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Molecularly imprinted polymers (MIPs) are artificial receptors equipped with selective recognition sites for target molecules. One of the most promising strategies for protein MIPs relies on the exploitation of short surface‐exposed protein fragments, termed epitopes, as templates to imprint binding sites in a polymer scaffold for a desired protein. However, the lack of high‐resolution structural data of flexible surface‐exposed regions challenges the selection of suitable epitopes. Here, we addressed this drawback by developing a polyscopoletin‐based MIP that recognizes recombinant proteins via imprinting of the widely used Strep‐tag II affinity peptide (Strep‐MIP). Electrochemistry, surface‐sensitive IR spectroscopy, and molecular dynamics simulations were employed to ensure an utmost control of the Strep‐MIP electrosynthesis. The functionality of this novel platform was verified with two Strep‐tagged enzymes: an O2‐tolerant [NiFe]‐hydrogenase, and an alkaline phosphatase. The enzymes preserved their biocatalytic activities after multiple utilization confirming the efficiency of Strep‐MIP as a general biocompatible platform to confine recombinant proteins for exploitation in biotechnology. [ABSTRACT FROM AUTHOR]

The first discovery of methanogens led to the formation of a new domain of life known as Archaea. The Archaea domain exhibits properties vastly different from previously known Bacteria and Eucarya domains. However, for a certain multi-step process, a syntrophic relationship between organisms from all domains is needed. This process is called methanogenesis and is defined as the biological production of methane. Different methanogenic pathways prevail depending on substrate availability and the employed order of methanogenic Archaea. Most methanogens reduce carbon dioxide to methane with hydrogen through a hydrogenotrophic pathway. For hydrogen activation, a group of enzymes called hydrogenases is required. Regardless of the methanogenic pathway, electrons are carried between microorganisms by hydrogen. Naturally occurring processes, such as methanogenesis, can be engineered for industrial use. With the growth and emergence of new industries, the amount of produced industrial waste is an ever-growing environmental problem. For successful wastewater remediation, a syntrophic correlation between various microorganisms is needed. The composition of microorganisms depends on wastewater type, organic loading rates, anaerobic reactor design, pH, and temperature. The last step of anaerobic wastewater treatment is production of biomethane by methanogenesis, which is thought to be a cost-effective means of energy production for this renewable biogas. [ABSTRACT FROM AUTHOR]

[NiFe] hydrogenases catalyze the reversible cleavage of molecular hydrogen into protons and electrons. Here, we have studied the impact of temperature and illumination on an oxygen‐tolerant and thermostable [NiFe] hydrogenase by IR and EPR spectroscopy. Equilibrium mixtures of two catalytic [NiFe] states, Nia‐C and Nia‐SR", were found to drastically change with temperature, indicating a thermal exchange of electrons between the [NiFe] active site and iron‐sulfur clusters of the enzyme. In addition, IR and EPR experiments performed under illumination revealed an unusual photochemical response of the enzyme. Nia‐SR", a fully reduced hydride intermediate of the catalytic cycle, was found to be reversibly photoconverted into another catalytic state, Nia‐L. In contrast to the well‐known photolysis of the more oxidized hydride intermediate Nia‐C, photoconversion of Nia‐SR" into Nia‐L is an active‐site redox reaction that involves light‐driven electron transfer towards the enzyme's iron‐sulfur clusters. Omitting the ground‐state intermediate Nia‐C, this direct interconversion of these two states represents a potential photochemical shortcut of the catalytic cycle that integrates multiple redox sites of the enzyme. In total, our findings reveal the non‐local redistribution of electrons via thermal and photochemical reaction channels and the potential of accelerating or controlling [NiFe] hydrogenases by light. [ABSTRACT FROM AUTHOR]

[NiFe]‐Hydrogenasen katalysieren die reversible Spaltung von molekularem Wasserstoff in Protonen und Elektronen. Hier untersuchen wir mittels IR‐ und EPR‐Spektroskopie den Einfluß von Temperatur und Licht auf eine sauerstofftolerante und thermostabile [NiFe]‐Hydrogenase. Dabei haben wir festgestellt, dass sich das Gleichgewicht zwischen zwei katalytischen [NiFe]‐Zuständen, Nia‐C und Nia‐SR′′, als Funktion der Temperatur drastisch ändert. Dies deutet auf einen thermischen Austausch von Elektronen zwischen dem aktiven [NiFe]‐Zentrum und den Eisen‐Schwefel‐Clustern des Enzyms hin. Darüber hinaus zeigten in situ IR‐ und EPR‐Experimente während eines Belichtungsexperiments eine ungewöhnliche photochemische Reaktion des Enzyms. Das vollständig reduzierte katalytische Hydrid‐Intermediat Nia‐SR′′ wandelt sich lichtinduziert reversibel in den katalytischen Nia‐L Zustand um. Im Gegensatz zur bekannten Photolyse des stärker oxidierten Hydrid‐Intermediates Nia‐C handelt es sich bei der Photokonversion von Nia‐SR′′ zu Nia‐L um eine Redoxreaktion am aktiven Zentrum, die einen lichtinduzierten Elektronentransfer zu den Eisen‐Schwefel‐Clustern des Enzyms beinhaltet. Durch das Auslassen des Nia‐C Intermediates stellt dieser direkte Reaktionsweg eine potenzielle photochemisch induzierte Abkürzung des katalytischen Zyklus dar, in die mehrere Redoxzentren des Enzyms involviert sind. Insgesamt deuten unsere Ergebnisse auf eine Umverteilung von Elektronen über thermische und photochemische Reaktionskanäle in diesem Enzym hin und zeigen die Möglichkeit auf, die Katalyse von [NiFe]‐Hydrogenasen durch Licht zu beschleunigen oder zu kontrollieren. [ABSTRACT FROM AUTHOR]

In this work, a series of DFT and DFT‐D methods is combined with double‐ζ basis sets to benchmark their performance in predicting the structures of five newly synthesized hexacarbonyl diiron complexes with a bridging ligand featuring a μ‐S2C3 motif in a ring‐containing unit functionalized with aromatic groups. Such complexes have been considered as [FeFe] hydrogenase catalytic site models with potential for eco‐friendly energetic applications. According to this assessment, r2SCAN is identified as the density functional recommended for the reliable description of the molecular and crystal structures of the herein studied models. However, the butterfly (μ‐S)2Fe2 core of the models demonstrates a minor deformation of its optimized geometry obtained from both molecular and periodic calculations. The FeFe bond length is slightly underestimated while the FeS bonds tend to be too long. Adding the D3(BJ) correction to r2SCAN does not lead to any improvement in the calculated structures. [ABSTRACT FROM AUTHOR]

