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Bacterial membranes shield the intracellular compartment by selectively allowing unwanted substances to enter in, which in turn reduces overall catalytic efficiency. This report presents a model system using the isolated plasma membranes of Citrobacter sp. S-77 that harbor oxygen-stable [NiFe]hydrogenase and [Mo]formate dehydrogenase, which are integrated into a natural catalytic nanodevice through an electron transfer relay. This naturally occurring nanodevice exhibited selectivity and efficiency in catalyzing the H2-driven conversion of CO2 to formate with the rate of 817 mmol·L-1·gprotein-1·h-1 under mild conditions of 30 °C, pH 7.0, and 0.1 MPa. When the isolated plasma membranes of Citrobacter sp. S-77 was immobilized with multi-walled carbon nanotubes and encapsulated in hydrogel beads of gellan-gum cross-linked with calcium ions, the catalyst for formate production remained stable over 10 repeated uses. This paper reports the first case of efficient and selective formate production from H2 and CO2 using bacterial plasma membranes.

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The splitting of hydrogen (H2) is an energy-yielding process, which is important for both biological systems and as a means of providing green energy. In biology, this reaction is mediated by enzymes called hydrogenases, which utilise complex nickel and iron cofactors to split H2 and transfer the resulting electrons to an electron-acceptor. These [NiFe]-hydrogenases have received considerable attention as catalysts in fuel cells, which utilise H2 to produce electrical current. [NiFe]-hydrogenases are a promising alternative to the platinum-based catalysts that currently predominate in fuel cells due to the abundance of nickel and iron, and the resistance of some family members to inhibition by gases, including carbon monoxide, which rapidly poison platinum-based catalysts. However, the majority of characterised [NiFe]-hydrogenases are inhibited by oxygen (O2), limiting their activity and stability. We recently reported the isolation and characterisation of the [NiFe]-hydrogenase Huc from Mycobacterium smegmatis, which is insensitive to inhibition by O2 and has an extremely high affinity, making it capable of oxidising H2 in air to below atmospheric concentrations. These properties make Huc a promising candidate for the development of enzyme-based fuel cells (EBFCs), which utilise H2 at low concentrations and in impure gas mixtures. In this review, we aim to provide context for the use of Huc for this purpose by discussing the advantages of [NiFe]-hydrogenases as catalysts and their deployment in fuel cells. We also address the challenges associated with using [NiFe]-hydrogenases for this purpose, and how these might be overcome to develop EBFCs that can be deployed at scale.

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Background: The toxic gas carbon monoxide (CO) is abundantly present in synthesis gas (syngas) and certain industrial waste gases that can serve as feedstocks for the biological production of industrially significant chemicals and fuels. For efficient bacterial growth to occur, and to increase productivity and titres, a high resistance to the gas is required. The aerobic bacterium Cupriavidus necator H16 can grow on CO2 + H2, although it cannot utilise CO as a source of carbon and energy. This study aimed to increase its CO resistance through adaptive laboratory evolution. Results: To increase the tolerance of C. necator to CO, the organism was continually subcultured in the presence of CO both heterotrophically and autotrophically. Ten individual cultures were evolved heterotrophically with fructose in this manner and eventually displayed a clear growth advantage over the wild type strain. Next-generation sequencing revealed several mutations, including a single point mutation upstream of a cytochrome bd ubiquinol oxidase operon (cydA2B2), which was present in all evolved isolates. When a subset of these mutations was engineered into the parental H16 strain, only the cydA2B2 upstream mutation enabled faster growth in the presence of CO. Expression analysis, mutation, overexpression and complementation suggested that cydA2B2 transcription is upregulated in the evolved isolates, resulting in increased CO tolerance under heterotrophic but not autotrophic conditions. However, through subculturing on a syngas-like mixture with increasing CO concentrations, C. necator could also be evolved to tolerate high CO concentrations under autotrophic conditions. A mutation in the gene for the soluble [NiFe]-hydrogenase subunit hoxH was identified in the evolved isolates. When the resulting amino acid change was engineered into the parental strain, autotrophic CO resistance was conferred. A strain constitutively expressing cydA2B2 and the mutated hoxH gene exhibited high CO tolerance under both heterotrophic and autotrophic conditions. Conclusion: C. necator was evolved to tolerate high concentrations of CO, a phenomenon which was dependent on the terminal respiratory cytochrome bd ubiquinol oxidase when grown heterotrophically and the soluble [NiFe]-hydrogenase when grown autotrophically. A strain exhibiting high tolerance under both conditions was created and presents a promising chassis for syngas-based bioproduction processes.

