NiFe Hydrogenase Research Articles

Harnessing Fermentation May Enhance the Performance of Biological Sulfate-Reducing Bioreactors.

Biological sulfate reduction (BSR) represents a promising strategy for bioremediation of sulfate-rich waste streams, yet the impact of metabolic interactions on performance is largely unexplored. Here, genome-resolved metagenomics was used to characterize 17 microbial communities in reactors treating synthetic sulfate-contaminated solutions. Reactors were supplemented with lactate or acetate and a small amount of fermentable substrate. Of the 163 genomes representing all the abundant bacteria, 130 encode 321 NiFe and FeFe hydrogenases and all genomes of the 22 sulfate-reducing microorganisms (SRM) encode genes for H2 uptake. We observed lactate oxidation solely in the first packed bed reactor zone, with propionate and acetate oxidation in the middle and predominantly acetate oxidation in the effluent zone. The energetics of these reactions are very different, yet sulfate reduction kinetics were unaffected by the type of electron donor available. We hypothesize that the comparable rates, despite the typically slow growth of SRM on acetate, are a result of the consumption of H2 generated by fermentation. This is supported by the sustained performance of a predominantly acetate-supplemented stirred tank reactor dominated by diverse fermentative bacteria encoding FeFe hydrogenase genes and SRM capable of acetate and hydrogen consumption and CO2 assimilation. Thus, addition of fermentable substrates to stimulate syntrophic relationships may improve the performance of BSR reactors supplemented with inexpensive acetate.

Exploiting hydrogenases for biocatalytic hydrogenations.

The ability of hydrogenase enzymes to activate H2 with excellent selectivity leads to many interesting possibilities for biotechnology driven by H2 as a clean reductant. Here, we review examples where hydrogenase enzymes have been used to drive native and non-native hydrogenation reactions in solution or as part of a redox cascade on a conductive support, with a focus on the developments we have contributed to this field. In all of the examples discussed, hydrogenation reactions are enabled by coupled redox reactions: the oxidation of H2 at a hydrogenase active site, linked electronically (via relay clusters in the enzyme and/or via conductive support) to the site of a reduction reaction, and we note how this parallels developments in site-separated reactivity in heterogeneous catalysis. We discuss the productivities achieved with biocatalytic hydrogenations, the scope for application of these approaches in industrial biotechnology, possibilities for scaling the production of hydrogenases, and future opportunities. Our focus is on NiFe hydrogenases, but we discuss briefly how FeFe hydrogenases might contribute to this field.

Combining Electrochemistry and Protein Engineering to Elucidate Outer-Sphere Effects in Hydrogenase Catalysis

Hydrogenases, the bacterial enzymes that oxidize and produce H2, come in two flavors, “NiFe hydrogenases” and “FeFe hydrogenases”, depending on the structure of their active site.In each of these two families of enzymes, the active site and its immediate environment are fully conserved, and yet the investigations of these enzymes using direct electrochemistry have shown that their catalytic properties vary greatly: this is true regarding catalytic bias and reversibility1, resistance to oxygen, and other properties which define whether or not a particular hydrogenase could be used under the operating conditions of a particular device.These functional variations within each family also tell us that structural features that are remote from the active site, the so-called “outer-sphere” effects, define, at least in part, the catalytic properties.2 In the FeFe hydrogenase from Clostridium beijerinckii, we have shown that oxygen resistance is related to a conformational change that depends on non-conserved residues that are up to 18 Å away from the active site. This conformational change occurs in two steps that can be resolved by combining site-directed mutagenesis and detailed protein film voltammetry experiments.3 These remote residues define an emerging family of O2-resistant FeFe hydrogenases. (a) A. Fasano, H. Land, V. Fourmond, G. Berggren, and C. Léger, « Reversible or irreversible catalysis of H+/H2 conversion by FeFe hydrogenases », J. Am. Chem. Soc. 143, 48, 20320-20325 (2021) (b) V. Fourmond, N. Plumeré, C. Léger, « Reversible catalysis », Nature Reviews Chemistry, 5, 348-360 (2021)S. Stripp, B. Duffus, V. Fourmond, C. Léger, S. Leimkühler, S. Hirota, Y. Hu, A. Jasniewski, H. Ogata, M. Ribbe, « Second and Outer Coordination Sphere Effects in Low Valent Metalloenzymes », Chemical Reviews, 122 11900 (2022) (a) M. Winkler, J. Duan, A. Rutz, C. Felbek, L. Scholtysek, O. Lampret, J. Jaenecke, U.-P.r Apfel, G. Gilardi, F. Valetti, V. Fourmond, E. Hofmann, C. Léger, T. Happe « A safety cap protects hydrogenase from oxygen attack » Nature Communications 12, 756 (2021) (b) A. Rutz, C. Das, A. Fasano, J. Jaenecke, S. Yadav, U.P. Apfel, V. Engelbrecht, V. Fourmond, C. Léger, L. Schäfer, T. Happe « Increasing the O2 resistance of the [FeFe]-hydrogenase CbA5H through enhanced protein flexibility », ACS Catalysis 13, 2, 856–865 (2023) Figure 1