Sophisticated enzymatic systems have evolved in nature to efficiently couple distinct biochemical reactions in form of cascades. As such, they serve as reference models to understand the indirect interactions of catalytic centers. Herein, we studied, in solution, the coupling of the reactions from membrane‐bound [NiFe] hydrogenase (MBH) from Cupriavidus necator (reversible H2 splitting into H+ and e−) and the molybdenum‐dependent formate dehydrogenase from Rhodobacter capsulatus (reversible formate to CO2 interconversion). To follow their interplay via the characteristic absorptions from the MBH's active site or the respective substrate and product bands of FDH, we utilized in situ IR spectrocopy and GC(‐MS), in the absence and presence of soluble redox mediators. Coarse‐grained molecular dynamics (CGMD) computations revealed the lack of productive enzyme complexes for direct electron transfer (ET). Thus, the observed minor amounts of H2 or formate were produced from transient interactions between the two enzymes. On the contrary, the significantly increased product formation in the presence of methyl viologen can be related to the putative multiple interaction sites of the redox mediator with FDH identified by CGMD. Our study represents a proof‐of‐concept approach that can be used in the future to develop novel coupled biocatalytic systems by identifying potential ET pathways. [ABSTRACT FROM AUTHOR]

The hox operon in Synechocystis sp. PCC 6803, encoding bidirectional hydrogenase responsible for H2 production, is transcriptionally upregulated under microoxic conditions. Although several regulators for hox transcription have been identified, their dynamics and higher-order DNA structure of hox region in microoxic conditions remain elusive. We focused on key regulators for the hox operon: cyAbrB2, a conserved regulator in cyanobacteria, and SigE, an alternative sigma factor. Chromatin immunoprecipitation sequencing revealed that cyAbrB2 binds to the hox promoter region under aerobic conditions, with its binding being flattened in microoxic conditions. Concurrently, SigE exhibited increased localization to the hox promoter under microoxic conditions. Genome-wide analysis revealed that cyAbrB2 binds broadly to AT-rich genome regions and represses gene expression. Moreover, we demonstrated the physical interactions of the hox promoter region with its distal genomic loci. Both the transition to microoxic conditions and the absence of cyAbrB2 influenced the chromosomal interaction. From these results, we propose that cyAbrB2 is a cyanobacterial nucleoid-associated protein (NAP), modulating chromosomal conformation, which blocks RNA polymerase from the hox promoter in aerobic conditions. We further infer that cyAbrB2, with altered localization pattern upon microoxic conditions, modifies chromosomal conformation in microoxic conditions, which allows SigE-containing RNA polymerase to access the hox promoter. The coordinated actions of this NAP and the alternative sigma factor are crucial for the proper hox expression in microoxic conditions. Our results highlight the impact of cyanobacterial chromosome conformation and NAPs on transcription, which have been insufficiently investigated. [ABSTRACT FROM AUTHOR]

Enzyme engineering is a powerful tool for improving or altering the properties of biocatalysts for industrial, research, and therapeutic applications. Fast and accurate screening of variant libraries is often the bottleneck of enzyme engineering and may be overcome by growth-based screening strategies with simple processes to enable high throughput. The currently available growth-based screening strategies have been widely employed for enzymes but not yet for catalytically potent and oxygen-sensitive metalloenzymes. Here, we present a screening system that couples the activity of an oxygen-sensitive formate dehydrogenase to the growth of Escherichia coli. This system relies on the complementation of the E. coli formate hydrogenlyase (FHL) complex by Mo-dependent formate dehydrogenase H (EcFDH-H). Using an EcFDH-Hdeficient strain, we demonstrate that growth inhibition by acidic glucose fermentation products can be alleviated by FHL complementation. This allows the identification of catalytically active EcFDH-H variants at a readily measurable cell density readout, reduced handling efforts, and a low risk of oxygen contamination. Furthermore, a good correlation between cell density and formate oxidation activity was established using EcFDH-H variants with variable catalytic activities. As proof of concept, the growth assay was employed to screen a library of 1,032 EcFDH-H variants and reduced the library size to 96 clones. During the subsequent colorimetric screening of these clones, the variant A12G exhibiting an 82.4% enhanced formate oxidation rate was identified. Since many metal-dependent formate dehydrogenases and hydrogenases form functional complexes resembling E. coli FHL, the demonstrated growth-based screening strategy may be adapted to components of such electron-transferring complexes. [ABSTRACT FROM AUTHOR]

The much‐needed global shift from fossil fuels to sustainable energy is driving significant attention towards hydrogen (H2) as a promising alternative. Proton reduction, a process central to H2 production, is a key area of research for this transition. Naturally‐occurring [FeFe] and [NiFe]‐hydrogenase enzymes play vital roles in the reversible production and oxidation of H2. These enzymes feature a proton‐relay unit comprising of pendant amine and thiol groups in the secondary coordination sphere at the active site. This unit accelerates the rate of H2 production/oxidation, making it a focal point for scientific exploration. Efforts are concentrated on mimicking the active sites of these enzymes both structurally and functionally. In this pursuit, many synthetic transition metal complexes with proton‐responsive units at the secondary coordination sphere of the active site mimic the enzyme's behavior. These units facilitate intramolecular metal‐hydride (M−H) generation and H2‐elimination via H+/H−s coupling, leveraging the proton from the pendant functional group and the hydride from the M−H intermediate. This review delves into electrocatalysts featuring pendant proton‐responsive units and their roles in the electrochemical hydrogen evolution reaction (eHER). [ABSTRACT FROM AUTHOR]

The enzyme CbA5H, a [FeFe]‐hydrogenase from Clostridium beijerinckii, has previously been shown to survive exposure to oxygen, making it a promising candidate for biotechnological applications. Thus far [NiFe]‐hydrogenases are typically considered for such applications, due to the superior O2‐tolerance and therefore simplified enzyme handling. However, methods for production of [FeFe]‐hydrogenases are generally more successful than for other classes of hydrogenases, therefore in this work we focus on demonstrating scalable CbA5H production, and report results with active enzyme prepared in bioreactors (up to 10 L) with >20‐fold improvement in purified enzyme yield. We then go on to confirm excellent H2/H+‐cycling activity of the air‐purified protein, highlighting that CbA5H can be prepared and isolated without the need for complex and expensive infrastructure. Next, we demonstrate good stability of the air‐purified CbA5H both in solution assays, and as a heterogenous catalyst system when immobilized on a carbon support. Finally, we successfully implement this enzyme within previously demonstrated biotechnologies for flavin and NADH recycling, highlighting its relevance in chemical synthesis, and we demonstrate production of an important API precursor, 3‐quinuclidinol at >0.4 g scale in standard benchtop hydrogenation infrastructure, with >100,000 CbA5H turnovers over 18 operational hours. [ABSTRACT FROM AUTHOR]

The fusion of hydrogenases and photosynthetic reaction centers (RCs) has proven to be a promising strategy for the production of sustainable biofuels. Type I (iron-sulfur-containing) RCs, acting as photosensitizers, are capable of promoting electrons to a redox state that can be exploited by hydrogenases for the reduction of protons to dihydrogen (H2). While both [FeFe] and [NiFe] hydrogenases have been used successfully, they tend to be limited due to either O2 sensitivity, binding specificity, or H2 production rates. In this study, we fuse a peripheral (stromal) subunit of Photosystem I (PS I), PsaE, to an O2-tolerant [FeFe] hydrogenase from Clostridium beijerinckii using a flexible [GGS]4 linker group (CbHydA1-PsaE). We demonstrate that the CbHydA1 chimera can be synthetically activated in vitro to show bidirectional activity and that it can be quantitatively bound to a PS I variant lacking the PsaE subunit. When illuminated in an anaerobic environment, the nanoconstruct generates H2 at a rate of 84.9 ± 3.1 µmol H2 mgchl -1 h-1. Further, when prepared and illuminated in the presence of O2, the nanoconstruct retains the ability to generate H2, though at a diminished rate of 2.2 ± 0.5 µmol H2 mgchl -1 h-1. This demonstrates not only that PsaE is a promising scaffold for PS I-based nanoconstructs, but the use of an O2-tolerant [FeFe] hydrogenase opens the possibility for an in vivo H2 generating system that can function in the presence of O2. [ABSTRACT FROM AUTHOR]