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Four Hyp proteins build a scaffold complex upon which the Fe(CN)2 CO group of the [NiFe]-cofactor of hydrogenases (Hyd) is made. Two of these Hyp proteins, the redox-active, [4Fe-4S]-containing HypD protein and the HypC chaperone, form the basis of this scaffold complex. Two different scaffold complexes exist in Escherichia coli, HypCD, and the paralogous HybG-HypD complex, both of which exhibit ATPase activity. Apart from a Rossmann fold, there is no obvious ATP-binding site in HypD. The aim of this study, therefore, was to identify amino acid motifs in HypD that are required for the ATPase activity of the HybG-HypD scaffold complex. Amino acid-exchange variants in three conserved motifs within HypD were generated. Variants in which individual cysteine residues coordinating the iron-sulfur ([4Fe-4S]) cluster were exchanged abolished Hyd enzyme activity and reduced ATPase activity but also destabilized the complex. Two conserved cysteine residues, C69 and C72, form part of HypD's Rossmann fold and play a role in HypD's thiol-disulfide exchange activity. Substitution of these two residues individually with alanine also abolished hydrogenase activity and strongly reduced ATPase activity, particularly the C72A exchange. Residues in a further conserved GFETT motif were exchanged, but neither hydrogenase enzyme activity nor ATPase activity of the isolated HybG-HypD complexes was significantly affected. Together, our findings identify a strong correlation between the redox activity of HypD, ATPase activity, and the ability of the complex to mature Hyd enzymes. These results further highlight the important role of thiol residues in the HybG-HypD scaffold complex during [NiFe]-cofactor biosynthesis.

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[NiFe]-hydrogenases are used by several human pathogens to catalyze the reversible conversion between molecular hydrogen and protons and electrons. Hydrogenases provide an increased metabolic flexibility for pathogens, such as Escherichia coli and Helicobacter pylori, by allowing the use of molecular hydrogen as an energy source to promote survival in anaerobic environments. With the rise of antimicrobial resistance and the desire for novel therapeutics, the [NiFe]-hydrogenases are alluring targets. Inhibiting the nickel insertion pathway of [NiFe]-hydrogenases is attractive as this pathway is required for the generation of functional enzymes and is orthogonal to human biochemistry. In this work, nickel availability for the production and function of E. coli [NiFe]-hydrogenase was explored through immunoblot and activity assays. Whole-cell hydrogenase activities were assayed in high throughput against a small molecule library of known bioactives. Iodoquinol was identified as a potential inhibitor of the nickel biosynthetic pathway of [NiFe]-hydrogenase through a two-step screening process, but further studies with immunoblot assays showed confounding effects dependent on the cell growth phase. This study highlights the significance of considering the growth phenotype for whole-cell based assays overall and its effects on various cellular processes influenced by metal trafficking and homeostasis.

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BACKGROUND: O2-tolerant [NiFe]-hydrogenases offer tremendous potential for applications in H2-based technology. As these metalloenzymes undergo a complicated maturation process that requires a dedicated set of multiple accessory proteins, their heterologous production is challenging, thus hindering their fundamental understanding and the development of related applications. Taking these challenges into account, we selected the comparably simple regulatory [NiFe]-hydrogenase (RH) from Cupriavidus necator as a model for the development of bioprocesses for heterologous [NiFe]-hydrogenase production. We already reported recently on the high-yield production of catalytically active RH in Escherichia coli by optimizing the culture conditions in shake flasks. RESULTS: In this study, we further increase the RH yield and ensure consistent product quality by a rationally designed high cell density fed-batch cultivation process. Overall, the bioreactor cultivations resulted in ˃130 mg L-1 of catalytically active RH which is a more than 100-fold increase compared to other RH laboratory bioreactor scale processes with C. necator. Furthermore, the process shows high reproducibility of the previously selected optimized conditions and high productivity. CONCLUSIONS: This work provides a good opportunity to readily supply such difficult-to-express complex metalloproteins economically and at high concentrations to meet the demand in basic and applied studies.

