contribution to the design of more efficient complexes for the production of hydrogen.