[FeFe] hydrogenases demonstrate remarkable catalytic efficiency in hydrogen evolution and oxidation processes. However, susceptibility of these enzymes to oxygen‐induced degradation impedes their practical deployment in hydrogen‐production devices and fuel cells. Recent investigations into the oxygen‐stable (Hinact) state of the H‐cluster revealed its inherent capacity to resist oxygen degradation. Herein, we present findings on Cl‐ and SH‐bound [2Fe‐2S] complexes, bearing relevance to the oxygen‐stable state within a biological context. A characteristic attribute of these complexes is the terminal Cl−/SH− ligation to the iron center bearing the CO bridge. Structural analysis of the t‐Cl demonstrates a striking resemblance to the Hinact state of DdHydAB and CbA5H. The t‐Cl/t‐SH exhibit reversible oxidation, with both redox species, electronically, being the first biomimetic analogs to the Htrans and Hinact states. These complexes exhibit notable resistance against oxygen‐induced decomposition, supporting the potential oxygen‐resistant nature of the Htrans and Hinact states. The swift reductive release of the Cl‐/SH‐group demonstrates its labile and kinetically controlled binding. The findings garnered from these investigations offer valuable insights into properties of the enzymatic O2‐stable state, and key factors governing deactivation and reactivation conversion. This work contributes to the advancement of bio‐inspired molecular catalysts and the integration of enzymes and artificial catalysts into H2‐evolution devices and fuel‐cell applications. [ABSTRACT FROM AUTHOR]

Plastics pose a considerable challenge to aquatic ecosystems because of their increasing global usage and non-biodegradable properties. Coastal plastic debris can persist in ecosystems; however, its effects on resident organisms remain unclear. A metagenomic analysis of the isopoda Ligia, collected from clean (Nae-do, ND) and plastic-contaminated sites (Maemul-do, MD) in South Korea, was conducted to clarify the effects of microplastic contamination on the gut microbiota. Ligia gut microbiota's total operational taxonomic units were higher in ND than in MD. Alpha diversity did not differ significantly between the two Ligia gut microbial communities collected from ND and MD, although richness (Observed species) was lower in MD than in ND. Proteobacteria (67.47%, ND; 57.30%, MD) and Bacteroidetes (13.63%, ND; 20.76%, MD) were the most abundant phyla found at both sites. Significant different genera in Ligia from EPS-polluted sites were observed. Functional gene analysis revealed that 19 plastic degradation-related genes, including those encoding hydrogenase, esterase, and carboxylesterase, were present in the gut microbes of Ligia from MD, indicating the potential role of the Ligia gut microbiota in plastic degradation. This study provides the first comparative field evidence of the gut microbiota dynamics of plastic detritus consumers in marine ecosystems. [ABSTRACT FROM AUTHOR]

This study unravels some of the complex microbial interactions established in the fermentation of sugarcane vinasse. The microbial communities of four thermophilic (55 °C) fixed-bed vinasse-fed reactors were analyzed by 16S rRNA gene amplicon sequencing and the marker gene data were aligned with reference genomes to predict metabolic functions of the microorganisms. The production (by Lactobacillus) and utilization (mainly by Thermoanaerobacterium and Caproiciproducens) of lactate explain the primary pathways associated with substrate fermentation towards biohydrogen, whilst only a single genus (Desulfotomaculum) and very low abundances of the enzymes involved in sulfate reduction were found. Although the abundant microbes in the inocula failed to grow during the continuous operation in all reactors, these fermentative consortia behave like microbial pantries containing bacteria that will grow according to the operating conditions, shaping lactate-accumulating (pH < 5.0) or lactate-consuming (pH > 5.0) and eventually sulfate-reducing (pH > 6.0) microbial communities in vinasse thermophilic fermentation. [Display omitted] • Metabolic functions of bacteria involved in vinasse fermentation were predicted. • Microbial composition depends on operating conditions, and not on inoculum source. • Lactobacillus is omnipresent in vinasse conversion as the main fermenting genus. • The ferredoxin hydrogenase gene was virtually absent in all four reactors. • Thermoanaerobacterium drives most of bioH 2 production utilizing lactate. [ABSTRACT FROM AUTHOR]

Fe3(CO)12 reacted with azadiethylthiols afforded [Fe2(μ‐S(CH2)2NR(CH2)2S)(CO)6]2 (R = nPr, 1A; iPr, 2A). Structure analysis revealed that 1A features the anti‐(a,e,a,e) configuration, while its iso‐propyl 2A adopts the syn‐(a,e,a,e) arrangement. NMR measurements at room temperature confirmed this structure, showing distinct signals for 2 NCH2 and 2 SCH2 methylene groups. Acid treatment of 1–2 yielded N‐protonated species (1A–H, 2A–H), maintaining the anti‐(a,e,a,e) configuration. Isolation of 1B–H revealed both the syn‐(a,e,e,a) and anti‐(a,e,e,a) configuration by X‐ray single‐crystallography. Protonation of 1A–H induced downfield shifts in methylene proton signals, with NH hydrogen recorded at 6.96 ppm. Furthermore, 1B–H displays similar NMR behavior, indicating the coexistence of two isomeric molecules of 1B–H, syn‐(a,e,e,a) and anti‐(a,e,e,a), which is consistent with the crystallographic results. Moreover, ion mobility‐mass spectrometry (IM‐MS) is harnessed for the swift identification and differentiation of these isomeric complexes in solution. By leveraging accurate mass measurements and MS/MS analyses, the molecular formula and constituents are validated. The sample mobility enables straightforward characterization of 1A, 1B, and 2A in relation to their stereo‐ and regio‐configurations, obviating the need for extensive time or sample quantities. Integration of findings from X‐ray crystallography, NMR, and IM‐MS furnishes precise and comprehensive structural insights into the coordination complexes. [ABSTRACT FROM AUTHOR]

The design of iron clusters featuring a bimetallic core and several protonation sites in the second coordination sphere of the metal centers is important for modeling the activity of polymetallic active sites such as the H-cluster of [FeFe]-hydrogenases. For this purpose, the syntheses of complexes [Fe3(CO)5(κ2-PPh2NR2)(μ-pdt)2] (R = Ph (1), Bn (2)) and [Fe3(CO)5(κ2-PPh2NR2)(μ-adtBn)(μ-pdt)] (R = Ph (3), Bn (4)) were carried out by reacting hexacarbonyl precursors [Fe2(CO)6(µ-xdt)] (xdt = pdt (propanedithiolate), adtBn (azadithiolate) with mononuclear complexes [Fe(κ2-pdt)(CO)2(κ2-PPh2NR2)] (PPh2NR2 = (PPhCH2NRCH2)2, R = Ph, Bn) in order to introduce amine functions, through well-known PPh2NR2 diphosphine, into the vicinity of the triiron core. The investigation of the reactivity of these triiron species towards the proton (in the presence of CF3SO3H) and the influence of the pendant amines on the redox properties of these complexes were explored using spectroscopic and electrochemical methods. The protonation sites in such triiron clusters and their relationships were identified. The orientation of the first and second protonation processes depends on the arrangement of the second coordination sphere. The similarities and differences, due to the extended metal nuclearity, with their dinuclear counterparts [Fe2(CO)4(κ2-PPh2NR2)(μ-pdt)], were highlighted. [ABSTRACT FROM AUTHOR]