A simple method for oriented immobilization of HydSL hydrogenase of Thiocapsa bogorovii on carbon electrodes

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.

The first cultivated representatives of the actinobacterial lineage OPB41 isolated from subsurface environments constitute a novel order Anaerosomatales.

The continental subsurface harbors microbial populations highly enriched in uncultured taxa. OPB41 is an uncultured order-level phylogenetic lineage within the actinobacterial class Coriobacteriia. OPB41 bacteria have a wide geographical distribution, but the physiology and metabolic traits of this cosmopolitan group remain elusive. From two contrasting subsurface environments, a terrestrial mud volcano and a deep subsurface aquifer, located in the central part of Eurasia, within the Caucasus petroleum region, we have isolated two pure cultures of anaerobic actinobacteria belonging to OPB41. The cells of both strains are small non-motile rods forming numerous pili-like appendages. Strain M08DHBT is mesophilic, while strain Es71-Z0120T is a true thermophile having a broad temperature range for growth (25-77°C). Strain M08DHBT anaerobically reduces sulfur compounds and utilizes an aromatic compound 3,4-dihydroxybenzoic acid. Strain Es71-Z0120T is an obligate dissimilatory Fe(III) reducer that is unable to utilize aromatic compounds. Both isolates grow lithotrophically and consume molecular hydrogen or formate using either thiosulfate, elemental sulfur, or Fe(III) as an electron acceptor. Genomes of the strains encode the putative reductive glycine pathway for autotrophic CO2 fixation, Ni-Fe hydrogenases, putative thiosulfate/polysulfide reductases, and multiheme c-type cytochromes presumably involved in dissimilatory Fe(III) reduction. We propose to assign the isolated strains to the novel taxa of the species-order levels and describe strain M08DHBT as Anaerosoma tenue gen. nov., sp. nov., and strain Es71-Z0120T as Parvivirga hydrogeniphila gen. nov., sp. nov., being members of Anaerosomatales ord. nov. This work expands the knowledge of the diversity, metabolic functions, and ecological role of the phylum Actinomycetota.

Zng1 is a GTP-dependent zinc transferase needed for activation of methionine aminopeptidase.

