Augmenting the Performance of Hydrogenase for Aerobic Photocatalytic Hydrogen Evolution via Solvent Tuning.

This work showcases the performance of [NiFeSe] hydrogenase from Desulfomicrobium baculatum for solar-driven hydrogen generation in a variety of organic-based deep eutectic solvents. Despite its well-known sensitivity towards air and organic solvents, the hydrogenase shows remarkable performance under an aerobic atmosphere in these solvents when paired with a TiO2 photocatalyst. Tuning the water content further increases hydrogen evolution activity to a TOF of 60±3 s-1 and quantum yield to 2.3±0.4 % under aerobic conditions, compared to a TOF of 4 s-1 in a purely aqueous solvent. Contrary to common belief, this work therefore demonstrates that placing natural hydrogenases into non-natural environments can enhance their intrinsic activity beyond their natural performance, paving the way for full water splitting using hydrogenases.

A Pyrene-Triazacyclononane Anchor Affords High Operational Stability for CO2 RR by a CNT-Supported Histidine-Tagged CODH.

An original 1-acetato-4-(1-pyrenyl)-1,4,7-triazacyclononane (AcPyTACN) was synthesized for the immobilization of a His-tagged recombinant CODH from Rhodospirillum rubrum (RrCODH) on carbon-nanotube electrodes. The strong binding of the enzyme at the Ni-AcPyTACN complex affords a high current density of 4.9 mA cm-2 towards electroenzymatic CO2 reduction and a high stability of more than 6×106  TON when integrated on a gas-diffusion bioelectrode.

Hybrid Amyloid-Based Redox Hydrogel for Bioelectrocatalytic H2 Oxidation.

An artificial amyloid-based redox hydrogel was designed for mediating electron transfer between a [NiFeSe] hydrogenase and an electrode. Starting from a mutated prion-forming domain of fungal protein HET-s, a hybrid redox protein containing a single benzyl methyl viologen moiety was synthesized. This protein was able to self-assemble into structurally homogenous nanofibrils. Molecular modeling confirmed that the redox groups are aligned along the fibril axis and are tethered to its core by a long, flexible polypeptide chain that allows close encounters between the fibril-bound oxidized or reduced redox groups. Redox hydrogel films capable of immobilizing the hydrogenase under mild conditions at the surface of carbon electrodes were obtained by a simple pH jump. In this way, bioelectrodes for the electrocatalytic oxidation of H2 were fabricated that afforded catalytic current densities of up to 270 µA cm-2 , with an overpotential of 0.33 V, under quiescent conditions at 45 °C.

The carbon monoxide dehydrogenase accessory protein CooJ is a histidine-rich multidomain dimer containing an unexpected Ni(II)-binding site.

Activation of nickel enzymes requires specific accessory proteins organized in multiprotein complexes controlling metal transfer to the active site. Histidine-rich clusters are generally present in at least one of the metallochaperones involved in nickel delivery. The maturation of carbon monoxide dehydrogenase in the proteobacterium Rhodospirillum rubrum requires three accessory proteins, CooC, CooT, and CooJ, dedicated to nickel insertion into the active site, a distorted [NiFe3S4] cluster coordinated to an iron site. Previously, CooJ from R. rubrum (RrCooJ) has been described as a nickel chaperone with 16 histidines and 2 cysteines at its C terminus. Here, the X-ray structure of a truncated version of RrCooJ, combined with small-angle X-ray scattering data and a modeling study of the full-length protein, revealed a homodimer comprising a coiled coil with two independent and highly flexible His tails. Using isothermal calorimetry, we characterized several metal-binding sites (four per dimer) involving the His-rich motifs and having similar metal affinity (KD = 1.6 µm). Remarkably, biophysical approaches, site-directed mutagenesis, and X-ray crystallography uncovered an additional nickel-binding site at the dimer interface, which binds Ni(II) with an affinity of 380 nm Although RrCooJ was initially thought to be a unique protein, a proteome database search identified at least 46 bacterial CooJ homologs. These homologs all possess two spatially separated nickel-binding motifs: a variable C-terminal histidine tail and a strictly conserved H(W/F)X2HX3H motif, identified in this study, suggesting a dual function for CooJ both as a nickel chaperone and as a nickel storage protein.

A Selective Sulfide Oxidation Catalyzed by Heterogeneous Artificial Metalloenzymes Iron@NikA.