Efficient hydrogen production in biocathode-driven microbial electrolysis cells (MECs) can be obtained by electroautotrophic microbes with the ability to regenerate biocatalytic activity. Inoculation and enrichment of a pure defined culture in the cathode are critical for high performance in hydrogen production. Zero-gap MECs with anion exchange membranes (AEMs) enhance hydrogen production due to low internal resistance and better pH balance. Engineered microbes and optimized microbe–electrode interactions can further increase MEC performance and accelerate its commercialization. Electroautotrophic microbes at biocathodes in microbial electrolysis cells (MECs) can catalyze the hydrogen evolution reaction with low energy demand, facilitating long-term stable performance through specific and renewable biocatalysts. However, MECs have not yet reached commercialization due to a lack of understanding of the optimal microbial strains and reactor configurations for achieving high performance. Here, we critically analyze the criteria for the inocula selection, with a focus on the effect of hydrogenase activity and microbe–electrode interactions. We also evaluate the impact of the reactor design and key parameters, such as membrane type, composition, and electrode surface area on internal resistance, mass transport, and pH imbalances within MECs. This analysis paves the way for advancements that could propel biocathode-assisted MECs toward scalable hydrogen gas production. [ABSTRACT FROM AUTHOR]

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. Many acetogens, including Acetobacterium woodii, cannot grow on carbon monoxide and thus cannot be used as platform to detoxify industrial waste gas streams. Here, the authors solve the problem by knocking out the two [FeFe] hydrogenases HydA2 and HydBA in A. woodii. [ABSTRACT FROM AUTHOR]

The reactivity of complexes [Fe2(μ‐S2)(CO)4L2] (L=CO (1), PPh3 (2)), with lithium alkynylide reagents generated in situ, was investigated. The behavior of the S2‐bridge in these compounds depends on the substitution at the diiron core. The reaction with the hexacarbonyl derivative 1 leads to the formation of the 1,2‐dithiolene bridged complex [Fe2(μ‐SCH=C(R)S)(CO)6] (3R) while the molecule [Fe2(μ‐SH)(μ‐SC≡CR)(CO)4(PPh3)2] (5R), with an open butterfly structure, is isolated when reacting the disubstituted derivative 2. The disubstituted dithiolene complex [Fe2(μ‐SCH=C(Ph)S)(CO)4(PPh3)2] (4Ph) can only be obtained by substitution of carbonyls with PPh3 in 3R. In the presence of piperidine, 5R isomerizes into the 1,1'‐dithiolene bridged derivative 6Ph. The novel compounds 4–6 were synthesized and characterized by IR and NMR spectroscopies. X‐ray crystallographic studies of the dithiolene complexes 3Ph–4Ph allowed their structural analysis. [ABSTRACT FROM AUTHOR]

The biohydrogen production in Dark fermentation (DF) could be enhanced by ZnFe 2 O 4 nanomaterials (NPs). The optimum conditions were obtained by a response surface method. Eventually, a biohydrogen production of 2645 mL/L was obtained after 48 h of batch culture, which is 116% higher than control without ZnFe 2 O 4 NPs. The mechanism was investigated by a multi-scale method. In bioreactor scale, ZnFe 2 O 4 NPs could enhance the glucose metabolic ability of C. pasteurianum to promote cell growth and biohydrogen production. The metabolic pathways of biohydrogen production were shifted from the butyric acid-type to the ethanol-type. In cellular scale, ZnFe 2 O 4 NPs could reduce bacterial flagella assembly. In macromolecule scale, the activities of hydrogenase and ethanol dehydrogenase were increased. In genetic scale, the differentially expressed genes mainly involved in biohydrogen production, like those encoded Fe-only hydrogenase, ethanol dehydrogenase, and nitrogenase were up-regulated. The results could provide theoretical and technical supports for the development of commercial biohydrogen process. • ZnFe2O4 NPs can effectively promote biohydrogen production. • Optimal enhancement conditions determined through response surface optimization. • ZnFe2O4 NPs enhance glucose utilization by C. pasteurianum. • ZnFe2O4 NPs boost hydrogenase & ethanol dehydrogenase in C. pasteurianum. • Mechanism of NPs promoting biohydrogen production revealed by multi-scale analysis. [ABSTRACT FROM AUTHOR]

Serpentinization, a geochemical process found on modern and ancient Earth, provides an ultra-reducing environment that can support microbial methanogenesis and acetogenesis. Several groups of archaea, such as the order Methanocellales, are characterized by their ability to produce methane. Here, we generate metagenomic sequences from serpentinized springs in The Cedars, California, and construct a circularized metagenome-assembled genome of a Methanocellales archaeon, termed Met12, that lacks essential methanogenesis genes. The genome includes genes for an acetyl-CoA pathway, but lacks genes encoding methanogenesis enzymes such as methyl-coenzyme M reductase, heterodisulfide reductases and hydrogenases. In situ transcriptomic analyses reveal high expression of a multi-heme c-type cytochrome, and heterologous expression of this protein in a model bacterium demonstrates that it is capable of accepting electrons. Our results suggest that Met12, within the order Methanocellales, is not a methanogen but a CO2-reducing, electron-fueled acetogen without electron bifurcation. Several groups of archaea, such as the order Methanocellales, are characterised by their ability to produce methane. Here, Suzuki et al. identify a Methanocellales archaeon that lacks essential methanogenesis genes and seems to be instead a CO2-reducing, electron-fueled acetogen. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Background: Ferredoxin 1 (Fd1) is the main electron donor to hydrogenase (HydA) for generating molecular hydrogen (H2) in green microalgae. In order to obtain an increased H2 production, the Fd1 of Chlamydomonas reinhardtii (CrFd1, encoded by crfd1) was therefore overexpressed in Chlorella sp. DT (DT) in this study. Results: The coding region of crfd1 was constructed into the p121-crfd1 plasmid, which was also constructed with a resistance gene to the antibiotic geneticin (G418) as a selection marker. The p121-crfd1 plasmid was transformed into DT cells by electroporation. Observation of the crfd1 gene fragment in the genomic DNA of DT-crfd1 mutants by PCR indicated that the transgene was successfully transformed. Using western blotting, the overexpressed CrFd1 protein, with a molecular weight of about 14 kDa, was found in DT-crfd1 mutants of DT-crfd1-4, DT-crfd1-22, and DT-crfd1-23. Using an in vitro assay, the H2 production of DT-crfd1-4, DT-crfd1-22, and DT-crfd1-23 mutants was about 4.4-, 5.0-, and 3.8-fold higher, respectively, than the DT wild type (DT-WT). Using an in vivo assay, the H2 production of DT-crfd1-4, DT-crfd1-22, and DT-crfd1-23 mutants was about 1.3-, 1.4-, and 1.2-fold higher, respectively, than the DT-WT. Conclusions: The results suggested that heterologous overexpression of CrFd1 could enhance H2 production in DT-crfd1 mutants even though in vitro H2 production of DT-crfd1-22 mutant was up to 5-fold higher than the DT-WT. [ABSTRACT FROM AUTHOR]

