Oxygen sensitivity of [FeFe]-hydrogenase: a comparative study of active site mimics inside vs. outside the enzyme.

[FeFe]-hydrogenase is nature's most efficient proton reducing and H2-oxidizing enzyme. However, biotechnological applications are hampered by the O2 sensitivity of this metalloenzyme, and the mechanism of aerobic deactivation is not well understood. Here, we explore the oxygen sensitivity of four mimics of the organometallic active site cofactor of [FeFe]-hydrogenase, [Fe2(adt)(CO)6-x(CN)x]x- and [Fe2(pdt)(CO)6-x(CN)x]x- (x = 1, 2) as well as the corresponding cofactor variants of the enzyme by means of infrared, Mössbauer, and NMR spectroscopy. Additionally, we describe a straightforward synthetic recipe for the active site precursor complex Fe2(adt)(CO)6. Our data indicate that the aminodithiolate (adt) complex, which is the synthetic precursor of the natural active site cofactor, is most oxygen sensitive. This observation highlights the significance of proton transfer in aerobic deactivation, and supported by DFT calculations facilitates an identification of the responsible reactive oxygen species (ROS). Moreover, we show that the ligand environment of the iron ions critically influences the reactivity with O2 and ROS like superoxide and H2O2 as the oxygen sensitivity increases with the exchange of ligands from CO to CN-. The trends in aerobic deactivation observed for the model complexes are in line with the respective enzyme variants. Based on experimental and computational data, a model for the initial reaction of [FeFe]-hydrogenase with O2 is developed. Our study underscores the relevance of model systems in understanding biocatalysis and validates their potential as important tools for elucidating the chemistry of oxygen-induced deactivation of [FeFe]-hydrogenase.

Engineering bionanoreactor in bacteria for efficient hydrogen production.

Hydrogen production through water splitting is a vital strategy for renewable and sustainable clean energy. In this study, we developed an approach integrating nanomaterial engineering and synthetic biology to establish a bionanoreactor system for efficient hydrogen production. The periplasmic space (20 to 30 nm) of an electroactive bacterium, Shewanella oneidensis MR-1, was engineered to serve as a bionanoreactor to enhance the interaction between electrons and protons, catalyzed by hydrogenases for hydrogen generation. To optimize electron transfer, we used the microbially reduced graphene oxide (rGO) to coat the electrode, which improved the electron transfer from the electrode to the cells. Native MtrCAB protein complex on S. oneidensis and self-assembled iron sulfide (FeS) nanoparticles acted in tandem to facilitate electron transfer from an electrode to the periplasm. To enhance proton transport, S. oneidensis MR-1 was engineered to express Gloeobacter rhodopsin (GR) and the light-harvesting antenna canthaxanthin. This led to efficient proton pumping when exposed to light, resulting in a 35.6% increase in the rate of hydrogen production. The overexpression of native [FeFe]-hydrogenase further improved the hydrogen production rate by 56.8%. The bionanoreactor engineered in S. oneidensis MR-1 achieved a hydrogen yield of 80.4 µmol/mg protein/day with a Faraday efficiency of 80% at a potential of -0.75 V. This periplasmic bionanoreactor combines the strengths of both nanomaterial and biological components, providing an efficient approach for microbial electrosynthesis.

Co-expression analysis reveals distinct alliances around two carbon fixation pathways in hydrothermal vent symbionts.