The evolution of zinc (Zn) as a protein cofactor altered the functional landscape of biology, but dependency on Zn also created an Achilles' heel, necessitating adaptive mechanisms to ensure Zn availability to proteins. A debated strategy is whether metallochaperones exist to prioritize essential Zn-dependent proteins. Here, we present evidence for a conserved family of putative metal transferases in human and fungi, which interact with Zn-dependent methionine aminopeptidase type I (MetAP1/Map1p/Fma1). Deletion of the putative metal transferase in Saccharomyces cerevisiae (ZNG1; formerly YNR029c) leads to defective Map1p function and a Zn-deficiency growth defect. Invitro, Zng1p can transfer Zn2+ or Co2+ to apo-Map1p, but unlike characterized copper chaperones, transfer is dependent on GTP hydrolysis. Proteomics reveal mis-regulation of the Zap1p transcription factor regulon because of loss of ZNG1 and Map1p activity, suggesting that Zng1p is required to avoid a compounding effect of Map1p dysfunction on survival during Zn limitation.

Nickel stress-tolerance in plant-bacterial associations.

Nickel (Ni) is an essential element for plant growth and is a constituent of several metalloenzymes, such as urease, Ni-Fe hydrogenase, Ni-superoxide dismutase. However, in high concentrations, Ni is toxic and hazardous to plants, humans and animals. High levels of Ni inhibit plant germination, reduce chlorophyll content, and cause osmotic imbalance and oxidative stress. Sustainable plant-bacterial native associations are formed under Ni-stress, such as Ni hyperaccumulator plants and rhizobacteria showed tolerance to high levels of Ni. Both partners (plants and bacteria) are capable to reduce the Ni toxicity and developed different mechanisms and strategies which they manifest in plant-bacterial associations. In addition to physical barriers, such as plants cell walls, thick cuticles and trichomes, which reduce the elevated levels of Ni entrance, plants are mitigating the Ni toxicity using their own antioxidant defense mechanisms including enzymes and other antioxidants. Bacteria in its turn effectively protect plants from Ni stress and can be used in phytoremediation. PGPR (plant growth promotion rhizobacteria) possess various mechanisms of biological protection of plants at both whole population and single cell levels. In this review, we highlighted the current understanding of the bacterial induced protective mechanisms in plant-bacterial associations under Ni stress.

The HydS C-terminal domain of the Thiocapsa bogorovii HydSL hydrogenase is involved in membrane anchoring and electron transfer

Thiocapsa bogorovii BBS (former name Thiocapsa roseopersicina) contains HydSL hydrogenase belonging to 1e subgroup of NiFe hydrogenases (isp-type). The operon of these hydrogenases contains gene for small subunit (hydS), gene for large subunit (hupL), and genes isp1 and isp2 between them. It is predicted that last two genes code electron transport careers for electron transfer from/to HydSL hydrogenase. However, the interaction between them is unclear. The aim of this study was to determine structural and functional role of T. bogorovii HydS C-terminal end. For this purpose, we modelled all subunits of the complex HydS-HydL-Isp1-Isp2. Hydrophobicity surface analysis of the Isp1 model revealed highly hydrophobic helices suggesting potential membrane localization, as well as the hydrophilic C-terminus, which is likely localized outside of membrane. Isp1 model was docked with models of full length and C-terminal truncated HydSL hydrogenases and results illustrate the possibility of HydSL membrane anchoring via transmembrane Isp1 with essential participation of C-terminal end of HydS in the interaction. C-terminal end of HydS subunit was deleted and our studies revealed that the truncated HydSL hydrogenase detached from cellular membranes in contrast to native hydrogenase. It is known that HydSL hydrogenase in T. bogorovii performs the reaction of elemental sulfur reduction (S0 + H2 = ≥H2S). Cells with truncated HydS produced much less H2S in the presence of H2 and S0. Thus, our data support the conclusion that C-terminal end of HydS subunit participates in interaction of HydSL hydrogenase with Isp1 protein for membrane anchoring and electron transfer.

Energetics for Proton Reduction in FeFe Hydrogenase.

