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The functional diversification of O 2 -tolerant [NiFe]-hydrogenases using orthogonal translation systems (OTSs) offers a promising strategy for developing advanced biocatalysts and biohybrid energy platforms. However, plasmid-based OTSs frequently impose metabolic burdens and suffer from plasmid instability during fermentation, particularly when co-produced with complex metalloenzymes. To overcome these bioprocess limitations, we employed CRISPR/Cas9-mediated genome editing to integrate a psychrophilic pyrrolysyl-tRNA synthetase/tRNA pair into the Escherichia coli BL21 genome. The resulting strain provided a plasmid-free orthogonal translation background that supported amber suppression-mediated expression of the regulatory [NiFe]-hydrogenase (RH) of Cupriavidus necator . Using this genomically integrated OTS, we achieved the production of a full-length, catalytically active RH variant. Our results demonstrate that chromosomal OTS is compatible with the efficient production and maturation of complex metalloenzymes. The present work lays the groundwork for the bio-orthogonal engineering of hydrogenases and related hybrid biocatalysts.

Carbon fixation via the Calvin cycle is constrained by the side activity of Rubisco with dioxygen, generating 2-phosphoglycolate. The metabolic recycling of phosphoglycolate was extensively studied in photoautotrophic organisms, including plants, algae, and cyanobacteria, where it is referred to as photorespiration. While receiving little attention so far, aerobic chemolithoautotrophic bacteria that operate the Calvin cycle independent of light must also recycle phosphoglycolate. As the term photorespiration is inappropriate for describing phosphoglycolate recycling in these nonphotosynthetic autotrophs, we suggest the more general term “phosphoglycolate salvage.” Here, we study phosphoglycolate salvage in the model chemolithoautotroph Cupriavidus necator H16 (Ralstonia eutropha H16) by characterizing the proxy process of glycolate metabolism, performing comparative transcriptomics of autotrophic growth under low and high CO₂ concentrations, and testing autotrophic growth phenotypes of gene deletion strains at ambient CO₂. We find that the canonical plant-like C₂ cycle does not operate in this bacterium, and instead, the bacterial-like glycerate pathway is the main route for phosphoglycolate salvage. Upon disruption of the glycerate pathway, we find that an oxidative pathway, which we term the malate cycle, supports phosphoglycolate salvage. In this cycle, glyoxylate is condensed with acetyl coenzyme A (acetyl-CoA) to give malate, which undergoes two oxidative decarboxylation steps to regenerate acetyl-CoA. When both pathways are disrupted, autotrophic growth is abolished at ambient CO₂. We present bioinformatic data suggesting that the malate cycle may support phosphoglycolate salvage in diverse chemolithoautotrophic bacteria. This study thus demonstrates a so far unknown phosphoglycolate salvage pathway, highlighting important diversity in microbial carbon fixation metabolism.

Hydrogenases are nature’s key catalysts involved in both microbial consumption and production of molecular hydrogen. H₂ exhibits a strongly bonded, almost inert electron pair and requires transition metals for activation. Consequently, all hydrogenases are metalloenzymes that contain at least one iron atom in the catalytic center. For appropriate interaction with H₂, the iron moiety demands for a sophisticated coordination environment that cannot be provided just by standard amino acids. This dilemma has been overcome by the introduction of unprecedented chemistry—that is, by ligating the iron with carbon monoxide (CO) and cyanide (or equivalent) groups. These ligands are both unprecedented in microbial metabolism and, in their free form, highly toxic to living organisms. Therefore, the formation of the diatomic ligands relies on dedicated biosynthesis pathways. So far, biosynthesis of the CO ligand in [NiFe]-hydrogenases was unknown. Here we show that the aerobic H₂ oxidizer Ralstonia eutropha, which produces active [NiFe]-hydrogenases in the presence of O₂, employs the auxiliary protein HypX (hydrogenase pleiotropic maturation X) for CO ligand formation. Using genetic engineering and isotope labeling experiments in combination with infrared spectroscopic investigations, we demonstrate that the α-carbon of glycine ends up in the CO ligand of [NiFe]-hydrogenase. The α-carbon of glycine is a building block of the central one-carbon metabolism intermediate, N 10-formyl-tetrahydrofolate (N 10-CHO-THF). Evidence is presented that the multidomain protein, HypX, converts the formyl group of N 10-CHO-THF into water and CO, thereby providing the carbonyl ligand for hydrogenase. This study contributes insights into microbial biosynthesis of metal carbonyls involving toxic intermediates.