Most autotrophic organisms possess a single carbon fixation pathway. The chemoautotrophic symbionts of the hydrothermal vent tubeworm Riftia pachyptila, however, possess two functional pathways: the Calvin-Benson-Bassham (CBB) and the reductive tricarboxylic acid (rTCA) cycles. How these two pathways are coordinated is unknown. Here we measured net carbon fixation rates, transcriptional/metabolic responses and transcriptional co-expression patterns of Riftia pachyptila endosymbionts by incubating tubeworms collected from the East Pacific Rise at environmental pressures, temperature and geochemistry. Results showed that rTCA and CBB transcriptional patterns varied in response to different geochemical regimes and that each pathway is allied to specific metabolic processes; the rTCA is allied to hydrogenases and dissimilatory nitrate reduction, whereas the CBB is allied to sulfide oxidation and assimilatory nitrate reduction, suggesting distinctive yet complementary roles in metabolic function. Furthermore, our network analysis implicates the rTCA and a group 1e hydrogenase as key players in the physiological response to limitation of sulfide and oxygen. Net carbon fixation rates were also exemplary, and accordingly, we propose that co-activity of CBB and rTCA may be an adaptation for maintaining high carbon fixation rates, conferring a fitness advantage in dynamic vent environments.

Characterization and potentiality of plant-derived silver nanoparticles for enhancement of biomass and hydrogen production in Chlorella sp. under nitrogen deprived condition.

Energy is a crucial entity for the development and it has various alternative forms of energy sources. Recently, the synthesis of nanoparticles using benign biocatalyst has attracted increased attention. In this study, silver nanoparticles were synthesized and characterized using Azadirachta indica plant-derived phytochemical as the reducing agent. Biomass of the microalga Chlorella sp. cultivated in BG11 medium increased after exposure to low concentrations of up to 0.48 mg L-1 AgNPs. In addition, algal cells treated with 0.24 mg L-1 AgNPs and cultivated in BG110 medium which contained no nitrogen source showed the highest hydrogen yield of 10.8 mmol L-1, whereas the untreated cells under the same conditions showed very low hydrogen yield of 0.003 mmol L-1. The enhanced hydrogen production observed in the treated cells was consistent with an increase in hydrogenase activity. Treatment of BG110 grown cells with low concentration of green synthesized AgNPs at 0.24 mg L-1 enhanced hydrogenase activity with a 5-fold increase of enzyme activity compared to untreated BG110 grown cells. In addition, to improve photolytic water splitting efficiency for hydrogen production, cells treated with AgNPs at 0.24 mg L-1 showed highest oxygen evolution signifying improvement in photosynthesis. The silver nanoparticles synthesized using phytochemicals derived from plant enhanced both microalgal biomass and hydrogen production with an added advantage of CO2 reduction which could be achieved due to an increase in biomass. Hence, treating microalgae with nanoparticles provided a promising strategy to reduce the atmospheric carbon dioxide as well as increasing production of hydrogen as clean energy.

Synergistic enhancement of pulsed light-induced H2 photoproduction in Chlamydomonas cells by optimal sulfite concentration and light waveform.

Pulsed light illumination stands out as a noteworthy technique for photosynthetic H2 production, playing a crucial role in eliminating O2 and activating hydrogenase enzymes. However, further improvements are essential to make H2 photoproduction suitable for future commercial applications. In our study, we observed a distinct enhancement in pulsed light-induced H2 photoproduction in the unicellular green alga Chlamydomonas reinhardtii when treated with the optimal concentration of the mild O2 scavenger Na2SO3. This improvement was a result of reduced O2 content, increased hydrogenase enzyme activity, and suppressed H2-uptake activity. Furthermore, our findings indicate that exposing Na2SO3-treated C. reinhardtii to optimal light waveform continues to significantly boost pulsed light-induced H2 photoproduction, attributed to the alleviation of impaired photosystem II activity. Altogether, the combined application of optimal sulfite concentration and light waveform effectively enhances pulsed light-induced photosynthetic H2 production in the green alga C. reinhardtii.

Mechanistic Principles of Hydrogen Evolution in the Membrane-Bound Hydrogenase.