Although great progress has been made in improving hydrogen production, highly efficient catalysts, which are able to produce hydrogen in a fast and steady way at ambient temperature and pressure, are still in large demand. Here, we report a [NiCo]-based hydrogenase mimic, NiCo2O4 nanozyme, that can catalyze robust hydrogen evolution spontaneously in water without external energy input at room temperature. This hydrogenase nanozyme facilitates water splitting reaction by forming a three-center Ni–OH–Co bond analogous to the [NiFe]-hydrogenase reaction by using aluminum as electron donor, and realizes hydrogen evolution with a high production rate of 915 L·h−1 per gram of nanozymes, which is hundreds of times higher than most of the natural hydrogenase or hydrogenase mimics. Furthermore, the NiCo2O4 nanozyme can robustly disrupt the adhesive oxidized layer of aluminum and enable the full consumption of electrons from aluminum. In contrast to the often-expensive synthetic catalysts that rely on rare elements and consume high energy, we envision that this NiCo2O4 nanozyme can potentially provide an upgrade for current hydrogen evolution, accelerate the development of scale-up hydrogen production, and generate a clean energy future. [ABSTRACT FROM AUTHOR]

Hydrogen gas is attractive as a clean fuel source if it can be produced efficiently without relying on fossil fuels. Biohydrogen production using photosynthetic bacteria may enable environmentally friendly hydrogen production but is currently limited by factors such as low oxygen tolerance. In this study, we isolate a new strain of bacteria that can produce hydrogen under aerial-phase conditions compared with those under liquid-phase conditions in a nitrogen gas or an argon gas atmosphere. Bacterial strains were cultured from scrapings taken from a steel signboard. Investigation of the hydrogen production of the strains under aerial- and liquid-phase conditions and subsequent DNA sequencing led to identification of the bacterium Cereibacter sp. KGU-NF001. Aerial-phase conditions were achieved by filter membranes with the bacterial strains and placing the membranes on medium-soaked cotton wool. The gas atmosphere affected the behavior of the isolated bacterial strains under both aerial- and liquid-phase conditions. Cereibacter sp. KGU-NF001 showed promising oxygen tolerance and was able to maintain hydrogen production of 1.33 mL/mg/d even when the atmosphere contained 12% oxygen. Our findings illustrate that biohydrogen production may be achieved by photosynthetic bacteria under oxygen-containing aerial-phase conditions, indicating a possible pathway to help lower our reliance on fossil fuels. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

The first discovery of methanogens led to the formation of a new domain of life known as Archaea. The Archaea domain exhibits properties vastly different from previously known Bacteria and Eucarya domains. However, for a certain multi-step process, a syntrophic relationship between organisms from all domains is needed. This process is called methanogenesis and is defined as the biological production of methane. Different methanogenic pathways prevail depending on substrate availability and the employed order of methanogenic Archaea. Most methanogens reduce carbon dioxide to methane with hydrogen through a hydrogenotrophic pathway. For hydrogen activation, a group of enzymes called hydrogenases is required. Regardless of the methanogenic pathway, electrons are carried between microorganisms by hydrogen. Naturally occurring processes, such as methanogenesis, can be engineered for industrial use. With the growth and emergence of new industries, the amount of produced industrial waste is an ever-growing environmental problem. For successful wastewater remediation, a syntrophic correlation between various microorganisms is needed. The composition of microorganisms depends on wastewater type, organic loading rates, anaerobic reactor design, pH, and temperature. The last step of anaerobic wastewater treatment is production of biomethane by methanogenesis, which is thought to be a cost-effective means of energy production for this renewable biogas.

Gases like H2, N2, CO2, and CO are increasingly recognized as critical feedstock in green energy conversion and as sources of nitrogen and carbon for the agricultural and chemical sectors. However, the industrial transformation of N2, CO2, and CO and the production of H2 require significant energy input, which renders processes like steam reforming and the Haber-Bosch reaction economically and environmentally unviable. Nature, on the other hand, performs similar tasks efficiently at ambient temperature and pressure, exploiting gas-processing metalloenzymes (GPMs) that bind low-valent metal cofactors based on iron, nickel, molybdenum, tungsten, and sulfur. Such systems are studied to understand the biocatalytic principles of gas conversion including N2 fixation by nitrogenase and H2 production by hydrogenase as well as CO2 and CO conversion by formate dehydrogenase, carbon monoxide dehydrogenase, and nitrogenase. In this review, we emphasize the importance of the cofactor/protein interface, discussing how second and outer coordination sphere effects determine, modulate, and optimize the catalytic activity of GPMs. These may comprise ionic interactions in the second coordination sphere that shape the electron density distribution across the cofactor, hydrogen bonding changes, and allosteric effects. In the outer coordination sphere, proton transfer and electron transfer are discussed, alongside the role of hydrophobic substrate channels and protein structural changes. Combining the information gained from structural biology, enzyme kinetics, and various spectroscopic techniques, we aim toward a comprehensive understanding of catalysis beyond the first coordination sphere.

The prevalence of bacterial persisters is related to their phenotypic diversity and is responsible for the relapse of chronic infections. Tolerance to antibiotic therapy is the hallmark of bacterial persistence. In this study, we have screened a transposon library of Mycobacterium smegmatis mc2 155 strain using antibiotic tolerance, survival in mouse macrophages, and biofilm-forming ability of the mutants. Out of 10 thousand clones screened, we selected ten mutants defective in all the three phenotypes. Six mutants showed significantly lower persister abundance under different stress conditions. Insertions in three genes belonging to the pathways of oxidative phosphorylation msmeg_3233 (cydA), biotin metabolism msmeg_3194 (bioB), and oxidative metabolism msmeg_0719, a flavoprotein monooxygenase, significantly reduced the number of live cells, suggesting their role in pathways promoting long-term survival. Another group that displayed a moderate reduction in CFU included a glycosyltransferase, msmeg_0392, a hydrogenase subunit, msmeg_2263 (hybC), and a DNA binding protein, msmeg_2211. The study has revealed potential candidates likely to facilitate the long-term survival of M. smegmatis. The findings offer new targets to develop antibiotics against persisters. Further, investigating the corresponding genes in M. tuberculosis may provide valuable leads in improving the treatment of chronic and persistent tuberculosis infections. [ABSTRACT FROM AUTHOR]