Performing a heterogeneous catalysis with proteins is still a challenge. Herein, we demonstrate the importance of cross-linked crystals for sulfoxide oxidation by an artificial enzyme. The biohybrid consists of the insertion of an iron complex into a NikA protein crystal. The heterogeneous catalysts displays a better efficiency-with higher reaction kinetics, a better stability and expand the substrate scope compared to its solution counterpart. Designing crystalline artificial enzymes represents a good alternative to soluble or supported enzymes for the future of synthetic biology.

A Solvent-Exposed Cysteine Forms a Peculiar NiII -Binding Site in the Metallochaperone CooT from Rhodospirillum rubrum.

In Rhodospirillum rubrum, the maturation of carbon monoxide dehydrogenase (CODH) requires three nickel chaperones, namely RrCooC, RrCooT and RrCooJ. Recently, the biophysical characterisation of the RrCooT homodimer and the X-ray structure of its apo form revealed the existence of a solvent-exposed NiII -binding site at the dimer interface, involving the strictly conserved Cys2. Here, a multifaceted approach that used NMR and X-ray absorption spectroscopies, complemented with structural bio-modelling methodologies, was used to characterise the binding mode of NiII in RrCooT. This study suggests that NiII adopts a square-planar geometry through a N2 S2 coordinating environment that comprises the two thiolate and amidate groups of both Cys2 residues at the dimer interface. The existence of a diamagnetic mononuclear NiII centre with bis-amidate/bis-thiolate ligands, coordinated by a single-cysteine motif, is unprecedented in biology and raises the question of its role in the activation of CODH at the molecular level.

Cross-Linked Artificial Enzyme Crystals as Heterogeneous Catalysts for Oxidation Reactions.

Designing systems that merge the advantages of heterogeneous catalysis, enzymology, and molecular catalysis represents the next major goal for sustainable chemistry. Cross-linked enzyme crystals display most of these essential assets (well-designed mesoporous support, protein selectivity, and molecular recognition of substrates). Nevertheless, a lack of reaction diversity, particularly in the field of oxidation, remains a constraint for their increased use in the field. Here, thanks to the design of cross-linked artificial nonheme iron oxygenase crystals, we filled this gap by developing biobased heterogeneous catalysts capable of oxidizing carbon-carbon double bonds. First, reductive O2 activation induces selective oxidative cleavage, revealing the indestructible character of the solid catalyst (at least 30 000 turnover numbers without any loss of activity). Second, the use of 2-electron oxidants allows selective and high-efficiency hydroxychlorination with thousands of turnover numbers. This new technology by far outperforms catalysis using the inorganic complexes alone, or even the artificial enzymes in solution. The combination of easy catalyst synthesis, the improvement of "omic" technologies, and automation of protein crystallization makes this strategy a real opportunity for the future of (bio)catalysis.

Evolution of Helicobacter: Acquisition by Gastric Species of Two Histidine-Rich Proteins Essential for Colonization.

Metal acquisition and intracellular trafficking are crucial for all cells and metal ions have been recognized as virulence determinants in bacterial pathogens. Virulence of the human gastric pathogen Helicobacter pylori is dependent on nickel, cofactor of two enzymes essential for in vivo colonization, urease and [NiFe] hydrogenase. We found that two small paralogous nickel-binding proteins with high content in Histidine (Hpn and Hpn-2) play a central role in maintaining non-toxic intracellular nickel content and in controlling its intracellular trafficking. Measurements of metal resistance, intracellular nickel contents, urease activities and interactomic analysis were performed. We observed that Hpn acts as a nickel-sequestration protein, while Hpn-2 is not. In vivo, Hpn and Hpn-2 form homo-multimers, interact with each other, Hpn interacts with the UreA urease subunit while Hpn and Hpn-2 interact with the HypAB hydrogenase maturation proteins. In addition, Hpn-2 is directly or indirectly restricting urease activity while Hpn is required for full urease activation. Based on these data, we present a model where Hpn and Hpn-2 participate in a common pathway of controlled nickel transfer to urease. Using bioinformatics and top-down proteomics to identify the predicted proteins, we established that Hpn-2 is only expressed by H. pylori and its closely related species Helicobacter acinonychis. Hpn was detected in every gastric Helicobacter species tested and is absent from the enterohepatic Helicobacter species. Our phylogenomic analysis revealed that Hpn acquisition was concomitant with the specialization of Helicobacter to colonization of the gastric environment and the duplication at the origin of hpn-2 occurred in the common ancestor of H. pylori and H. acinonychis. Finally, Hpn and Hpn-2 were found to be required for colonization of the mouse model by H. pylori. Our data show that during evolution of the Helicobacter genus, acquisition of Hpn and Hpn-2 by gastric Helicobacter species constituted a decisive evolutionary event to allow Helicobacter to colonize the hostile gastric environment, in which no other bacteria persistently thrives. This acquisition was key for the emergence of one of the most successful bacterial pathogens, H. pylori.