The membrane-bound hydrogenase (Mbh) from Pyrococcus furiosus is an archaeal member of the Complex I superfamily. It catalyzes the reduction of protons to H2 gas powered by a [NiFe] active site and transduces the free energy into proton pumping and Na+/H+ exchange across the membrane. Despite recent structural advances, the mechanistic principles of H2 catalysis and ion transport in Mbh remain elusive. Here, we probe how the redox chemistry drives the reduction of the proton to H2 and how the catalysis couples to conformational dynamics in the membrane domain of Mbh. By combining large-scale quantum chemical density functional theory (DFT) and correlated ab initio wave function methods with atomistic molecular dynamics simulations, we show that the proton transfer reactions required for the catalysis are gated by electric field effects that direct the protons by water-mediated reactions from Glu21L toward the [NiFe] site, or alternatively along the nearby His75L pathway that also becomes energetically feasible in certain reaction steps. These local proton-coupled electron transfer (PCET) reactions induce conformational changes around the active site that provide a key coupling element via conserved loop structures to the ion transport activity. We find that H2 forms in a heterolytic proton reduction step, with spin crossovers tuning the energetics along key reaction steps. On a general level, our work showcases the role of electric fields in enzyme catalysis and how these effects are employed by the [NiFe] active site of Mbh to drive PCET reactions and ion transport.

Redirecting electron flow in Acetobacterium woodii enables growth on CO and improves growth on formate.

Anaerobic, acetogenic bacteria are well known for their ability to convert various one-carbon compounds, promising feedstocks for a future, sustainable biotechnology, to products such as acetate and biofuels. The model acetogen Acetobacterium woodii can grow on CO2, formate or methanol, but not on carbon monoxide, an important industrial waste product. Since hydrogenases are targets of CO inhibition, here, we genetically delete the two [FeFe] hydrogenases HydA2 and HydBA in A. woodii. We show that the ∆hydBA/hydA2 mutant indeed grows on CO and produces acetate, but only after a long adaptation period. SNP analyzes of CO-adapted cells reveal a mutation in the HycB2 subunit of the HydA2/HydB2/HydB3/Fdh-containing hydrogen-dependent CO2 reductase (HDCR). We observe an increase in ferredoxin-dependent CO2 reduction and vice versa by the HDCR in the absence of the HydA2 module and speculate that this is caused by the mutation in HycB2. In addition, the CO-adapted ∆hydBA/hydA2 mutant growing on formate has a final biomass twice of that of the wild type.

Minimal and hybrid hydrogenases are active from archaea.

Microbial hydrogen (H2) cycling underpins the diversity and functionality of diverse anoxic ecosystems. Among the three evolutionarily distinct hydrogenase superfamilies responsible, [FeFe] hydrogenases were thought to be restricted to bacteria and eukaryotes. Here, we show that anaerobic archaea encode diverse, active, and ancient lineages of [FeFe] hydrogenases through combining analysis of existing and new genomes with extensive biochemical experiments. [FeFe] hydrogenases are encoded by genomes of nine archaeal phyla and expressed by H2-producing Asgard archaeon cultures. We report an ultraminimal hydrogenase in DPANN archaea that binds the catalytic H-cluster and produces H2. Moreover, we identify and characterize remarkable hybrid complexes formed through the fusion of [FeFe] and [NiFe] hydrogenases in ten other archaeal orders. Phylogenetic analysis and structural modeling suggest a deep evolutionary history of hybrid hydrogenases. These findings reveal new metabolic adaptations of archaea, streamlined H2 catalysts for biotechnological development, and a surprisingly intertwined evolutionary history between the two major H2-metabolizing enzymes.

The H-cluster of [FeFe] Hydrogenases: Its Enzymatic Synthesis and Parallel Inorganic Semisynthesis.