The energetics for proton reduction in FeFe-hydrogenase has been reinvestigated by theoretical modeling, in light of recent experiments. Two different mechanisms have been considered. In the first one, the bridging hydride position was blocked by the enzyme, which is the mechanism that has been supported by a recent spectroscopic study by Cramer et al. A major difficulty in the present study to agree with experimental energetics was to find the right position for the added proton in the first reduction step. It was eventually found that the best position was as a terminal hydride on the distal iron, which has not been suggested in any of the recent, experimentally based mechanisms. The lowest transition state was surprisingly found to be a bond formation between a proton on a cysteine and the terminal hydride. This type of TS is similar to the one for heterolytic H2 cleavage in NiFe hydrogenase. The second mechanism investigated here is not supported by the present calculations or the recent experiments by Cramer et al., but was still studied as an interesting comparison. In that mechanism, the formation of the bridging hydride was allowed. The H–H formation barrier is only 3.6 kcal/mol higher than for the first mechanism, but there are severe problems concerning the motion of the protons.

(Invited) Photoinduced Hydrogen Evolution with Ni-Fe Hydrogenase-Viologen-Porphyrin Immobilized ITO Electrode

Hydrogenase-viologen-porphyrin triad was immobilized on ITO electrode and carried out photoinduced hydrogen evolution. This system can reductively generate hydrogen by electron donation from the photoexcited porphyrin to hydrogenase via viologen.Hydrogen production by water splitting (electrochemical, photochemical, or thermal) offers an obvious way to capture solar energy. Catalysts are a crucial issue for Hydrogen production, and there is intense interest in finding alternatives to noble metals that are capable of converting water into hydrogen close to the thermodynamic potential.As a biological alternative, enzymes known as hydrogenases catalyze the reduction of protons into hydrogen at active sites composed of iron- or nickel/iron complexes: importantly, the electrochemical reaction is reversible with a small overpotential. Determination of the structures of hydrogenases from several organisms paired with detailed spectroscopic and electrochemical investigations have considerably improved our understanding of these enzymes in recent years.Hydrogenases also show high H2 production yields in photocatalytic schemes within sacrificial electron donors in pH neutral aqueous solution. In these systems with hydrogenase, a photoexcited photosensitizer provides electrons to the active site where proton reduction occurs via an internal electron transfer iron sulfur clusters. Examples are the immobilization of a hydrogenase on Ru-sensitised TiO2, on Cd-based, quantum dotsas well as homogeneous systems using the hydrogenases with a covalently linked photosystem I or in combination with an organic dye,and multi-component systems with a dye and a soluble redox mediator.In this study, we report on ITO electrode system with hydrogenase, where Ni-Fe hydrogenase immobilized on ITO surface using a linker contains both a viologen and a porphyrin. [NiFe]-Hydrogenase from Desulfovibrio vulgaris (Miyazaki) is approximately 90 kDa in size, with large and small subunits of 60 kDa and 30 kDa, respectively. The Ni-Fe active site is located to the surface of the enzyme. Three Fe-S clusters from the putative electron transfer pathway between the active site. The proximal [4Fe4S] cluster lies closest to the active site while the distal [4Fe4S] cluster is found near the surface of the enzyme. A [3Fe4S] cluster is located in between the two [4Fe4S] clusters and is known as the medial cluster. The distal [4Fe4S] cluster works as the first electron acceptor of the [NiFe]-hydrogenase in the case of hydrogen evolution.Figure 1 shows the ITO electrode system prepared in this study. Hydrogenase was immobilized via the 4,4’-(3-aminopropyl)-bipyridinium (DAPV) by a glutamate-rich patch of hydrogenase where the distal Fe-S cluster is located.The photoexcited state of Tetrakis(4-carboxyphenyl)porphyrin (TCPP) donates electrons to [NiFe]-hydrogenase via the 4,4’-(3-aminopropyl)-bipyridinium (DAPV) bridging between the two. DAPV was linked to acidic amino acid near the distal [4Fe4S] cluster, electron entrance, of [NiFe]-Hydrogenase in order to efficiently donate electrons to the [NiFe]-Hydrogenase.Photoinduced hydrogen evolution were carried out under applied -0.3V vs.SHE. During light irradiation, cathodic continuous current was observed due to the hydrogen evolution. The photoexcited state of TCPP donate electrons to hydrogenase via the viologen, and ITO electrode play an electron donor to HOMO of the TCPP. Therefore, photoinduced hydrogen evolution occur in TCPP-DAPV-hydrogenase triads on ITO electrode. Figure 1