Relationship between Ni(II) and Zn(II) coordination and nucleotide binding by the Helicobacter pylori [NiFe]-hydrogenase and urease maturation factor HypB.

The pathogen Helicobacter pylori requires two nickel-containing enzymes, urease and [NiFe]-hydrogenase, for efficient colonization of the human gastric mucosa. These enzymes possess complex metallocenters that are assembled by teams of proteins in multistep pathways. One essential accessory protein is the GTPase HypB, which is required for Ni(II) delivery to [NiFe]-hydrogenase and participates in urease maturation. Ni(II) or Zn(II) binding to a site embedded in the GTPase domain of HypB modulates the enzymatic activity, suggesting a mechanism of regulation. In this study, biochemical and structural analyses of H. pylori HypB (HpHypB) revealed an intricate link between nucleotide and metal binding. HpHypB nickel coordination, stoichiometry, and affinity were modulated by GTP and GDP, an effect not observed for zinc, and biochemical evidence suggests that His-107 coordination to nickel toggles on and off in a nucleotide-dependent manner. These results are consistent with the crystal structure of HpHypB loaded with Ni(II), GDP, and Pi, which reveals a nickel site distinct from that of zinc-loaded Methanocaldococcus jannaschii HypB as well as subtle changes to the protein structure. Furthermore, Cys-142, a metal ligand from the Switch II GTPase motif, was identified as a key component of the signal transduction between metal binding and the enzymatic activity. Finally, potassium accelerated the enzymatic activity of HpHypB but had no effect on the other biochemical properties of the protein. Altogether, this molecular level information about HpHypB provides insight into its cellular function and illuminates a possible mechanism of metal ion discrimination.

Structure-function relationships of anaerobic gas-processing metalloenzymes.

Reactions involving H(2), N(2), CO, CO(2) and CH(4) are likely to have been central to the origin of life. This is indicated by the active-site structures of the enzymes involved, which are often reminiscent of minerals. Through the combined efforts of protein crystallography, various types of spectroscopy, theoretical calculations and model chemistry, it has been possible to put forward plausible mechanisms for gas-based metabolism by extant microorganisms. Although the reactions are based on metal centres, the protein matrix regulates reactivity and substrate and product trafficking through internal pathways, specific ligation and dielectricity.

Cobaloxime-based artificial hydrogenases.

Cobaloximes are popular H2 evolution molecular catalysts but have so far mainly been studied in nonaqueous conditions. We show here that they are also valuable for the design of artificial hydrogenases for application in neutral aqueous solutions and report on the preparation of two well-defined biohybrid species via the binding of two cobaloxime moieties, {Co(dmgH)2} and {Co(dmgBF2)2} (dmgH2 = dimethylglyoxime), to apo Sperm-whale myoglobin (SwMb). All spectroscopic data confirm that the cobaloxime moieties are inserted within the binding pocket of the SwMb protein and are coordinated to a histidine residue in the axial position of the cobalt complex, resulting in thermodynamically stable complexes. Quantum chemical/molecular mechanical docking calculations indicated a coordination preference for His93 over the other histidine residue (His64) present in the vicinity. Interestingly, the redox activity of the cobalt centers is retained in both biohybrids, which provides them with the catalytic activity for H2 evolution in near-neutral aqueous conditions.

Photocatalytic hydrogen production using polymeric carbon nitride with a hydrogenase and a bioinspired synthetic Ni catalyst.

Solar-light-driven H2 production in water with a [NiFeSe]-hydrogenase (H2ase) and a bioinspired synthetic nickel catalyst (NiP) in combination with a heptazine carbon nitride polymer, melon (CN(x)), is reported. The semibiological and purely synthetic systems show catalytic activity during solar light irradiation with turnover numbers (TONs) of more than 50,000 mol H2(mol H2ase)(-1) and approximately 155 mol H2 (mol NiP)(-1) in redox-mediator-free aqueous solution at pH 6 and 4.5, respectively. Both systems maintained a reduced photoactivity under UV-free solar light irradiation (λ>420 nm).

The structure of the periplasmic nickel-binding protein NikA provides insights for artificial metalloenzyme design.