ConspectusNature's prototypical hydrogen-forming catalysts─hydrogenases─have attracted much attention because they catalyze hydrogen evolution at near zero overpotential and ambient conditions. Beyond any possible applications in the energy sphere, the hydrogenases feature complicated active sites, which implies novel biosynthetic pathways. In terms of the variety of cofactors, the [FeFe]-hydrogenase is among the most complex.For more than a decade, we have worked on the biosynthesis of the active site of [FeFe] hydrogenases. This site, the H-cluster, is a six-iron ensemble consisting of a [4Fe-4S]H cluster linked to a [2Fe]H cluster that is coordinated to CO, cyanide, and a unique organic azadithiolate ligand. Many years ago, three enzymes, namely, HydG, HydE, and HydF, were shown to be required for the biosynthesis and the in vitro maturation of [FeFe] hydrogenases. The structures of the maturases were determined crystallographically, but still little progress was made on the biosynthetic pathway. As described in this Account, the elucidation of the biosynthetic pathway began in earnest with the identification of a molecular iron-cysteinate complex produced within HydG.In this Account, we present our most recent progress toward the molecular mechanism of [2Fe]H biosynthesis using a collaborative approach involving cell-free biosynthesis, isotope and element-sensitive spectroscopies, as well as inorganic synthesis of purported biosynthetic intermediates. Our study starts from the radical SAM enzyme HydG that lyses tyrosine into CO and cyanide and forms an Fe(CO)2(CN)-containing species. Crystallographic identification of a unique auxiliary 5Fe-4S cluster in HydG leads to a proposed catalytic cycle in which a free cysteine-chelated "dangler" Fe serves as the platform for the stepwise formation of a [4Fe-4S][Fe(CO)(CN)(cysteinate)] intermediate, which releases the [Fe(CO)2(CN)(cysteinate)] product, Complex B. Since Complex B is unstable, we applied synthetic organometallic chemistry to make an analogue, syn-B, and showed that it fully replaces HydG in the in vitro maturation of the H-cluster. Syn-B serves as the substrate for the next radical SAM enzyme HydE, where the low-spin Fe(II) center is activated by 5'-dAdo• to form an adenosylated Fe(I) intermediate. We propose that this Fe(I) species strips the carbon backbone and dimerizes in HydE to form a [Fe2(SH)2(CO)4(CN)2]2- product. This mechanistic scenario is supported by the use of a synthetic version of this dimer complex, syn-dimer, which allows for the formation of active hydrogenase with only the HydF maturase. Further application of this semisynthesis strategy shows that an [Fe2(SCH2NH2)2(CO)4(CN)2]2- complex can activate the apo hydrogenase, marking it as the last biosynthetic intermediate en route to the H-cluster. This combined enzymatic and semisynthetic approach greatly accelerates our understanding of H-cluster biosynthesis. We anticipate additional mechanistic details regarding H-cluster biosynthesis to be gleaned, and this methodology may be further applied in the study of other complex metallocofactors.

A Conserved Binding Pocket in HydF is Essential for Biological Assembly and Coordination of the Diiron Site of [FeFe]-Hydrogenases.

The active site cofactor of [FeFe]-hydrogenases consists of a cubane [4Fe-4S]-cluster and a unique [2Fe-2S]-cluster, harboring unusual CO- and CN--ligands. The biosynthesis of the [2Fe-2S]-cluster requires three dedicated maturation enzymes called HydG, HydE and HydF. HydG and HydE are both involved in synthesizing a [2Fe-2S]-precursor, still lacking parts of the azadithiolate (adt) moiety that bridge the two iron atoms. This [2Fe-2S]-precursor is then finalized within the scaffold protein HydF, which binds and transfers the [2Fe-2S]-precursor to the hydrogenase. However, its exact binding mode within HydF is still elusive. Herein, we identified the binding location of the [2Fe-2S]-precursor by altering size and charge of a highly conserved protein pocket via site directed mutagenesis (SDM). Moreover, we identified two serine residues that are essential for binding and assembling the [2Fe-2S]-precursor. By combining SDM and molecular docking simulations, we provide a new model on how the [2Fe-2S]-cluster is bound to HydF and demonstrate the important role of one highly conserved aspartate residue, presumably during the bioassembly of the adt moiety.

Replacing a Cysteine Ligand by Selenocysteine in a [NiFe]-Hydrogenase Unlocks Hydrogen Production Activity and Addresses the Role of Concerted Proton-Coupled Electron Transfer in Electrocatalytic Reversibility.