[NiFe] hydrogenases catalyze the reversible splitting of H₂ into protons and electrons at a deeply buried active site. The catalytic center can be accessed by gas molecules through a hydrophobic tunnel network. While most [NiFe] hydrogenases are inactivated by O₂, a small subgroup, including the membrane-bound [NiFe] hydrogenase (MBH) of Ralstonia eutropha, is able to overcome aerobic inactivation by catalytic reduction of O₂ to water. This O₂ tolerance relies on a special [4Fe3S] cluster that is capable of releasing two electrons upon O₂ attack. Here, the O₂ accessibility of the MBH gas tunnel network has been probed experimentally using a “soak-and-freeze” derivatization method, accompanied by protein X-ray crystallography and computational studies. This combined approach revealed several sites of O₂ molecules within a hydrophobic tunnel network leading, via two tunnel entrances, to the catalytic center of MBH. The corresponding site occupancies were related to the O₂ concentrations used for MBH crystal derivatization. The examination of the O₂-derivatized data furthermore uncovered two unexpected structural alterations at the [4Fe3S] cluster, which might be related to the O₂ tolerance of the enzyme.

[NiFe]‐hydrogenases catalyze the reversible conversion of molecular hydrogen into protons end electrons. This reaction takes place at a NiFe(CN)2(CO) cofactor located in the large subunit of the bipartite hydrogenase module. The corresponding apo‐protein carries usually a C‐terminal extension that is cleaved off by a specific endopeptidase as soon as the cofactor insertion has been accomplished by the maturation machinery. This process triggers complex formation with the small, electron‐transferring subunit of the hydrogenase module, revealing catalytically active enzyme. The role of the C‐terminal extension in cofactor insertion, however, remains elusive. We have addressed this problem by using genetic engineering to remove the entire C‐terminal extension from the apo‐form of the large subunit of the membrane‐bound [NiFe]‐hydrogenase (MBH) from Ralstonia eutropha. Unexpectedly, the MBH holoenzyme derived from this precleaved large subunit was targeted to the cytoplasmic membrane, conferred H2‐dependent growth of the host strain, and the purified protein showed exactly the same catalytic activity as native MBH. The only difference was a reduced hydrogenase content in the cytoplasmic membrane. These results suggest that in the case of the R. eutropha MBH, the C‐terminal extension is dispensable for cofactor insertion and seems to function only as a maturation facilitator. The apo‐forms of [NiFe]‐hydrogenase large subunits are usually synthesized with a C‐terminal peptide extension that is proteolytically cleaved off upon incorporation of the catalytic metal center. Although to a limited amount, an artificially precleaved large subunit, which was deleted for the C‐terminal extension by genetic engineering, still received the active site components delivered by the dedicated maturation machinery. This suggests that the C‐terminal extension optimizes metal center incorporation, but is not essential for the formation of catalytically active [NiFe]‐hydrogenase.

The functional diversification of O2-tolerant [NiFe]-hydrogenases using orthogonal translation systems (OTSs) offers a promising strategy for developing advanced biocatalysts and biohybrid energy platforms. However, plasmid-based OTSs frequently impose metabolic burdens and suffer from plasmid instability during fermentation, particularly when co-produced with complex metalloenzymes. To overcome these bioprocess limitations, we employed CRISPR/Cas9-mediated genome editing to integrate a psychrophilic pyrrolysyl-tRNA synthetase/tRNA pair into the Escherichia coli BL21 genome. The resulting strain provided a plasmid-free orthogonal translation background that supported amber suppression-mediated expression of the regulatory [NiFe]-hydrogenase (RH) of Cupriavidus necator. Using this genomically integrated OTS, we achieved the production of a full-length, catalytically active RH variant. Our results demonstrate that chromosomal OTS is compatible with the efficient production and maturation of complex metalloenzymes. The present work lays the groundwork for the bio-orthogonal engineering of hydrogenases and related hybrid biocatalysts.The functional diversification of O2-tolerant [NiFe]-hydrogenases using orthogonal translation systems (OTSs) offers a promising strategy for developing advanced biocatalysts and biohybrid energy platforms. However, plasmid-based OTSs frequently impose metabolic burdens and suffer from plasmid instability during fermentation, particularly when co-produced with complex metalloenzymes. To overcome these bioprocess limitations, we employed CRISPR/Cas9-mediated genome editing to integrate a psychrophilic pyrrolysyl-tRNA synthetase/tRNA pair into the Escherichia coli BL21 genome. The resulting strain provided a plasmid-free orthogonal translation background that supported amber suppression-mediated expression of the regulatory [NiFe]-hydrogenase (RH) of Cupriavidus necator. Using this genomically integrated OTS, we achieved the production of a full-length, catalytically active RH variant. Our results demonstrate that chromosomal OTS is compatible with the efficient production and maturation of complex metalloenzymes. The present work lays the groundwork for the bio-orthogonal engineering of hydrogenases and related hybrid biocatalysts.