Growing concerns over greenhouse gas emissions and energy insecurity caused by the depletion of conventional fuels have led to a search for sustainable fuel alternatives. As an alternative energy carrier, hydrogen (H 2) is particularly attractive as only water is released during combustion. The process of H 2 production from genetically engineered phototrophic microorganisms through biophotolysis leads the way to solve energy shortages. Genetically engineered cyanobacteria species are potential candidates due to their superior properties for reducing greenhouse gases and using solar energy as an energy source. The review discusses the mechanisms and enzymes involved in H 2 production by cyanobacteria and applications of genetic engineering. A critical analysis of the fundamental issues attributed to the technical advancement of photobiological cyanobacteria-based H 2 production is provided, as well as the perspectives for future research to reduce carbon dioxide emissions through the creation of waste-free technology. • Genetic engineering findings of cyanobacterial nitrogenase enzyme are described. • Recent genetic engineering methods to obtain hydrogenase mutants are discussed. • Engineering photosynthetic H 2 production is shown as an efficient method. • Development of a non-waste H 2 production technology is explained. [ABSTRACT FROM AUTHOR]

[FeFe]‐hydrogenases are capable of reducing protons at a high rate. However, molecular oxygen (O2) induces the degradation of their catalytic cofactor, the H‐cluster, which consists of a cubane [4Fe4S] subcluster (4FeH) and a unique diiron moiety (2FeH). Previous attempts to prevent O2‐induced damage have focused on enhancing the protein's sieving effect for O2 by blocking the hydrophobic gas channels that connect the protein surface and the 2FeH. In this study, we aimed to block an O2 diffusion pathway and shield 4FeH instead. Molecular dynamics (MD) simulations identified a novel water channel (WH) surrounding the H‐cluster. As this hydrophilic path may be accessible for O2 molecules we applied site‐directed mutagenesis targeting amino acids along WH in proximity to 4FeH to block O2 diffusion. Protein film electrochemistry experiments demonstrate increased O2 stabilities for variants G302S and S357T, and MD simulations based on high‐resolution crystal structures confirmed an enhanced local sieving effect for O2 in the environment of the 4FeH in both cases. The results strongly suggest that, in wild type proteins, O2 diffuses from the 4FeH to the 2FeH. These results reveal new strategies for improving the O2 stability of [FeFe]‐hydrogenases by focusing on the O2 diffusion network near the active site. [ABSTRACT FROM AUTHOR]

Green algae, such as Chlamydomonas reinhardtii , can produce H 2 efficiently using their hydrogenases, but these enzymes are O 2 -sensitive and compete with CO 2 -fixation for electrons. To overcome these limitations, we previously developed an anaerobic, carbon-limited protocol that keeps the Calvin-Benson cycle inactive, and the evolved O 2 is scavenged by an absorbent. The PROTON-GRADIENT REGULATION5 (PGR5)-deficient mutant performs better than the wild type in this system, as it produces at least 2.5-fold more H 2 and maintains its photosynthetic apparatus and hydrogenase activity even at the intensity of sunlight. However, as the pgr5 mutant is known to be sensitive to fluctuating light conditions, it is of paramount interest to determine how it reacts to changes in light intensity during H 2 production. Therefore, we developed an automated system to monitor H 2 production in thin layer cultures under simulated daily light conditions. We found that the pgr5 mutant outperformed the wild type strain by 100 % when light intensity was varied stepwise between 0 and 1000 μmol photons m−2s−1 during the day. Photosynthetic subunits, including PsbA, PSBO, CP47, PetB and PsaA, were fully preserved in the pgr5 mutant, and approximately 29 % of its original hydrogenase activity was sustained after 85 h of H 2 production in simulated daily light conditions. Hence, the pgr5 mutant is a promising candidate for H 2 production under the adverse light conditions that algae may encounter in bioindustry settings. [Display omitted] • An automated thin-cell layer H 2 production system for Chlamydomonas was developed. • The pgr5 mutant outperformed the wild type in simulated daily light conditions. • The photosynthetic and hydrogenase activities of the pgr5 were better preserved. • The pgr5 mutant shows promise for bioindustry settings with adverse conditions. [ABSTRACT FROM AUTHOR]

The energy of cyanobacterial hydrogen (H 2) produced via bio-photolysis is being investigated as a potential solution to early-century environmental challenges. The main limiting factors of cyanobacterial H 2 photoproduction are the availability of electrons for [NiFe]-hydrogenase (H 2 ase) and the suppression of bidirectional H 2 ase activity induced by O 2 acquired from water molecules splitting in photosystem II. The current study investigated how photosynthetic inhibitors (PIs) affected H 2 production in non-N 2 -fixing cyanobacteria. Study findings revealed a rather high H 2 yield in Synechocystis sp. PSU 1262, as well as a beneficial (14.2-fold) influence of 500 μmol KCN on the H 2 production by the aforesaid strain. A 12/6-h light/dark cycle increased H 2 production by 80.3% in cells supplemented with 500 μmol KCN. Under the optimised conditions, the photobiological H 2 production of Synechocystis sp. PSU 1262 increased from 49.6 to 1552 nmol H 2 mg−1 Chl a h−1. PIs suppressed chlorophyll a concentration under illumination, lowering the O 2 levels, which enhanced bidirectional H 2 ase activity in Synechocystis sp. PSU 1262 cells. Applying varied light modes, preceded by the incorporation of PIs at optimal concentrations in H 2 production by research cyanobacterial strains, improved the H 2 yield and contributed significantly to the research originality. [Display omitted] • Six species of cyanobacteria from different ecosystems were isolated and identified. • The H 2 -producing activity of the strains was studied in the dark and in the light. • The effect of four inhibitors of photosynthesis on H 2 production was studied. • The effect of light and dark modes was evaluated on H 2 production by cyanobacteria. [ABSTRACT FROM AUTHOR]

Abio/bio hybrids, which incorporate biocatalysts that promote efficient and selective material conversions under mild conditions into existing catalytic reactions, have attracted considerable attention for developing new catalytic systems. This study constructed a H2‐forming biocathode based on a carbon material combined with whole‐cell biocatalysis of genetically‐engineered‐hydrogenase‐overproducing Escherichia coli for the photoelectrochemical water splitting for clean H2 production. Low‐cost and abundant carbon materials are generally not suitable for H2‐forming cathode due to their high overpotential for proton reduction; however, the combination of the reduction of an organic electron mediator on the carbon electrode and the H2 formation with the reduced mediator by the redox enzyme hydrogenase provides a H2‐forming cathodic reaction comparable to that of the noble metal electrode. The present study demonstrates that the recombinant E. coli whole cell can be employed as a part of the H2‐forming biocathode system, and the biocathode system wired with TiO2 photoanode can be a photoelectrochemical water‐splitting system without external voltage assistance under natural pH. The findings of this study expand the feasibility of applications of whole‐cell biocatalysis and contribute to obtaining solar‐to‐chemical conversions by abio/bio hybrid systems, especially for low‐cost, noble‐metal‐free, and clean H2 production. [ABSTRACT FROM AUTHOR]