Hydrogenases catalyze hydrogen/proton interconversion that is normally electrochemically reversible (having minimal overpotential requirement), a special property otherwise almost exclusive to platinum metals. The mechanism of [NiFe]-hydrogenases includes a long-range proton-coupled electron-transfer process involving a specific Ni-coordinated cysteine and the carboxylate of a nearby glutamate. A variant in which this cysteine has been exchanged for selenocysteine displays two distinct changes in electrocatalytic properties, as determined by protein film voltammetry. First, proton reduction, even in the presence of H2 (a strong product inhibitor), is greatly enhanced relative to H2 oxidation: this result parallels a characteristic of natural [NiFeSe]-hydrogenases which are superior H2 production catalysts. Second, an inflection (an S-shaped "twist" in the trace) appears around the formal potential, the small overpotentials introduced in each direction (oxidation and reduction) signaling a departure from electrocatalytic reversibility. Concerted proton-electron transfer offers a lower energy pathway compared to stepwise transfers. Given the much lower proton affinity of Se compared to that of S, the inflection provides compelling evidence that concerted proton-electron transfer is important in determining why [NiFe]-hydrogenases are reversible electrocatalysts.

The formate-hydrogen axis and its impact on the physiology of enterobacterial fermentation.

Formic acid (HCOOH) and dihydrogen (H2) are characteristic products of enterobacterial mixed-acid fermentation, with H2 generation increasing in conjunction with a decrease in extracellular pH. Formate and acetyl-CoA are generated by radical-based and coenzyme A-dependent cleavage of pyruvate catalysed by pyruvate formate-lyase (PflB). Formate is also the source of H2, which is generated along with carbon dioxide through the action of the membrane-associated, cytoplasmically-oriented formate hydrogenlyase (FHL-1) complex. Synthesis of the FHL-1 complex is completely dependent on the cytoplasmic accumulation of formate. Consequently, formate determines its own disproportionation into H2 and CO2 by the FHL-1 complex. Cytoplasmic formate levels are controlled by FocA, a pentameric channel that translocates formic acid/formate bidirectionally between the cytoplasm and periplasm. Each protomer of FocA has a narrow hydrophobic pore through which neutral formic acid can pass. Two conserved amino acid residues, a histidine and a threonine, at the center of the pore control directionality of translocation. The histidine residue is essential for pH-dependent influx of formic acid. Studies with the formate analogue hypophosphite and amino acid variants of FocA suggest that the mechanisms of formic acid efflux and influx differ. Indeed, current data suggest, depending on extracellular formate levels, two separate uptake mechanisms exist, both likely contributing to maintain pH homeostasis. Bidirectional formate/formic acid translocation is dependent on PflB and influx requires an active FHL-1 complex. This review describes the coupling of formate and H2 production in enterobacteria.

Characterization of the Bottlenecks and Pathways for Inhibitor Dissociation from [NiFe] Hydrogenase.

[NiFe] hydrogenases can act as efficient catalysts for hydrogen oxidation and biofuel production. However, some [NiFe] hydrogenases are inhibited by gas molecules present in the environment, such as O2 and CO. One strategy to engineer [NiFe] hydrogenases and achieve O2- and CO-tolerant enzymes is by introducing point mutations to block the access of inhibitors to the catalytic site. In this work, we characterized the unbinding pathways of CO in the complex with the wild-type and 10 different mutants of [NiFe] hydrogenase from Desulfovibrio fructosovorans using τ-random accelerated molecular dynamics (τRAMD) to enhance the sampling of unbinding events. The ranking provided by the relative residence times computed with τRAMD is in agreement with experiments. Extensive data analysis of the simulations revealed that from the two bottlenecks proposed in previous studies for the transit of gas molecules (residues 74 and 122 and residues 74 and 476), only one of them (residues 74 and 122) effectively modulates diffusion and residence times for CO. We also computed pathway probabilities for the unbinding of CO, O2, and H2 from the wild-type [NiFe] hydrogenase, and we observed that while the most probable pathways are the same, the secondary pathways are different. We propose that introducing mutations to block the most probable paths, in combination with mutations to open the main secondary path used by H2, can be a feasible strategy to achieve CO and O2 resistance in the [NiFe] hydrogenase from Desulfovibrio fructosovorans.