Repurposing a Bio-Inspired NiFe Hydrogenase Model for CO2 Reduction with Selective Production of Methane as the Unique C-Based Product

In the current environmental and economic context, there is an urgent need to develop catalytic systems to efficiently activate and transform abundant small molecules while demonstrating selectivit...

Preferential use of carbon central metabolism and anaerobic respiratory chains in porcine extraintestinal pathogenic Escherichia coli during bloodstream infection

Porcine extraintestinal pathogenic Escherichia coli (ExPEC) is occurring with increasing frequency in China, and leads to significant economic and welfare costs in the swine industry. The underlying mechanisms of porcine ExPEC in blood colonization during systematic infection is poorly understood. Here we measured the gene expression of porcine ExPEC in infected animal bloodstream in vivo and fresh swine blood in vitro. Using comparisons with P values of ≤ 0.01, we identified 354 and 313 genes as being significantly up- or down-regulated at least 2-fold change during bloodstream infection, respectively. Excepting for an array of iron acquisition systems, numerous genes involved in carbon central metabolism and anaerobic respiratory chains were upregulated here. These genes were categorized into several clusters including the TCA-cycle (frdABCD, citCEFXG), d-ribose transporter (rbsDACB), nickel transporter (nikABCDER), NiFe hydrogenase (hybOABCDEF, hycBCDEFG), Hyp-complex (hypABCDE), DMSO reductase (dmsABC and ynfEFGHI), format dehydrogenase (fdnGHI) and NADH dehydrogenase I (nuoA-N). The mutant with simultaneous inactivation of ribose and citrate imports showed significant reduced fitness in host blood, suggesting these two carbohydrates are utilized by central metabolism network as important carbon-source during bloodstream infection. Similar deficiency was also observed in the mutant double deleted NiFe hydrogenase 2 and 3 anaerobic respiratory chains. Further study found that FNR (a global regulator facilitating bacterial adaptation to anaerobic conditions) is an important regulator in response to bloodstream to activate center metabolism and anaerobic respiratory chains, thus contribute to the full-virulence of porcine ExPEC. These findings provide compelling evidence to support the notion that carbon central metabolism network and anaerobic respiratory chains play key roles for porcine ExPEC fitness within host bloodstream.

Electricity from anaerobic methane oxidation by a single methanogenic archaeon Methanosarcina barkeri

Anaerobic oxidation of methane (AOM) coupled with electron transfer to electrodes has recently showed great promise for energy harvesting in microbial electrochemical reactors. To date this process has been reported limited to the syntrophic cooperation of archaea and bacteria in mixed cultures. It remains unknown whether pure archaea alone can catalyze electricity production from methane. Here we report the performance of AOM-driven electricity generation by a single methanogen Methanosarcina barkeri. At a poised electrode potential of +300 mV vs. SHE, a maximum current of 649.7 mA m−2 was achieved from methane with an appreciable Coulombic efficiency up to 86.9%. The electrogenic mechanism analysis demonstrated that M. barkeri donated most of electrons to electrodes directly and secreted soluble redox-active compounds to facilitate indirect extracellular electron transfer (EET). Electrochemical in situ Fourier transform infrared spectra and metabolic inhibitions suggested the methyl-coenzyme M reductase α-subunit (McrA), NiFe hydrogenases and cytochrome proteins were involved in the AOM-EET process. This work provides the first proof of concept for AOM-powered electricity production by a single archaeon via reversed methanogenesis and suggests methanogens may play an important role in coupling AOM with the reduction of solid electron acceptors in natural environments.