Understanding the interaction of a protein with a relevant ligand is crucial for the design of an artificial metalloenzyme. Our own interest is focused on the synthesis of artificial monooxygenases. In an initial effort, we have used the periplasmic nickel-binding protein NikA from Escherichia coli and iron complexes in which N(2)Py(2) ligands (where Py is pyridine) have been varied in terms of charge, aromaticity, and size. Six "NikA/iron complex" hybrids have been characterized by X-ray crystallography, and their interactions and solution properties have been studied. The hybrids are stable as indicated by their K (d) values, which are all in the micromolar range. The X-ray structures show that the ligands interact with NikA through salt bridges with arginine residues and π-stacking with a tryptophan residue. We have further characterized these interactions using quantum mechanical calculations and determined that weak CH/π hydrogen bonds finely modulate the stability differences between hybrids. We emphasize the important role of the tryptophan residues. Thus, our study aims at the complete characterization of the factors that condition the interaction of an artificial ligand and a protein and their implications for catalysis. Besides its potential usefulness in the synthesis of artificial monooxygenases, our approach should be generally applicable in the field of artificial metalloenzymes.

Relating diffusion along the substrate tunnel and oxygen sensitivity in hydrogenase.

In hydrogenases and many other redox enzymes, the buried active site is connected to the solvent by a molecular channel whose structure may determine the enzyme's selectivity with respect to substrate and inhibitors. The role of these channels has been addressed using crystallography and molecular dynamics, but kinetic data are scarce. Using protein film voltammetry, we determined and then compared the rates of inhibition by CO and O2 in ten NiFe hydrogenase mutants and two FeFe hydrogenases. We found that the rate of inhibition by CO is a good proxy of the rate of diffusion of O2 toward the active site. Modifying amino acids whose side chains point inside the tunnel can slow this rate by orders of magnitude. We quantitatively define the relations between diffusion, the Michaelis constant for H2 and rates of inhibition, and we demonstrate that certain enzymes are slowly inactivated by O2 because access to the active site is slow.

Proteomic analysis of Rhodospirillum rubrum after carbon monoxide exposure reveals an important effect on metallic cofactor biosynthesis.

Some carboxydotrophs like Rhodospirillum rubrum are able to grow with CO as their sole source of energy using a Carbone monoxide dehydrogenase (CODH) and an Energy conserving hydrogenase (ECH) to perform anaerobically the so called water-gas shift reaction (WGSR) (CO + H2O → CO2 + H2). Several studies have focused at the biochemical and biophysical level on this enzymatic system and a few OMICS studies on CO metabolism. Knowing that CO is toxic in particular due to its binding to heme iron atoms, and is even considered as a potential antibacterial agent, we decided to use a proteomic approach in order to analyze R. rubrum adaptation in term of metabolism and management of the toxic effect. In particular, this study allowed highlighting a set of proteins likely implicated in ECH maturation, and important perturbations in term of cofactor biosynthesis, especially metallic cofactors. This shows that even this CO tolerant microorganism cannot avoid completely CO toxic effects associated with its interaction with metallic ions. SIGNIFICANCE: This proteomic study highlights the fact that even in a microorganism able to handle carbon monoxide and in some way detoxifying it via the intrinsic action of the carbon monoxide dehydrogenase (CODH), CO has important effects on metal homeostasis, metal cofactors and metalloproteins. These effects are direct or indirect via transcription regulation, and amplified by the high interdependency of cofactors biosynthesis.

Formaldehyde--a rapid and reversible inhibitor of hydrogen production by [FeFe]-hydrogenases.

Dihydrogen (H(2)) production by [FeFe]-hydrogenases is strongly inhibited by formaldehyde (methanal) in a reaction that is rapid, reversible, and specific to this type of hydrogenase. This discovery, using three [FeFe]-hydrogenases that are homologous about the active site but otherwise structurally distinct, was made by protein film electrochemistry, which measures the activity (as electrical current) of enzymes immobilized on an electrode; importantly, the inhibitor can be removed after addition. Formaldehyde causes rapid loss of proton reduction activity which is restored when the solution is exchanged. Inhibition is confirmed by conventional solution assays. The effect depends strongly on the direction of catalysis: inhibition of H(2) oxidation is much weaker than for H(2) production, and formaldehyde also protects against CO and O(2) inactivation. By contrast, inhibition of [NiFe]-hydrogenases is weak. The results strongly suggest that formaldehyde binds at, or close to, the active site of [FeFe]-hydrogenases at a site unique to this class of enzyme--highly conserved lysine and cysteine residues, the bridgehead atom of the dithiolate ligand, or the reduced Fe(d) that is the focal center of catalysis.

Revisiting the CooJ family, a potential chaperone for nickel delivery to [NiFe]­carbon monoxide dehydrogenase.