Properties of the iron-sulfur cluster electron transfer relay in an [FeFe]-hydrogenase that is tuned for H2 oxidation catalysis.

[FeFe]-hydrogenases catalyze the reversible oxidation of H2 from electrons and protons at an organometallic active site cofactor named the H-cluster. In addition to the H-cluster, most [FeFe]-hydrogenases possess accessory FeS cluster (F-cluster) relays that function in mediating electron transfer with catalysis. There is significant variation in the structural properties of F-cluster relays among the [FeFe]-hydrogenases; however, it is unknown how this variation relates to the electronic and thermodynamic properties, and thus the electron transfer properties, of enzymes. Clostridium pasteurianum [FeFe]-hydrogenase II (CpII) exhibits a large catalytic bias for H2 oxidation (compared to H2 production), making it a notable system for examining if F-cluster properties contribute to the overall function and efficiency of the enzyme. By applying a combination of multifrequency and potentiometric electron paramagnetic resonance, we resolved two electron paramagnetic resonance signals with distinct power- and temperature-dependent properties at g = 2.058 1.931 1.891 (F2.058) and g = 2.061 1.920 1.887 (F2.061), with assigned midpoint potentials of -140 ± 18 mV and -406 ± 12 mV versus normal hydrogen electrode, respectively. Spectral analysis revealed features consistent with spin-spin coupling between the two [4Fe-4S] F-clusters, and possible functional models are discussed that account for the contribution of coupling to the electron transfer landscape. The results signify the interplay of electronic coupling and free energy properties and parameters of the FeS clusters to the electron transfer mechanism through the relay and provide new insight as to how relays functionally complement the catalytic directionality of active sites to achieve highly efficient catalysis.

Predicting the Structure of Enzymes with Metal Cofactors: The Example of [FeFe] Hydrogenases.

The advent of deep learning algorithms for protein folding opened a new era in the ability of predicting and optimizing the function of proteins once the sequence is known. The task is more intricate when cofactors like metal ions or small ligands are essential to functioning. In this case, the combined use of traditional simulation methods based on interatomic force fields and deep learning predictions is mandatory. We use the example of [FeFe] hydrogenases, enzymes of unicellular algae promising for biotechnology applications to illustrate this situation. [FeFe] hydrogenase is an iron-sulfur protein that catalyzes the chemical reduction of protons dissolved in liquid water into molecular hydrogen as a gas. Hydrogen production efficiency and cell sensitivity to dioxygen are important parameters to optimize the industrial applications of biological hydrogen production. Both parameters are related to the organization of iron-sulfur clusters within protein domains. In this work, we propose possible three-dimensional structures of Chlorella vulgaris 211/11P [FeFe] hydrogenase, the sequence of which was extracted from the recently published genome of the given strain. Initial structural models are built using: (i) the deep learning algorithm AlphaFold; (ii) the homology modeling server SwissModel; (iii) a manual construction based on the best known bacterial crystal structure. Missing iron-sulfur clusters are included and microsecond-long molecular dynamics of initial structures embedded into the water solution environment were performed. Multiple-walkers metadynamics was also used to enhance the sampling of structures encompassing both functional and non-functional organizations of iron-sulfur clusters. The resulting structural model provided by deep learning is consistent with functional [FeFe] hydrogenase characterized by peculiar interactions between cofactors and the protein matrix.

Cascade reactions with two non-physiological partners for NAD(P)H regeneration via renewable hydrogen.

