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Diverse aerobic bacteria use atmospheric H 2 as an energy source for growth and survival 1 . This globally significant process regulates the composition of the atmosphere, enhances soil biodiversity and drives primary production in extreme environments 2 , 3 . Atmospheric H 2 oxidation is attributed to uncharacterized members of the [NiFe] hydrogenase superfamily 4 , 5 . However, it remains unresolved how these enzymes overcome the extraordinary catalytic challenge of oxidizing picomolar levels of H 2 amid ambient levels of the catalytic poison O 2 and how the derived electrons are transferred to the respiratory chain 1 . Here we determined the cryo-electron microscopy structure of the Mycobacterium smegmatis hydrogenase Huc and investigated its mechanism. Huc is a highly efficient oxygen-insensitive enzyme that couples oxidation of atmospheric H 2 to the hydrogenation of the respiratory electron carrier menaquinone. Huc uses narrow hydrophobic gas channels to selectively bind atmospheric H 2 at the expense of O 2 , and 3 [3Fe–4S] clusters modulate the properties of the enzyme so that atmospheric H 2 oxidation is energetically feasible. The Huc catalytic subunits form an octameric 833 kDa complex around a membrane-associated stalk, which transports and reduces menaquinone 94 Å from the membrane. These findings provide a mechanistic basis for the biogeochemically and ecologically important process of atmospheric H 2 oxidation, uncover a mode of energy coupling dependent on long-range quinone transport, and pave the way for the development of catalysts that oxidize H 2 in ambient air. Structural and biochemical studies of the Mycobacterium smegmatis hydrogenase Huc provides insights into how [NiFe] hydrogenases oxidize trace amounts of atmospheric hydrogen and transfer the electrons liberated via quinone transport.

Recent physiological and ecological studies have challenged the long-held belief that microbial metabolism of molecular hydrogen (H 2 ) is a niche process. To gain a broader insight into the importance of microbial H 2 metabolism, we comprehensively surveyed the genomic and metagenomic distribution of hydrogenases, the reversible enzymes that catalyse the oxidation and evolution of H 2 . The protein sequences of 3286 non-redundant putative hydrogenases were curated from publicly available databases. These metalloenzymes were classified into multiple groups based on (1) amino acid sequence phylogeny, (2) metal-binding motifs, (3) predicted genetic organisation and (4) reported biochemical characteristics. Four groups (22 subgroups) of [NiFe]-hydrogenase, three groups (6 subtypes) of [FeFe]-hydrogenases and a small group of [Fe]-hydrogenases were identified. We predict that this hydrogenase diversity supports H 2 -based respiration, fermentation and carbon fixation processes in both oxic and anoxic environments, in addition to various H 2 -sensing, electron-bifurcation and energy-conversion mechanisms. Hydrogenase-encoding genes were identified in 51 bacterial and archaeal phyla, suggesting strong pressure for both vertical and lateral acquisition. Furthermore, hydrogenase genes could be recovered from diverse terrestrial, aquatic and host-associated metagenomes in varying proportions, indicating a broad ecological distribution and utilisation. Oxygen content ( p O 2 ) appears to be a central factor driving the phylum- and ecosystem-level distribution of these genes. In addition to compounding evidence that H 2 was the first electron donor for life, our analysis suggests that the great diversification of hydrogenases has enabled H 2 metabolism to sustain the growth or survival of microorganisms in a wide range of ecosystems to the present day. This work also provides a comprehensive expanded system for classifying hydrogenases and identifies new prospects for investigating H 2 metabolism.