Photosynthetic fermentation for hydrogen production can convert some organic compounds into hydrogen, thus realizing the dual benefits of waste utilization and energy production. However, the low H 2 production rate is currently a major constraint restricting its further development. ATP synthesis during photophosphorylation could be a key factor on hydrogen production. In this work, the vector pRK415 and three endogenous promoters P pufQ , P pucB and P phaC were used to optimize the expression intensity of the fbcFBC gene, which encodes cytochrome bc 1 , a key protein in the photosynthetic electron transport chain. The effect of fbcFBC expression level on the hydrogen production performance of R. sphaeroides HY01 and its hydrogenase gene deletion mutant strain WH04 was studied. The hydrogen yields of pRKfbc/HY01 and pRKfbc/WH04 increased by 15.5% and 5.1% compared with the control. Substitutions of the fbcFBC promoter with promoters P pufQ , P pucB and P phaC , the transcription level of fbcFBC and ATP content were significantly improved. In particular, the transcription level of fbcFBC and ATP content in WHpuc were respectively increased by 734% and 57.0% compared with that in WH04. The results demonstrate that Cyt bc 1 is a limiting factor affecting ATP synthesis in the process of photo-fermentative hydrogen production. The mutants overexpressing fbcFBC are more efficient in hydrogen production than the naturally isolated strains under the medium light intensity of 5000 lux. [Display omitted] • fbcFBC gene can be overexpressed by the overexpression vector pRK415. • fbcFBC gene was overexpressed by inserting promoters P pufQ , P pucB and P phaC. • Overexpressing fbcFBC raised photosynthetic electron transport and ATP synthesis. • Overexpression of fbcFBC promoted hydrogen production in photosynthetic bacteria. • Overexpression of fbcFBC had the optimal value for hydrogen production promotion. [ABSTRACT FROM AUTHOR]

Green hydrogen, produced during microalgal photosynthesis, is regarded as one of the most promising sustainable energy sources. It utilizes sunlight and water, which are essentially unlimited, and its combustion results in only water as a waste product. In microalgal hydrogen energy production systems, the sensitivity of hydrogenase to O2 poses a significant challenge, limiting sustained photosynthetic H2 production in microalgae. Additionally, efficient photosynthetic H2 production in anaerobic microalgal cells is hindered by impaired electron source (photosystem II) and electron loss through the Calvin‐Benson cycle, cyclic electron transfer around photosystem I, and O2 photoreduction, which are identified as the other key challenges. Over the past eight decades, considerable progress has been made in addressing these challenges and regulating electron transfer to achieve sustainable and efficient photosynthetic H2 production in microalgae. In this review, we discuss a range of regulatory methods for achieving sustainable and efficient photosynthetic H2 production in microalgae. Emphasizing the significant progress made over the past eight decades, we also address current challenges and propose potential future solutions. [ABSTRACT FROM AUTHOR]

Environmental damage is caused by the waste produced by slaughterhouses. The leftovers from slaughterhouses make a solid base for the production of clean energy. In the present study, biohydrogen was produced using the cattle rumen fluid (RF) collected from the slaughterhouse. The RF serves as the only substrate, and 21.13 ± 0.176 µmol H2 ml-1rumen fluid (RF) was found to be produced during biohydrogen synthesis. After 72 h, the rumen fluid was collected and a pure culture of bacteria that produce biohydrogen was isolated. After characterization, the organism was identified as Staphylococcus sciuri MK898925.1. The Sterilised RF was used as the sole substrate for biohydrogen production by Staphylococcus sciuri MK898925.1, and biohydrogen production was found to be 142.4 ± 2.37 µmol H2 ml-1 RF. Staphylococcus sciuri MK898925.1 was screened for the presence of the gene hydrogenases and it was discovered that the gene was present in the bacteria. The bacteria Staphylococcus sciuri MK898925.1 isolated from the rumen fluid of cattle can be used to produce biohydrogen. A large amount of slaughterhouse waste rumen fluid can be recycled and converted into energy at a very low cost [ABSTRACT FROM AUTHOR]

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HydSL hydrogenase from Thiocapsa bogorovii BBS (formerly Thiocapsa roseopersicina BBS) is NiFe hydrogenase belonging to 1e subgroup. Recently it was shown that C-terminal domain of HydS subunit is important for hydrogenase docking to transmembrane protein Isp1 for electron transfer (https://doi.org/10.1016/j.bbabio.2021.148492.) It is suggested that this C-domain is a hydrophobic transmembrane alpha helix located near distal FeS claster. Using this peculiarity, a simple method for oriented HydSL hydrogenase immobilization on a hydrophobic carbon electrode is developed. The method is based on the incubation of the hydrogenase with carbon paper Freudenberg H24C3 or Vulcan nanoparticles in the solution of MOPS buffer and isopropanol alcohol with ratio 1/2 (v/v) with slow (15 h) evaporation of the solution in humidified atmosphere. The developed electrodes were applied to the fuel cell with Pt catalyst as the oxygen electrode and produced 2 mW cm−2 power with current at this power more than 6 mA cm−2. Furthermore, the fuel cell was stable after 49 days of incubation under the air atmosphere with periodic measurements with some power and current increase. [Display omitted] • HydSL hydrogenase from Thiocapsa bogorovii has hydrophobic C-terminus near the distal FeS claster. • A simple method for oriented immobilization of HydSL hydrogenase of on hydrophobic carbon electrodes was developed. • FC with hydrogenase electrode and Pt oxygen electrode showed power ∼2 mW cm−2 at currents above 6 mA cm−2. • The FC with hydrogen electrode was stable for 49 days. [ABSTRACT FROM AUTHOR]

Several species of microalgae can convert light energy into molecular hydrogen (H2) by employing enzymes of early phylogenetic origin, [FeFe]-hydrogenases, coupled to the photosynthetic electron transport chain. Bacterial [FeFe]-hydrogenases consist of a conserved domain that harbors the active site cofactor, the H-domain, and an additional domain that binds electron-conducting FeS clusters, the F-domain. In contrast, most algal hydrogenases characterized so far have a structurally reduced, so-termed M1-type architecture, which consists only of the H-domain that interacts directly with photosynthetic ferredoxin PetF as an electron donor. To date, only a few algal species are known to contain bacterial-type [FeFe]-hydrogenases, and no M1-type enzymes have been identified in these species. Here, we show that the chlorophycean alga Uronema belkae possesses both bacterial-type and algal-type [FeFe]-hydrogenases. Both hydrogenase genes are transcribed, and the cells produce H2 under hypoxic conditions. The biochemical analyses show that the two enzymes show features typical for each of the two [FeFe]-hydrogenase types. Most notable in the physiological context is that the bacterial-type hydrogenase does not interact with PetF proteins, suggesting that the two enzymes are integrated differently into the alga's metabolism. [ABSTRACT FROM AUTHOR]

Chemists have long sought to regulate the reactivity of H2, to yield hydride ions, hydrogen atoms, or electrons on demand. One source of inspiration for achieving this control is [NiFe]hydrogenase ([NiFe]H2ase), which reacts with H2 to form various hydrogen active species such as NiIII hydride species, NiII hydride species, and NiI low‐valent species. Chemists have attempted to synthesize these hydrogen active species not only as models for the active species of [NiFe]H2ase, but also as electron transfer catalysts. However, the synthesis of NiI complex directly from H2 has not been reported. This paper reports the first example of a single‐step synthesis of a NiI complex, via reaction of a NiII complex with H2, stable for over 3 months at room temperature and we further demonstrate a reductive coupling of acridinium ions as part of a reaction cycle. [ABSTRACT FROM AUTHOR]