The rise of diversity in metabolic platforms across the Candidate Phyla Radiation

BackgroundA 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.ResultsHere, 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.ConclusionsWe 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.

Time-Course Transcriptome of Parageobacillus thermoglucosidasius DSM 6285 Grown in the Presence of Carbon Monoxide and Air.

Parageobacillus thermoglucosidasius is a metabolically versatile, facultatively anaerobic thermophile belonging to the family Bacillaceae. Previous studies have shown that this bacterium harbours co-localised genes coding for a carbon monoxide (CO) dehydrogenase (CODH) and Ni-Fe hydrogenase (Phc) complex and oxidises CO and produces hydrogen (H2) gas via the water-gas shift (WGS) reaction. To elucidate the genetic events culminating in the WGS reaction, P. thermoglucosidasius DSM 6285 was cultivated under an initial gas atmosphere of 50% CO and 50% air and total RNA was extracted at ~8 (aerobic phase), 20 (anaerobic phase), 27 and 44 (early and late hydrogenogenic phases) hours post inoculation. The rRNA-depleted fraction was sequenced using Illumina NextSeq, v2.5, 1x75bp chemistry. Differential expression revealed that at 8 vs.. 20, 20 vs.. 27 and 27 vs.. 44 h post inoculation, 2190, 2118 and 231 transcripts were differentially (FDR < 0.05) expressed. Cluster analysis revealed 26 distinct gene expression trajectories across the four time points. Of these, two similar clusters, showing overexpression at 20 relative to 8 h and depletion at 27 and 44 h, harboured the CODH and Phc transcripts, suggesting possible regulation by O2. The transition between aerobic respiration and anaerobic growth was marked by initial metabolic deterioration, as reflected by up-regulation of transcripts linked to sporulation and down-regulation of transcripts linked to flagellar assembly and metabolism. However, the transcriptome and growth profiles revealed the reversal of this trend during the hydrogenogenic phase.

Hydrogen-uptake genes improve symbiotic efficiency in common beans (Phaseolus vulgaris L.).

Hydrogen-uptake (Hup) activity is implicated in the mitigation of energy losses associated with the biological nitrogen fixation process, and has been related to productivity increases in some legume hosts. However, in common bean (Phaseolus vulgaris L.) the expression of hydrogenase is rare. In this study an 18-kb hup gene cluster from Rhizobium leguminosarum bv. viciae encoding a NiFe hydrogenase was successfully transferred to three common bean rhizobial strains lacking hydrogenase activity (Hup-) but symbiotically very effective and used in commercial inoculants in Brazil: one strain originally from Colombia (Rhizobium tropici CIAT 899), and two strains from Brazil (R. tropici H 12 and Rhizobium freirei PRF 81). The inclusion of NiCl2 in the nutrient solution did not increase hydrogenase activity, indicating that common bean plants allow efficient nickel provision for hydrogenase synthesis in the bacteroids. The symbiotic performance-evaluated by nodulation, plant growth, N accumulation and seed production-of wild-type and Hup+ derivative strains was compared in experiments performed with cultivar Carioca under greenhouse conditions, in sterile substrate and in non-sterile soil. Statistically significant increases in one or more parameters were observed for all three Hup+ derivatives when compared to the respective wild-type strain. Differences were found mainly with the Brazilian strains, reaching impressive increases in nodule efficiency and seed total N content. The results highlight the potential of using Rhizobium Hup+ strains for the design of more energy-efficient inoculants for the common bean crop.