Green algae such as Chlamydomonas reinhardtii synthesize an [FeFe] hydrogenase that is highly active in hydrogen evolution. However, the extreme sensitivity of [FeFe] hydrogenases to oxygen presents a major challenge for exploiting these organisms to achieve sustainable photosynthetic hydrogen production. In this study, the mechanism of oxygen inactivation of the [FeFe] hydrogenase CrHydAI from G reinhardtii has been investigated. X-ray absorption spectroscopy shows that reaction with oxygen results in destruction of the [4Fe-4S] domain of the active site H-cluster while leaving the di-iron domain $(2Fe_H )$essentially intact. By protein film electrochemistry we were able to determine the order of events leading up to this destruction. Carbon monoxide, a competitive inhibitor of CrHydAI which binds to an Fe atom of the $(2Fe_yH )$domain and is otherwise not known to attack FeS clusters in proteins, reacts nearly two orders of magnitude faster than oxygen and protects the enzyme against oxygen damage. These results therefore show that destruction of the [4Fe-4S] cluster is initiated by binding and reduction of oxygen at the di-iron domain—a key step that is blocked by carbon monoxide. The relatively slow attack by oxygen compared to carbon monoxide suggests that a very high level of discrimination can be achieved by subtle factors such as electronic effects (specific orbital overlap requirements) and steric constraints at the active site.

Ni-Fe clusters are inserted into the large subunit of [NiFe] hydrogenases by maturation proteins such as the Ni chaperone HypA via an unknown mechanism. We determined crystal structures of an immature large subunit HyhL complexed with HypA from Thermococcus kodakarensis. Structure analysis revealed that the N-terminal region of HyhL extends outwards and interacts with the Ni-binding domain of HypA. Intriguingly, the C-terminal extension of immature HyhL, which is cleaved in the mature form, adopts a β-strand adjacent to its N-terminal β-strands. The position of the C-terminal extension corresponds to that of the N-terminal extension of a mature large subunit, preventing the access of endopeptidases to the cleavage site of HyhL. These findings suggest that Ni insertion into the active site induces spatial rearrangement of both the N- and C-terminal tails of HyhL, which function as a key checkpoint for the completion of the Ni-Fe cluster assembly.

Hydrogenases (H 2ases) catalyze the reversible oxidation of molecular hydrogen and play a central role in microbial energy metabolism. Most of these enzymes are found in Archaea and Bacteria, but a few are present in Eucarya as well. They can be distributed into three classes: the [Fe]-H 2ases, the [NiFe]-H 2ases, and the metal-free H 2ases. The vast majority of known H 2ases belong to the first two classes, and over 100 of these enzymes have been characterized genetically and/or biochemically. Compelling evidence from sequences and structures indicates that the [NiFe]- and [Fe]-H 2ases are phylogenetically distinct classes of proteins. The catalytic core of the [NiFe]-H 2ases is a heterodimeric protein, although additional subunits are present in many of these enzymes. Functional classes of [NiFe]-H 2ases have been defined, and they are consistent with categories defined by sequence similarity of the catalytic subunits. The catalytic core of the [Fe]-H 2ases is a ca. 350-residue domain that accommodates the active site (H-cluster). A few monomeric [Fe]-H 2ases are barely larger than the H-cluster domain. Many others are monomeric as well, but possess additional domains that contain redox centers, mostly iron–sulfur. Some [Fe]-H 2ases are oligomeric. The modular structure of H 2ases is strikingly illustrated in recently unveiled sequences and structures. It is also remarkable that most of the accessory domains and subunits of H 2ases have counterparts in other redox complexes, in particular NADH-ubiquinone oxidoreductase (Complex I) of respiratory chains. Microbial genome sequences are bringing forth a significant body of additional H 2ase sequence data and contribute to the understanding of H 2ase distribution and evolution. Altogether, the available data suggest that [Fe]-H 2ases are restricted to Bacteria and Eucarya, while [NiFe]-H 2ases, with one possible exception, seem to be present only in Archaea and Bacteria. H 2ase processing and maturation involve the products of several genes which have been identified and are currently being characterized in the case of the [NiFe]-H 2ases. In contrast, near to nothing is known regarding the maturation of the [Fe]-H 2ases. Inspection of the currently available genome sequences suggests that the [NiFe]-H 2ase maturation proteins have no similar counterparts in the genomes of organisms possessing [Fe]-H 2ases only. This observation, if confirmed, would be consistent with the phylogenetic distinctiveness of the two classes of H 2ases. Sequence alignments of catalytic subunits of H 2ases have been implemented to construct phylogenetic trees that were found to be consistent, in the main, with trees derived from other data. On the basis of the comparisons performed and discussed here, proposals are made to simplify and rationalize the nomenclature of H 2ase-encoding genes.