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Hydrogenases are biotechnologically relevant metalloenzymes that catalyze the reversible conversion of molecular hydrogen into protons and electrons. The O2-tolerant [NiFe]-hydrogenases from Cupriavidus necator (formerly Ralstonia eutropha) are of particular interest as they maintain catalysis even in the presence of molecular oxygen. However, to meet the demands of biotechnological applications and scientific research, a heterologous production strategy is required to overcome the low production yields in their native host. We have previously used the regulatory hydrogenase (RH) from C. necator as a model for the development of such a heterologous hydrogenase production process in E. coli. Although high protein yields were obtained, the purified enzyme was inactive due to the lack of the catalytic center, which contains an inorganic nickel-iron cofactor. In the present study, we significantly improved the production process to obtain catalytically active RH. We optimized important factors such as O2 content, metal availability, production temperature and time as well as the co-expression of RH-specific maturase genes. The RH was successfully matured during aerobic cultivation of E. coli by co-production of seven hydrogenase-specific maturases and a nickel permease, which was confirmed by activity measurements and spectroscopic investigations of the purified enzyme. The improved production conditions resulted in a high yield of about 80 mg L-1 of catalytically active RH and an up to 160-fold space-time yield in E. coli compared to that in the native host C. necator [<0.1 U (L d) -1]. Our strategy has important implications for the use of E. coli K-12 and B strains in the recombinant production of complex metalloenzymes, and provides a blueprint for the production of catalytically active [NiFe]-hydrogenases in biotechnologically relevant quantities.

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The biosynthesis of the NiFe(CN)2CO organometallic cofactor of [NiFe]-hydrogenase (Hyd) involves several discreet steps, including the synthesis of the Fe(CN)2CO group on a HypD-HypC scaffold complex. HypC has an additional function in transferring the Fe(CN)2CO group to the apo-precursor of the Hyd catalytic subunit. Bacteria that synthesize more than one Hyd enzyme often have additional HypC-type chaperones specific for each precursor. The specificity determinants of this large chaperone family are not understood. Escherichia coli synthesizes two HypC paralogs, HypC and HybG. HypC delivers the Fe(CN)2CO group to pre-HycE, the precursor of the H2-evolving Hyd-3 enzyme, while HybG transfers the group to the pre-HybC of the H2-oxidizing Hyd-2 enzyme. We could show that a conserved histidine residue around the amino acid position 50 in both HypC and HybG, when exchanged for an alanine, resulted in a severe reduction in the activity of its cognate Hyd enzyme. This reduction in enzyme activity proved to be due to the impaired ability of the chaperones to interact with HypD. Surprisingly, and only in the case of the HybG H52A variant, its co-synthesis with HypD improved its interaction with pre-HycE, resulting in the maturation of Hyd-3. This study demonstrates that the conserved histidine residue helps enhance the interaction of the chaperone with HypD, but additionally, and in E. coli only for HybG, acts as a determinant to prevent the inadvertent maturation of the wrong large-subunit precursor. This study identifies a new level of control exerted by a bacterium synthesizing multiple [NiFe]-Hyd to ensure the correct enzyme is matured only under the appropriate physiological conditions.

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There are ten nickel enzymes found across biological systems, each with a distinct active site and reactivity that spans reductive, oxidative, and redox-neutral processes. We focus on the reductive enzymes, which catalyze reactions that are highly germane to the modern-day climate crisis: [NiFe] hydrogenase, carbon monoxide dehydrogenase, acetyl coenzyme A synthase, and methyl coenzyme M reductase. The current mechanistic understanding of each enzyme system is reviewed along with existing knowledge gaps, which are addressed through the development of protein-derived models, as described here. This opinion is intended to highlight the advantages of using robust protein scaffolds for modeling multiscale contributions to reactivity and inspire the development of novel artificial metalloenzymes for other small molecule transformations.