Nickel insertion into nickel-dependent carbon monoxide dehydrogenase (CODH) represents a key step in the enzyme activation. This is the last step of the biosynthesis of the active site, which contains an atypical heteronuclear NiFe4S4 cluster known as the C-cluster. The enzyme maturation is performed by three accessory proteins, namely CooC, CooT and CooJ. Among them, CooJ from Rhodospirillum rubrum is a histidine-rich protein containing two distinct and spatially separated Ni(II)-binding sites: a N-terminal high affinity site (HAS) and a histidine tail at the C-terminus. In 46 CooJ homologues, the HAS motif was found to be strictly conserved with a H(W/F)XXHXXXH sequence. Here, a proteome database search identified at least 150 CooJ homologues and revealed distinct motifs for HAS, featuring 2, 3 or 4 histidines. The purification and biophysical characterization of three representative members of this protein family showed that they are all homodimers able to bind Ni(II) ions via one or two independent binding sites. Initially thought to be present only in R. rubrum, this study strongly suggests that CooJ could play a significant role in CODH maturation or in nickel homeostasis.

The Helicobacter pylori GroES cochaperonin HspA functions as a specialized nickel chaperone and sequestration protein through its unique C-terminal extension.

The transition metal nickel plays a central role in the human gastric pathogen Helicobacter pylori because it is required for two enzymes indispensable for colonization, the nickel metalloenzyme urease and [NiFe] hydrogenase. To sustain nickel availability for these metalloenzymes while providing protection from the metal's harmful effects, H. pylori is equipped with several specific nickel-binding proteins. Among these, H. pylori possesses a particular chaperone, HspA, that is a homolog of the highly conserved and essential bacterial heat shock protein GroES. HspA contains a unique His-rich C-terminal extension and was demonstrated to bind nickel in vitro. To investigate the function of this extension in H. pylori, we constructed mutants carrying either a complete deletion or point mutations in critical residues of this domain. All mutants presented a decreased intracellular nickel content measured by inductively coupled plasma mass spectrometry (ICP-MS) and reduced nickel tolerance. While urease activity was unaffected in the mutants, [NiFe] hydrogenase activity was significantly diminished when the C-terminal extension of HspA was mutated. We conclude that H. pylori HspA is involved in intracellular nickel sequestration and detoxification and plays a novel role as a specialized nickel chaperone involved in nickel-dependent maturation of hydrogenase.

Structure, function, and biosynthesis of nickel-dependent enzymes.

Nickel enzymes, present in archaea, bacteria, plants, and primitive eukaryotes are divided into redox and nonredox enzymes and play key functions in diverse metabolic processes, such as energy metabolism and virulence. They catalyze various reactions by using active sites of diverse complexities, such as mononuclear nickel in Ni-superoxide dismutase, glyoxylase I and acireductone dioxygenase, dinuclear nickel in urease, heteronuclear metalloclusters in [NiFe]-carbon monoxide dehydrogenase, acetyl-CoA decarbonylase/synthase and [NiFe]-hydrogenase, and even more complex cofactors in methyl-CoM reductase and lactate racemase. The presence of metalloenzymes in a cell necessitates a tight regulation of metal homeostasis, in order to maintain the appropriate intracellular concentration of nickel while avoiding its toxicity. As well, the biosynthesis and insertion of nickel active sites often require specific and elaborated maturation pathways, allowing the correct metal to be delivered and incorporated into the target enzyme. In this review, the phylogenetic distribution of nickel enzymes will be briefly described. Their tridimensional structures as well as the complexity of their active sites will be discussed. In view of the latest findings on these enzymes, a special focus will be put on the biosynthesis of their active sites and nickel activation of apo-enzymes.

Visible light-driven H(2) production by hydrogenases attached to dye-sensitized TiO(2) nanoparticles.

A study of hybrid, enzyme-modified nanoparticles able to produce H(2) using visible light as the energy source has been carried out to establish per-site performance standards for H(2) production catalysts able to operate under ambient conditions. The [NiFeSe]-hydrogenase from Desulfomicrobium baculatum (Db [NiFeSe]-H) is identified as a particularly proficient catalyst. The optimized system consisting of Db [NiFeSe]-H attached to Ru dye-sensitized TiO(2), with triethanolamine as a sacrificial electron donor, produces H(2) at a turnover frequency of approximately 50 (mol H(2)) s(-1) (mol total hydrogenase)(-1) at pH 7 and 25 degrees C, even under the typical solar irradiation of a northern European sky. The system shows high electrocatalytic stability not only under anaerobic conditions but also after prolonged exposure to air, thus making it sufficiently robust for benchtop applications.