Oxygen-tolerant hydrogenases Hydrogenases are metalloprotein enzymes that catalyse the reversible oxidation of dihydrogen to protons and electrons, a critical pathway in anaerobic metabolism. This reaction is of particular interest for hydrogen-based applications, in fuel cells for instance, but many applications are hindered by the high oxygen sensitivity that is an intrinsic feature of most hydrogenases. Two groups report the structures of oxygen-tolerant hydrogenases, one from the soil bacterium Ralstonia eutropha and the other from the marine bacterium Hydrogenovibrio marinus . The structures shed light on how redox-sensitive active-site intermediates are protected from destruction. Both enzymes feature a novel iron-sulphur centre at the active site, coordinated by a group of cysteine residues. Hydrogenases are abundant enzymes that catalyse the reversible interconversion of H 2 into protons and electrons at high rates 1 . Those hydrogenases maintaining their activity in the presence of O 2 are considered to be central to H 2 -based technologies, such as enzymatic fuel cells and for light-driven H 2 production 2 . Despite comprehensive genetic, biochemical, electrochemical and spectroscopic investigations 3 , 4 , 5 , 6 , 7 , 8 , the molecular background allowing a structural interpretation of how the catalytic centre is protected from irreversible inactivation by O 2 has remained unclear. Here we present the crystal structure of an O 2 -tolerant [NiFe]-hydrogenase from the aerobic H 2 oxidizer Ralstonia eutropha H16 at 1.5 Å resolution. The heterodimeric enzyme consists of a large subunit harbouring the catalytic centre in the H 2 -reduced state and a small subunit containing an electron relay consisting of three different iron-sulphur clusters. The cluster proximal to the active site displays an unprecedented [4Fe-3S] structure and is coordinated by six cysteines. According to the current model, this cofactor operates as an electronic switch depending on the nature of the gas molecule approaching the active site. It serves as an electron acceptor in the course of H 2 oxidation and as an electron-delivering device upon O 2 attack at the active site. This dual function is supported by the capability of the novel iron-sulphur cluster to adopt three redox states at physiological redox potentials 7 , 8 , 9 . The second structural feature is a network of extended water cavities that may act as a channel facilitating the removal of water produced at the [NiFe] active site. These discoveries will have an impact on the design of biological and chemical H 2 -converting catalysts that are capable of cycling H 2 in air.

[NiFe]-hydrogenases are biotechnologically relevant enzymes catalyzing the reversible splitting of H 2 into 2 e − and 2H + under ambient conditions. Catalysis takes place at the heterobimetallic NiFe(CN) 2 (CO) center, whose multistep biosynthesis involves careful handling of two transition metals as well as potentially harmful CO and CN − molecules. Here, we investigated the sequential assembly of the [NiFe] cofactor, previously based on primarily indirect evidence, using four different purified maturation intermediates of the catalytic subunit, HoxG, of the O 2 -tolerant membrane-bound hydrogenase from Cupriavidus necator . These included the cofactor-free apo-HoxG, a nickel-free version carrying only the Fe(CN) 2 (CO) fragment, a precursor that contained all cofactor components but remained redox inactive and the fully mature HoxG. Through biochemical analyses combined with comprehensive spectroscopic investigation using infrared, electronic paramagnetic resonance, Mössbauer, X-ray absorption and nuclear resonance vibrational spectroscopies, we obtained detailed insight into the sophisticated maturation process of [NiFe]-hydrogenase. The multistep incorporation process of the catalytic NiFe(CN) 2 (CO) cofactor into [NiFe]-hydrogenase was deciphered by isolating key maturation intermediates, which were characterized by biochemical and a variety of spectroscopic techniques.