Chemists have long sought to regulate the reactivity of H2, to yield hydride ions, hydrogen atoms, or electrons on demand. One source of inspiration for achieving this control is [NiFe]hydrogenase ([NiFe]H2ase), which reacts with H2 to form various hydrogen active species such as NiIII hydride species, NiII hydride species, and NiI low‐valent species. Chemists have attempted to synthesize these hydrogen active species not only as models for the active species of [NiFe]H2ase, but also as electron transfer catalysts. However, the synthesis of NiI complex directly from H2 has not been reported. This paper reports the first example of a single‐step synthesis of a NiI complex, via reaction of a NiII complex with H2, stable for over 3 months at room temperature and we further demonstrate a reductive coupling of acridinium ions as part of a reaction cycle. [ABSTRACT FROM AUTHOR]

The Wood–Ljungdahl pathway (WLP) is a key metabolic component of acetogenic bacteria where it acts as an electron sink. In Archaea, despite traditionally being linked to methanogenesis, the pathway has been found in several Thermoproteota and Asgardarchaeota lineages. In Bathyarchaeia and Lokiarchaeia, its presence has been linked to a homoacetogenic metabolism. Genomic evidence from marine hydrothermal genomes suggests that lineages of Korarchaeia could also encode the WLP. In this study, we reconstructed 50 Korarchaeia genomes from marine hydrothermal vents along the Arctic Mid‐Ocean Ridge, substantially expanding the Korarchaeia class with several taxonomically novel genomes. We identified a complete WLP in several deep‐branching lineages, showing that the presence of the WLP is conserved at the root of the Korarchaeia. No methyl‐CoM reductases were encoded by genomes with the WLP, indicating that the WLP is not linked to methanogenesis. By assessing the distribution of hydrogenases and membrane complexes for energy conservation, we show that the WLP is likely used as an electron sink in a fermentative homoacetogenic metabolism. Our study confirms previous hypotheses that the WLP has evolved independently from the methanogenic metabolism in Archaea, perhaps due to its propensity to be combined with heterotrophic fermentative metabolisms. [ABSTRACT FROM AUTHOR]

It has been reported that the frhAGB-encoded hydrogenase from Thermococcus onnurineus NA1 is homologous to the F420-reducing hydrogenase from methanogens and can reduce thioredoxin reductase (TrxR) via direct electron transfer from H2 oxidation. In this study, to find other interaction targets of frhAGB-encoded hydrogenase, we searched for structural homologs of TrxR using the Position-Specific Iterative Basic Local Alignment Search Tool (PSI-BLAST) protein database search program and AlphaFold Protein Structure Database. Fdh3B (TON_0542), a subunit of the formate dehydrogenase 3 (Fdh3), showed the most similar structure to TrxR in its domains. The interaction potential of Fdh3B with frhAGB-encoded hydrogenase was demonstrated by measuring H2-dependent NADP+ reduction by a mixture of purified proteins. Similarly, the H2-dependent NADP+ -reducing activity of Fdh3 whole complex and two other TrxR homologs encoded by TON_0702 and TON_1376 was determined. It was also demonstrated that the Fdh3 complex could reduce CO2 to formate in the presence of the frhAGB-encoded hydrogenase and H2, as shown for the H2-dependent CO2 reductase (HDCR) of acetogen. The in vivo contribution of the frhAGB-encoded hydrogenase and Fdh3 to H2-dependent CO2 reduction was investigated using a resting cell assay, and formate production was significantly decreased in fdh3A or frhA deletion mutants. In conclusion, the frhAGB-encoded hydrogenase was identified to directly transfer electrons to Fdh3 without an electron carrier, similar to the mechanism observed for TrxR. These results expand our understanding of the functional role of the frhAGB-encoded hydrogenase in non-methanogenic Thermococcus species. IMPORTANCE The strategy using structural homology with the help of structure prediction by AlphaFold was very successful in finding potential targets for the frhAGBencoded hydrogenase of Thermococcus onnurineus NA1. The finding that the hydrogenase can interact with FdhB to reduce the cofactor NAD(P)+ is significant in that the enzyme can function to supply reducing equivalents, just as F420-reducing hydrogenases in methanogens use coenzyme F420 as an electron carrier. Additionally, it was identified that T. onnurineus NA1 could produce formate from H2 and CO2 by the concerted action of frhAGB-encoded hydrogenase and formate dehydrogenase Fdh3. [ABSTRACT FROM AUTHOR]

Objectives: Research on hydrogenases from Cupriavidus necator has been ongoing for more than two decades and still today the common methods for culture inoculation are used. These methods were never adapted to the requirements of modified bacterial strains, resulting in different physiological states of the bacteria in the precultures, which in turn lead prolonged and different lag-phases. Results: In order to obtain uniform and always equally fit precultures for inoculation, we have established in this study an optimized protocol for precultures of the derivative of C. necator HF210 (C. necator HP80) which is used for homologous overexpression of the genes for the NAD+-reducing soluble hydrogenase (SH). We compared different media for preculture growth and determined the optimal time point for harvest. The protocol obtained in this study is based on two subsequent precultures, the first one in complex nutrient broth medium (NB) and a second one in fructose –nitrogen mineral salt medium (FN). Conclusion: Despite having two subsequent precultures our protocol reduces the preculture time to less than 30 h and provides reproducible precultures for cultivation of C. necator HP80. [ABSTRACT FROM AUTHOR]

Proteus mirabilis is a common uropathogen and a leading cause of catheter-associated urinary tract infections (CAUTIs), which are often polymicrobial. Through a genome-wide screen, we previously identified two [NiFe] hydrogenases as candidate fitness factors for P. mirabilis CAUTI: a Hyb-type Group 1c H2-uptake hydrogenase and a Hyf-type Group 4a H2-producing hydrogenase. In this study, we disrupted one gene of each system (hyfE and hybC) and also generated a double mutant to examine the contribution of flexible H2 metabolism to P. mirabilis growth and fitness in vitro and during experimental CAUTI. Since P. mirabilis is typically present as part of a polymicrobial community in the urinary tract, we also examined the impact of two common co-colonization partners, Providencia stuartii and Enterococcus faecalis, on the expression and contribution of each hydrogenase to fitness. Our data demonstrate that neither system alone is critical for P. mirabilis growth in vitro or fitness during experimental CAUTI. However, perturbation of flexible H2 metabolism in the ∆hybC∆hyfE double mutant decreased P. mirabilis fitness in vitro and during infection. The Hyf system alone contributed to the generation of proton motive force and swarming motility, but only during anaerobic conditions. Unexpectedly, both systems contributed to benzyl viologen reduction in TYET medium, and disruption of either system increased expression of the other. We further demonstrate that polymicrobial interactions with P. stuartii and E. faecalis alter the expression of Hyb and Hyf in vitro as well as the contribution of each system to P. mirabilis fitness during CAUTI. [ABSTRACT FROM AUTHOR]

The biosynthesis of the active site of the [FeFe]-hydrogenases (HydA1), the H-cluster, is of interest because these enzymes are highly efficient catalysts for the oxidation and production of H2. The biosynthesis of the [2Fe]H subcluster of the H-cluster proceeds from simple precursors, which are processed by three maturases: HydG, HydE, and HydF. Previous studies established that HydG produces an Fe(CO)2(CN) adduct of cysteine, which is the substrate for HydE. In this work, we show that by using the synthetic cluster [Fe2(μ-SH)2(CN)2(CO)4]2- active HydA1 can be biosynthesized without maturases HydG and HydE.