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Hydrogenases are abundant metalloenzymes that catalyze the reversible conversion of molecular H2 into protons and electrons. Important achievements have been made over the past two decades in the understanding of these highly complex enzymes. However, most hydrogenases have low production yields requiring many efforts and high costs for cultivation limiting their investigation. Heterologous production of these hydrogenases in a robust and genetically tractable expression host is an attractive strategy to make these enzymes more accessible. In the present study, we chose the oxygen-tolerant H2-sensing regulatory [NiFe]-hydrogenase (RH) from Ralstonia eutropha H16 owing to its relatively simple architecture compared to other [NiFe]-hydrogenases as a model to develop a heterologous hydrogenase production system in Escherichia coli. Using screening experiments in 24 deep-well plates with 3 mL working volume, we investigated relevant cultivation parameters, including inducer concentration, expression temperature, and expression time. The RH yield could be increased from 14 mg/L up to >250 mg/L by switching from a batch to an EnPresso B-based fed-batch like cultivation in shake flasks. This yield exceeds the amount of RH purified from the homologous host R. eutropha by several 100-fold. Additionally, we report the successful overproduction of the RH single subunits HoxB and HoxC, suitable for biochemical and spectroscopic investigations. Even though both RH and HoxC proteins were isolated in an inactive, cofactor free apo-form, the proposed strategy may powerfully accelerate bioprocess development and structural studies for both basic research and applied studies. These results are discussed in the context of the regulation mechanisms governing the assembly of large and small hydrogenase subunits.

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To study metalloenzymes in detail, we developed a new experimental setup allowing the controlled preparation of catalytic intermediates for characterization by various spectroscopic techniques. The in situ monitoring of redox transitions by infrared spectroscopy in enzyme lyophilizate, crystals, and solution during gas exchange in a wide temperature range can be accomplished as well. Two O2 -tolerant [NiFe]-hydrogenases were investigated as model systems. First, we utilized our platform to prepare highly concentrated hydrogenase lyophilizate in a paramagnetic state harboring a bridging hydride. This procedure proved beneficial for 57 Fe nuclear resonance vibrational spectroscopy and revealed, in combination with density functional theory calculations, the vibrational fingerprint of this catalytic intermediate. The same in situ IR setup, combined with resonance Raman spectroscopy, provided detailed insights into the redox chemistry of enzyme crystals, underlining the general necessity to complement X-ray crystallographic data with spectroscopic analyses.

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The microbial H2-producing (hydrogenogenic) carbon monoxide (CO)-oxidizing activity by the membrane-associated CO dehydrogenase (CODH)/energy-converting hydrogenase (ECH) complex is an important metabolic process in the microbial community. However, the studies on hydrogenogenic carboxydotrophs had to rely on inherently cultivation and isolation methods due to their rare abundance, which was a bottleneck in ecological study. Here, we provided gene-targeted sequencing method for the diversity estimation of thermophilic hydrogenogenic carboxydotrophs. We designed six new degenerate primer pairs which effectively amplified the coding regions of CODH genes forming gene clusters with ECH genes (CODHech genes) in Firmicutes which includes major thermophilic hydrogenogenic carboxydotrophs in terrestrial thermal habitats. Amplicon sequencing by these primers using DNAs from terrestrial hydrothermal sediments and CO-gas-incubated samples specifically detected multiple CODH genes which were identical or phylogenetically related to the CODHech genes in Firmictes. Furthermore, we found that phylogenetically distinct CODHech genes were enriched in CO-gas-incubated samples, suggesting that our primers detected uncultured hydrogenogenic carboxydotrophs as well. The new CODH-targeted primers provided us with a fine-grained (~ 97.9% in nucleotide sequence identity) diversity analysis of thermophilic hydrogenogenic carboxydotrophs by amplicon sequencing and will bolster the ecological study of these microorganisms.

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BACKGROUND: A unifying feature of the bacterial Candidate Phyla Radiation (CPR) is a limited and highly variable repertoire of biosynthetic capabilities. However, the distribution of metabolic traits across the CPR and the evolutionary processes underlying them are incompletely resolved. RESULTS: Here, we selected ~ 1000 genomes of CPR bacteria from diverse environments to construct a robust internal phylogeny that was consistent across two unlinked marker sets. Mapping of glycolysis, the pentose phosphate pathway, and pyruvate metabolism onto the tree showed that some components of these pathways are sparsely distributed and that similarity between metabolic platforms is only partially predicted by phylogenetic relationships. To evaluate the extent to which gene loss and lateral gene transfer have shaped trait distribution, we analyzed the patchiness of gene presence in a phylogenetic context, examined the phylogenetic depth of clades with shared traits, and compared the reference tree topology with those of specific metabolic proteins. While the central glycolytic pathway in CPR is widely conserved and has likely been shaped primarily by vertical transmission, there is evidence for both gene loss and transfer especially in steps that convert glucose into fructose 1,6-bisphosphate and glycerate 3P into pyruvate. Additionally, the distribution of Group 3 and Group 4-related NiFe hydrogenases is patchy and suggests multiple events of ancient gene transfer. CONCLUSIONS: We infer that patterns of gene gain and loss in CPR, including acquisition of accessory traits in independent transfer events, could have been driven by shifts in host-derived resources and led to sparse but varied genetic inventories.