Structural characterization of HypX responsible for CO biosynthesis in the maturation of NiFe-hydrogenase

Several accessory proteins are required for the assembly of the metal centers in hydrogenases. In NiFe-hydrogenases, CO and CN− are coordinated to the Fe in the NiFe dinuclear cluster of the active center. Though these diatomic ligands are biosynthesized enzymatically, detail mechanisms of their biosynthesis remain unclear. Here, we report the structural characterization of HypX responsible for CO biosynthesis to assemble the active site of NiFe hydrogenase. CoA is constitutionally bound in HypX. Structural characterization of HypX suggests that the formyl-group transfer will take place from N10-formyl-THF to CoA to form formyl-CoA in the N-terminal domain of HypX, followed by decarbonylation of formyl-CoA to produce CO in the C-terminal domain though the direct experimental results are not available yet. The conformation of CoA accommodated in the continuous cavity connecting the N- and C-terminal domains will interconvert between the extended and the folded conformations for HypX catalysis.

Metagenomics Reveals Bacterial and Archaeal Adaptation to Urban Land-Use: N Catabolism, Methanogenesis, and Nutrient Acquisition.

Urbanization results in the systemic conversion of land-use, driving habitat and biodiversity loss. The “urban convergence hypothesis” posits that urbanization represents a merging of habitat characteristics, in turn driving physiological and functional responses within the biotic community. To test this hypothesis, we sampled five cities (Baltimore, MD, United States; Helsinki and Lahti, Finland; Budapest, Hungary; Potchefstroom, South Africa) across four different biomes. Within each city, we sampled four land-use categories that represented a gradient of increasing disturbance and management (from least intervention to highest disturbance: reference, remnant, turf/lawn, and ruderal). Previously, we used amplicon sequencing that targeted bacteria/archaea (16S rRNA) and fungi (ITS) and reported convergence in the archaeal community. Here, we applied shotgun metagenomic sequencing and QPCR of functional genes to the same soil DNA extracts to test convergence in microbial function. Our results suggest that urban land-use drives changes in gene abundance related to both the soil N and C metabolism. Our updated analysis found taxonomic convergence in both the archaeal and bacterial community (16S amplicon data). Convergence of the archaea was driven by increased abundance of ammonia oxidizing archaea and genes for ammonia oxidation (QPCR and shotgun metagenomics). The proliferation of ammonia-oxidizers under turf and ruderal land-use likely also contributes to the previously documented convergence of soil mineral N pools. We also found a higher relative abundance of methanogens (amplicon sequencing), a higher relative abundance of gene sequences putatively identified as Ni-Fe hydrogenase and nickel uptake (shotgun metagenomics) under urban land-use; and a convergence of gene sequences putatively identified as contributing to the nickel transport function under urban turf sites. High levels of disturbance lead to a higher relative abundance of gene sequences putatively identified as multiple antibiotic resistance protein marA and multidrug efflux pump mexD, but did not lead to an overall convergence in antibiotic resistance gene sequences.

An Abrupt Change of Configuration of Surface Ligands Affects the H2 Production Efficiency of Mediator-Based CdS Nanorod/ Hydrogenase Assemblies