Significance The isolation of an active formate hydrogenlyase is a breakthrough in understanding the molecular basis of bacterial hydrogen production. For over 100 years, Escherichia coli has been known to evolve H ₂ when cultured under fermentative conditions. Glucose is metabolized to formate, which is then oxidized to CO ₂ with the concomitant reduction of protons to H ₂ by a single complex called formate hydrogenlyase, which had been genetically, but never biochemically, characterized. In this study, innovative molecular biology and electrochemical experiments reveal a hydrogenase enzyme with the unique ability to sustain H ₂ production even under high partial pressures of H ₂. Harnessing bacterial H ₂ production offers the prospect of a source of fully renewable H ₂ energy, freed from any dependence on fossil fuel. Under anaerobic conditions, Escherichia coli can carry out a mixed-acid fermentation that ultimately produces molecular hydrogen. The enzyme directly responsible for hydrogen production is the membrane-bound formate hydrogenlyase (FHL) complex, which links formate oxidation to proton reduction and has evolutionary links to Complex I, the NADH:quinone oxidoreductase. Although the genetics, maturation, and some biochemistry of FHL are understood, the protein complex has never been isolated in an intact form to allow biochemical analysis. In this work, genetic tools are reported that allow the facile isolation of FHL in a single chromatographic step. The core complex is shown to comprise HycE (a [NiFe] hydrogenase component termed Hyd-3), FdhF (the molybdenum-dependent formate dehydrogenase-H), and three iron-sulfur proteins: HycB, HycF, and HycG. A proportion of this core complex remains associated with HycC and HycD, which are polytopic integral membrane proteins believed to anchor the core complex to the cytoplasmic side of the membrane. As isolated, the FHL complex retains formate hydrogenlyase activity in vitro. Protein film electrochemistry experiments on Hyd-3 demonstrate that it has a unique ability among [NiFe] hydrogenases to catalyze production of H ₂ even at high partial pressures of H ₂. Understanding and harnessing the activity of the FHL complex is critical to advancing future biohydrogen research efforts.

Uptake hydrogenase (Hup) recycles H2 formed by nitrogenase during nitrogen fixation, thereby preserving energy. Among root nodule bacteria, most rhizobial strains examined are Hup−, while only one Hup− Frankia inoculum had been identified. Previous analyses had led to the identification of two different [NiFe] hydrogenase syntons. We analysed the distribution of different types of [NiFe] hydrogenase in the genomes of different Frankia species. Our results show that Frankia strains can contain four different [NiFe] hydrogenase syntons representing groups 1f, 1h, 2a and 3b according to Søndergaard et al. (2016); no more than three types were found in any individual genome. The phylogeny of the structural proteins of groups 1f, 1h and 2a follows Frankia phylogeny; the phylogeny of the accessory proteins does not consistently. An analysis of different [NiFe] hydrogenase types in Actinomycetia shows that under the most parsimonious assumption, all four types were present in the ancestral Frankia strain. Based on Hup activities analysed and the losses of syntons in different lineages of genome reduction, we can conclude that groups 1f and 2a are involved in recycling H2 formed by nitrogenase while group 1h and group 3b are not.

Abstract Cyanobacteria may possess two distinct nickel-iron (NiFe)-hydrogenases: an uptake enzyme found in N 2 -fixing strains, and a bidirectional one present in both non-N 2 -fixing and N 2 -fixing strains. The uptake hydrogenase (encoded by hupSL ) catalyzes the consumption of the H 2 produced during N 2 fixation, while the bidirectional enzyme ( hoxEFUYH ) probably plays a role in fermentation and/or acts as an electron valve during photosynthesis. hupSL constitute a transcriptional unit, and are essentially transcribed under N 2 -fixing conditions. The bidirectional hydrogenase consists of a hydrogenase and a diaphorase part, and the corresponding five hox genes are not always clustered or cotranscribed. The biosynthesis/maturation of NiFe-hydrogenases is highly complex, requiring several core proteins. In cyanobacteria, the genes that are thought to affect hydrogenases pleiotropically ( hyp ), as well as the genes presumably encoding the hydrogenase-specific endopeptidases ( hupW and hoxW ) have been identified and characterized. Furthermore, NtcA and LexA have been implicated in the transcriptional regulation of the uptake and the bidirectional enzyme respectively. Recently, the phylogenetic origin of cyanobacterial and algal hydrogenases was analyzed, and it was proposed that the current distribution in cyanobacteria reflects a differential loss of genes according to their ecological needs or constraints. In addition, the possibilities and challenges of cyanobacterial-based H 2 production are addressed.