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HypD and HypC, or its paralogue HybG in Escherichia coli, form the core of the scaffold complex that synthesizes the Fe(CN)2 CO component of the bimetallic NiFe-cofactor of [NiFe]-hydrogenase. We show here that purified HypC-HypD and HybG-HypD complexes catalyse hydrolysis of ATP to ADP (kcat  â‰… 0.85·s-1 ); the ATPase activity of the individual proteins was between 5- and 10-fold lower than that of the complex. Pre-incubation of HypD with ATP was necessary to restore full activity upon addition of HybG. The conserved Cys41 residue on HypD was essential for full ATPase activity of the complex. Together, our data suggest that HypD undergoes ATP-dependent conformational activation to facilitate complex assembly in preparation for substrate reduction.

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[NiFe] hydrogenases are complex model enzymes for the reversible cleavage of dihydrogen (H2 ). However, structural determinants of efficient H2 binding to their [NiFe] active site are not properly understood. Here, we present crystallographic and vibrational-spectroscopic insights into the unexplored structure of the H2 -binding [NiFe] intermediate. Using an F420 -reducing [NiFe]-hydrogenase from Methanosarcina barkeri as a model enzyme, we show that the protein backbone provides a strained chelating scaffold that tunes the [NiFe] active site for efficient H2 binding and conversion. The protein matrix also directs H2 diffusion to the [NiFe] site via two gas channels and allows the distribution of electrons between functional protomers through a subunit-bridging FeS cluster. Our findings emphasize the relevance of an atypical Ni coordination, thereby providing a blueprint for the design of bio-inspired H2 -conversion catalysts.

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The reaction of Ni(TPAP)(COD) {where TPAP = [(NC5H4)CH2]3P(NC2H4)3N} with Fe(CO)5 resulted in the isolation of the title heterobimetallic NiFe(TPAP)(CO)5 complex di-µ-carbonyl-tricarbon-yl[2,8,9-tris-(pyridin-2-yl-meth-yl)-2,5,8,9-tetra-aza-1-phosphabi-cyclo-[3.3.3]undeca-ne]ironnickel, [FeNi(C24H30N7P)(CO)5]. Characterization of the complex by 1H and 31P NMR as well as IR spectroscopy are presented. The structure of NiFe(TPAP)(CO)5 reveals three terminally bound CO mol-ecules on Fe0, two bridging CO mol-ecules between Ni0 and Fe0, and TPAP coordinated to Ni0. The Ni-Fe bond length is 2.4828 (4) Å, similar to that of the reduced form of the active site of NiFe hydrogenase (∼2.5 Å). Additionally, a proximal pendant base from one of the non-coordinating pyridine groups of TPAP is also present. Although involvement of a pendant base has been cited in the mechanism of NiFe hydrogenase, this moiety has yet to be incorporated in a structurally characterized synthetic mimic with key structural motifs (terminally bound CO or CN ligands on Fe). Thus, the title complex NiFe(TPAP)(CO)5 is an unique synthetic model for NiFe hydrogenase. In the crystal, the complex mol-ecules are linked by C-H⋯O hydrogen bonds, forming undulating layers parallel to (100). Within the layers, there are offset π-π [inter-centroid distance = 3.2739 (5) Å] and C-H⋯π inter-actions present. The layers are linked by further C-H⋯π inter-actions, forming a supra-molecular framework.