An attractive application of hydrogenases, combined with the availability of cheap and renewable hydrogen (i.e., from solar and wind powered electrolysis or from recycled wastes), is the production of high-value electron-rich intermediates such as reduced nicotinamide adenine dinucleotides. Here, the capability of a very robust and oxygen-resilient [FeFe]-hydrogenase (CbA5H) from Clostridium beijerinckii SM10, previously identified in our group, combined with a reductase (BMR) from Bacillus megaterium (now reclassified as Priestia megaterium) was tested. The system shows a good stability and it was demonstrated to reach up to 28 ± 2 nmol NADPH regenerated s-1 mg of hydrogenase-1 (i.e., 1.68 ± 0.12 U mg-1, TOF: 126 ± 9 min-1) and 0.46 ± 0.04 nmol NADH regenerated s-1 mg of hydrogenase-1 (i.e., 0.028 ± 0.002 U mg-1, TOF: 2.1 ± 0.2 min-1), meaning up to 74 mg of NADPH and 1.23 mg of NADH produced per hour by a system involving 1 mg of CbA5H. The TOF is comparable with similar systems based on hydrogen as regenerating molecule for NADPH, but the system is first of its kind as for the [FeFe]-hydrogenase and the non-physiological partners used. As a proof of concept a cascade reaction involving CbA5H, BMR and a mutant BVMO from Acinetobacter radioresistens able to oxidize indole is presented. The data show how the cascade can be exploited for indigo production and multiple reaction cycles can be sustained using the regenerated NADPH.

Final Stages in the Biosynthesis of the [FeFe]-Hydrogenase Active Site.

The paper aims to elucidate the final stages in the biosynthesis of the [2Fe]H active site of the [FeFe]-hydrogenases. The recently hypothesized intermediate [Fe2(SCH2NH2)2(CN)2(CO)4]2- ([1]2-) was prepared by a multistep route from [Fe2(S2)(CN)(CO)5]-. The following synthetic intermediates were characterized in order: [Fe2(SCH2NHFmoc)2(CNBEt3)(CO)5]-, [Fe2(SCH2NHFmoc)2(CN)-(CO)5]-, and [Fe2(SCH2NHFmoc)2(CN)2(CO)4]2-, where Fmoc is fluorenylmethoxycarbonyl). Derivatives of these anions include [K(18-crown-6)]+, PPh4 + and PPN+ salts as well as the 13CD2-isotopologues. These Fe2 species exist as a mixture of two isomers attributed to diequatorial (ee) and axial-equatorial (ae) stereochemistry at sulfur. In vitro experiments demonstrate that [1]2- maturates HydA1 in the presence of HydF and a cocktail of reagents. HydA1 can also be maturated using a highly simplified cocktail, omitting HydF and other proteins. This result is consistent with HydA1 participating in the maturation process and refines the roles of HydF.

Isolation of an H2-dependent electron-bifurcating CO2-reducing megacomplex with MvhB polyferredoxin from Methanothermobacter marburgensis.

In the hydrogenotrophic methanogenic pathway, formylmethanofuran dehydrogenase (Fmd) catalyzes the formation of formylmethanofuran through reducing CO2. Heterodisulfide reductase (Hdr) provides two low potential electrons for the Fmd reaction using a flavin-based electron-bifurcating mechanism. [NiFe]-hydrogenase (Mvh) or formate dehydrogenase (Fdh) complexes with Hdr and provides electrons to Hdr from H2 and formate, or the reduced form of F420, respectively. Recently, an Fdh-Hdr complex was purified as a 3-MDa megacomplex that contained Fmd, and its three-dimensional structure was elucidated by cryo-electron microscopy. In contrast, the Mvh-Hdr complex has been characterized only as a complex without Fmd. Here, we report the isolation and characterization of a 1-MDa Mvh-Hdr-Fmd megacomplex from Methanothermobacter marburgensis. After anion-exchange and hydrophobic chromatography was performed, the proteins with Hdr activity eluted in the 1- and 0.5-MDa fractions during size exclusion chromatography. Considering the apparent molecular mass and the protein profile in the fractions, the 1-MDa megacomplex was determined to be a dimeric Mvh-Hdr-Fmd complex. The megacomplex fraction contained a polyferredoxin subunit MvhB, which contains 12 [4Fe-4S]-clusters. MvhB polyferredoxin has never been identified in the previously purified Mvh-Hdr and Fmd preparations, suggesting that MvhB polyferredoxin is stabilized by the binding between Mvh-Hdr and Fmd in the Mvh-Hdr-Fmd complex. The purified Mvh-Hdr-Fmd megacomplex catalyzed electron-bifurcating reduction of [13C]-CO2 to form [13C]-formylmethanofuran in the absence of extrinsic ferredoxin. These results demonstrated that the subunits in the Mvh-Hdr-Fmd megacomplex are electronically connected for the reduction of CO2, which likely involves MvhB polyferredoxin as an electron relay.