Tracking the route of substrates, intermediates, and inhibitors in proteins is fundamental in understanding their specific function. However, following the route of gases like molecular oxygen within enzymes has always been challenging. In protein X-ray crystallography, gases can be mimicked using krypton or xenon (with a higher electron count); however, these have a different physical behavior compared to true substrates/inhibitors. In our crystal structure of the O 2 -tolerant membrane-bound [NiFe] hydrogenase (MBH) from Ralstonia eutropha , we were able to show the direct path of molecular oxygen between the enzyme exterior and the active site with the “soak-and-freeze” derivatization method. This technique might be useful to detect O 2 traveling routes in many other enzymes. [NiFe] hydrogenases catalyze the reversible splitting of H 2 into protons and electrons at a deeply buried active site. The catalytic center can be accessed by gas molecules through a hydrophobic tunnel network. While most [NiFe] hydrogenases are inactivated by O 2 , a small subgroup, including the membrane-bound [NiFe] hydrogenase (MBH) of Ralstonia eutropha , is able to overcome aerobic inactivation by catalytic reduction of O 2 to water. This O 2 tolerance relies on a special [4Fe3S] cluster that is capable of releasing two electrons upon O 2 attack. Here, the O 2 accessibility of the MBH gas tunnel network has been probed experimentally using a “soak-and-freeze” derivatization method, accompanied by protein X-ray crystallography and computational studies. This combined approach revealed several sites of O 2 molecules within a hydrophobic tunnel network leading, via two tunnel entrances, to the catalytic center of MBH. The corresponding site occupancies were related to the O 2 concentrations used for MBH crystal derivatization. The examination of the O 2 -derivatized data furthermore uncovered two unexpected structural alterations at the [4Fe3S] cluster, which might be related to the O 2 tolerance of the enzyme.

Activation of dihydrogen is by far not a trivial catalytic reaction. Microbes have evolved sophisticated hydrogenases with complex transition metal centers to get access to H 2 . A recurring feature of these centers is the presence of iron atoms equipped with carbon monoxide ligands. In case of [NiFe]-hydrogenases, which contain a NiFe(CN) 2 CO catalytic center, biosynthesis of the toxic CO ligand remained elusive. We show that [NiFe]-hydrogenases that are catalytically active in the presence of dioxygen use a dedicated maturase for CO ligand synthesis under aerobic conditions. CO is derived from the most oxidized intermediate of the central one-carbon metabolism, formyl-tetrahydrofolate. This discovery contributes a so far unknown reaction to the one-carbon metabolism and opens perspectives for chemical and of bioinspired catalysis. Hydrogenases are nature’s key catalysts involved in both microbial consumption and production of molecular hydrogen. H 2 exhibits a strongly bonded, almost inert electron pair and requires transition metals for activation. Consequently, all hydrogenases are metalloenzymes that contain at least one iron atom in the catalytic center. For appropriate interaction with H 2 , the iron moiety demands for a sophisticated coordination environment that cannot be provided just by standard amino acids. This dilemma has been overcome by the introduction of unprecedented chemistry—that is, by ligating the iron with carbon monoxide (CO) and cyanide (or equivalent) groups. These ligands are both unprecedented in microbial metabolism and, in their free form, highly toxic to living organisms. Therefore, the formation of the diatomic ligands relies on dedicated biosynthesis pathways. So far, biosynthesis of the CO ligand in [NiFe]-hydrogenases was unknown. Here we show that the aerobic H 2 oxidizer Ralstonia eutropha , which produces active [NiFe]-hydrogenases in the presence of O 2 , employs the auxiliary protein HypX (hydrogenase pleiotropic maturation X) for CO ligand formation. Using genetic engineering and isotope labeling experiments in combination with infrared spectroscopic investigations, we demonstrate that the α-carbon of glycine ends up in the CO ligand of [NiFe]-hydrogenase. The α-carbon of glycine is a building block of the central one-carbon metabolism intermediate, N 10 -formyl-tetrahydrofolate ( N 10 -CHO-THF). Evidence is presented that the multidomain protein, HypX, converts the formyl group of N 10 -CHO-THF into water and CO, thereby providing the carbonyl ligand for hydrogenase. This study contributes insights into microbial biosynthesis of metal carbonyls involving toxic intermediates.