Farmed ruminants are the largest source of anthropogenic methane emissions globally. The methanogenic archaea responsible for these emissions use molecular hydrogen (H 2 ), produced during bacterial and eukaryotic carbohydrate fermentation, as their primary energy source. In this work, we used comparative genomic, metatranscriptomic and co-culture-based approaches to gain a system-wide understanding of the organisms and pathways responsible for ruminal H 2 metabolism. Two-thirds of sequenced rumen bacterial and archaeal genomes encode enzymes that catalyse H 2 production or consumption, including 26 distinct hydrogenase subgroups. Metatranscriptomic analysis confirmed that these hydrogenases are differentially expressed in sheep rumen. Electron-bifurcating [FeFe]-hydrogenases from carbohydrate-fermenting Clostridia (e.g., Ruminococcus ) accounted for half of all hydrogenase transcripts. Various H 2 uptake pathways were also expressed, including methanogenesis ( Methanobrevibacter ), fumarate and nitrite reduction ( Selenomonas ), and acetogenesis ( Blautia ). Whereas methanogenesis-related transcripts predominated in high methane yield sheep, alternative uptake pathways were significantly upregulated in low methane yield sheep. Complementing these findings, we observed significant differential expression and activity of the hydrogenases of the hydrogenogenic cellulose fermenter Ruminococcus albus and the hydrogenotrophic fumarate reducer Wolinella succinogenes in co-culture compared with pure culture. We conclude that H 2 metabolism is a more complex and widespread trait among rumen microorganisms than previously recognised. There is evidence that alternative hydrogenotrophs, including acetogenic and respiratory bacteria, can prosper in the rumen and effectively compete with methanogens for H 2 . These findings may help to inform ongoing strategies to mitigate methane emissions by increasing flux through alternative H 2 uptake pathways, including through animal selection, dietary supplementation and methanogenesis inhibitors.

The anaerobic, hyperthermophlic, cellulolytic bacterium Caldicellulosiruptor bescii grows optimally at ∼80 °C and effectively degrades plant biomass without conventional pretreatment. It utilizes a variety of carbohydrate carbon sources, including both C5 and C6 sugars, released from plant biomass and produces lactate, acetate, CO₂, and H₂ as primary fermentation products. The C. bescii genome encodes two hydrogenases, a bifurcating [Fe-Fe] hydrogenase and a [Ni-Fe] hydrogenase. The [Ni-Fe] hydrogenase is the most widely distributed in nature and is predicted to catalyze hydrogen production and to pump protons across the cellular membrane creating proton motive force. Hydrogenases are the key enzymes in hydrogen metabolism and their crystal structure reveals complexity in the organization of their prosthetic groups suggesting extensive maturation of the primary protein. Here, we report the deletion of a cluster of genes, hypABFCDE, required for maturation of the [Ni-Fe] hydrogenase. These proteins are specific for the hydrogenases they modify and are required for hydrogenase activity. The deletion strain grew more slowly than the wild type or the parent strain and produced slightly less hydrogen overall, but more hydrogen per mole of cellobiose. Acetate yield per mole of cellobiose was increased ∼67 % and ethanol yield per mole of cellobiose was decreased ∼39 %. These data suggest that the primary role of the [Ni-Fe] hydrogenase is to generate a proton gradient in the membrane driving ATP synthesis and is not the primary enzyme for hydrogen catalysis. In its absence, ATP is generated from increased acetate production resulting in more hydrogen produced per mole of cellobiose.