Inactivation of hydrogenase-3 leads to enhancement of 1,3-propanediol and 2,3-butanediol production by Klebsiella pneumoniae.

Klebsiella pneumoniae can use glucose or glycerol as carbon sources to produce 1,3-propanediol or 2,3-butanediol, respectively. In the metabolism of Klebsiella pneumoniae, hydrogenase-3 is responsible for H2 production from formic acid, but it is not directly related to the synthesis pathways for 1,3-propanediol and 2,3-butanediol. In the first part of this research, hycEFG, which encodes subunits of the enzyme hydrogenase-3, was knocked out, so K. pneumoniae ΔhycEFG lost the ability to produce H2 during cultivation using glycerol as a carbon source. As a consequence, the concentration of 1,3-propanediol increased and the substrate (glycerol) conversion ratio reached 0.587 mol/mol. Then, K. pneumoniae ΔldhAΔhycEFG was constructed to erase lactic acid synthesis which led to the further increase of 1,3-propanediol concentration. A substrate (glycerol) conversion ratio of 0.628 mol/mol in batch conditions was achieved, which was higher compared to the wild type strain (0.545 mol/mol). Furthermore, since adhE encodes an alcohol dehydrogenase that catalyzes ethanol production from acetaldehyde, K. pneumoniae ΔldhAΔadhEΔhycEFG was constructed to prevent ethanol production. Contrary to expectations, this did not lead to a further increase, but to a decrease in 1,3-propanediol production. In the second part of this research, glucose was used as the carbon source to produce 2,3-butanediol. Knocking out hycEFG had distinct positive effect on 2,3-butanediol production. Especially in K. pneumoniae ΔldhAΔadhEΔhycEFG, a substrate (glucose) conversion ratio of 0.730 mol/mol was reached, which is higher compared to wild type strain (0.504 mol/mol). This work suggests that the inactivation of hydrogenase-3 may have a global effect on the metabolic regulation of K. pneumoniae, leading to the improvement of the production of two industrially important bulk chemicals, 1,3-propanediol and 2,3-butanediol.

H2 production under stress: [FeFe]­hydrogenases reveal strong stability in high pressure environments.

Hydrogenases are a diverse group of metalloenzymes that catalyze the conversion of H2 into protons and electrons and the reverse reaction. A subgroup is formed by the [FeFe]­hydrogenases, which are the most efficient enzymes of microbes for catalytic H2 conversion. We have determined the stability and activity of two [FeFe]­hydrogenases under high temperature and pressure conditions employing FTIR spectroscopy and the high-pressure stopped-flow methodology in combination with fast UV/Vis detection. Our data show high temperature stability and an increase in activity up to the unfolding temperatures of the enzymes. Remarkably, both enzymes reveal a very high pressure stability of their structure, even up to pressures of several kbars. Their high pressure-stability enables high enzymatic activity up to 2 kbar, which largely exceeds the pressure limit encountered by organisms in the deep sea and sub-seafloor on Earth.