Assemblies of inorganic nanocrystals and natural enzymes are promising systems that combine both the unique and tunable photophysical properties of nanocrystals as well as the high catalytic efficiency and selectivity of bioreaction centers. The key challenge in design of these assemblies is to favor the electron/energy transfer (ET) between the abiotic and biotic materials while retarding the undesired charge recombination. The solution to this challenge relies on both rational design of materials with proper energetic and other physical properties, and, equally important if not more so, the surface condition of each component and the configuration of their assemblies. In this study, we investigate a recently developed nanorod and NiFe hydrogenase (H2ase) assemblies which utilize a redox-mediated approach to shuttle the electron transfer between the nanorods and the H2ase enzymes with an impressive H2 production quantum yield up to 77 %.1 We study the impact of alkyl chain lengths of a common type of capping ligand for nanocrystals, mercaptoalkylcarboxylate, on the H2 production quantum yield of the system and elaborate the ligand’s impact on the underlying ET transfer between NRs and the electron acceptors. We observed the existence of an abrupt decrease of the quantum yield for H2 production of the system when increasing the alkyl chain length of the ligands from n= 7 to 10 (35 % and 12 %, respectively), whereas only a minor performance decrease is observed when n is below 7 (35 % and 42 % for n=7 and 2, respectively). These results are further shown in good agreement with the sudden decrease of the yield of the reduced mediator, propyl-bridged 2-2’-bipyridinium (PDQ2+), during the steady-state photoreduction experiments, suggesting that generation efficiency of the redox equivalents control the overall efficiency of the current redox-mediated systems. Further transient spectroscopic measurements revealed that the intrinsic ET transfer rates from the NR to the mediator PDQ are all on the order of 108 s-1 regardless of the length of the capping ligands. Instead, the amount of the average surface attached PDQ2+ molecules decreases dramatically when increasing the length of n above 7, with a saturated surface coverage of σ=40 to σ =0.5 for n=2 and n= 7, respectively. These results cannot be explained by the commonly perceived ligand length dependent ET transfer by tunneling through a barrier: k = exp (- ) on the nanorod surface, with the distance dependent constants, , reported to between 0.3 to 0.8 Å-1.2,3 Instead, these results are rationalized by the accessibility of CdS surface to electron acceptors, probably due to the change of the ligand configuration from the disordered to ordered phases. These results characterize quantitively the efficiency limiting step in the mediator based CdS nanorod/Hydrogenases assemblies, demonstrate impact of configurational arrangements of ligands on NR surface on its ET behavior, and therefore are important for the design of biotic and abiotic systems for various applications. Acknowledgment Wenxing Yang acknowledges the financial support from the Swedish Research Council for an International Postdoc Fellowship. Reference (1) Chica, B.; Wu, C. H.; Liu, Y.; Adams, M. W. W.; Lian, T.; Dyer, R. B. Balancing Electron Transfer Rate and Driving Force for Efficient Photocatalytic Hydrogen Production in CdSe/CdS Nanorod-[NiFe] Hydrogenase Assemblies. Energy Environ. Sci. 2017, 10 (10), 2245–2255. (2) Tagliazucchi, M.; Tice, D. B.; Sweeney, C. M.; Morris-Cohen, A. J.; Weiss, E. A. Ligand-Controlled Rates of Photoinduced Electron Transfer in Hybrid CdSe Nanocrystal/Poly(Viologen) Films. ACS Nano 2011, 5 (12), 9907–9917. (3) Wilker, M. B.; Utterback, J. K.; Greene, S.; Brown, K. A.; Mulder, D. W.; King, P. W.; Dukovic, G. Role of Surface-Capping Ligands in Photoexcited Electron Transfer between CdS Nanorods and [FeFe] Hydrogenase and the Subsequent H 2 Generation. J. Phys. Chem. C 2018, 122 (1), 741–750. Figure 1

Shotgun proteomic analysis of nanoparticle-synthesizing Desulfovibrio alaskensis in response to platinum and palladium

Platinum and palladium are much sought-after metals of critical global importance in terms of abundance and availability. At the nano-scale these metals are of even higher value due to their catalytic abilities for industrial applications. Desulfovibrio alaskensis is able to capture ionic forms of both of these metals, reduce them and synthesize elemental nanoparticles. Despite this ability, very little is known about the biological pathways involved in the formation of these nanoparticles. Proteomic analysis of D. alaskensis in response to platinum and palladium has highlighted those proteins involved in both the reductive pathways and the wider stress-response system. A core set of 13 proteins was found in both treatments and consisted of proteins involved in metal transport and reduction. There were also seven proteins that were specific to either platinum or palladium. Overexpression of one of these platinum-specific genes, a NiFe hydrogenase small subunit (Dde_2137), resulted in the formation of larger nanoparticles. This study improves our understanding of the pathways involved in the metal resistance mechanism of Desulfovibrio and is informative regarding how we can tailor the bacterium for nanoparticle production, enhancing its application as a bioremediation tool and as a way to capture contaminant metals from the environment.