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Hydrogenase enzymes are currently under the international research spotlight due to emphasis on biologically produced hydrogen as one potential energy carrier to relinquish the requirement for 'fossil fuel' derived energy. Three major classes of hydrogenase exist in microbes all able to catalyze the reversible oxidation of dihydrogen to protons and electrons. These classes are defined by their active site metal content: [NiFe]-; [FeFe]- and [Fe]-hydrogenases. Of these the [NiFe]-hydrogenases have links to ancient forms of metabolism, utilizing hydrogen as the original source of reductant on Earth. This review progresses to highlight the Group 4 [NiFe]-hydrogenase enzymes that preferentially generate hydrogen exploiting various partner enzymes or ferredoxin, while in some cases translocating ions across biological membranes. Specific focus is paid to Group 4A, the Formate hydrogenlyase complexes. These are the combination of a six or nine subunit [NiFe]-hydrogenase with a soluble formate dehydrogenase to derived electrons from formate oxidation for proton reduction. The incidence, physiology, structure and biotechnological application of these complexes will be explored with attention on Escherichia coli Formate Hydrogenlyase-1 (FHL-1).

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Biological H2 production has potential to address energy security and environmental concerns if produced from renewable or waste sources. The purple non-sulfur photosynthetic bacterium Rubrivivax gelatinosus CBS produces H2 while oxidizing CO, a component of synthesis gas (Syngas). CO-linked H2 production is facilitated by an energy-converting hydrogenase (Ech), while a subsequent H2 oxidation reaction is catalyzed by a membrane-bound hydrogenase (MBH). Both hydrogenases contain [NiFe] active sites requiring 6 maturation factors (HypA-F) for assembly, but it is unclear which of the two annotated sets of hyp genes are required for each in R. gelatinosus CBS. Herein, we report correlated expression of hyp1 genes with Ech genes and hyp2 expression with MBH genes. Moreover, we find that while Ech H2 evolving activity is only delayed when hyp1 is deleted, hyp2 deletion completely disrupts MBH H2 uptake, providing a platform for a biologically driven water-gas shift reaction to produce H2 from CO.

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Enzyme activity is typically assayed by quantitatively measuring the initial and final concentrations of the substrates and/or products over a defined time period. For enzymatic reactions involving gaseous substrates, the substrate concentrations can be estimated either directly by gas chromatography or mass spectrometry, or indirectly by absorption spectroscopy, if the catalytic reactions involve electron transfer with electron mediators that exhibit redox-dependent spectral changes. We have developed a new assay system for measuring the time course of enzymatic reactions involving gaseous substrates based on Raman spectroscopy. This system permits continuous monitoring of the gas composition in the reaction cuvette in a non-invasive manner over a prolonged time period. We have applied this system to the kinetic study of the [NiFe] hydrogenase from Desulfovibrio vulgaris Miyazaki F. This enzyme physiologically catalyzes the reversible oxidation of H2 and also possesses the nonphysiological functions of H/D exchange and nuclear spin isomer conversion reactions. The proposed system has the additional advantage of enabling us to measure all of the hydrogenase-mediated reactions simultaneously. Using the proposed system, we confirmed that H2 (the fully exchanged product) is concomitantly produced alongside HD by the H/D exchange reaction in the D2 /H2 O system. Based on a kinetic model, the ratio of the rate constants of the H/D exchange reaction (k) at the active site and product release rate (kout ) was estimated to be 1.9 ± 0.2. The proposed assay method based on Raman spectroscopy can be applied to the investigation of other enzymes involving gaseous substrates.

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Nickel is essential for the survival of many pathogenic bacteria. E. coli and H. pylori require nickel for [NiFe]-hydrogenases. H. pylori also requires nickel for urease. At high concentrations nickel can be toxic to the cell, therefore, nickel concentrations are tightly regulated. Metalloregulators help to maintain nickel concentration in the cell by regulating the expression of the genes associated with nickel import and export. Nickel import into the cell, delivery of nickel to target proteins, and export of nickel from the cell is a very intricate and well-choreographed process. The delivery of nickel to [NiFe]-hydrogenase and urease is complex and involves several chaperones and accessory proteins. A combination of biochemical, crystallographic, and spectroscopic techniques has been utilized to study the structures of these proteins, as well as protein-protein interactions resulting in an expansion of our knowledge regarding how these proteins sense and bind nickel. In this review, recent advances in the field will be discussed, focusing on the metal site structures of nickel bound to metalloregulators and chaperones.