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 (pO 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.

H2 metabolism is proposed to be the most ancient and diverse mechanism of energy-conservation. The metalloenzymes mediating this metabolism, hydrogenases, are encoded by over 60 microbial phyla and are present in all major ecosystems. We developed a classification system and web tool, HydDB, for the structural and functional analysis of these enzymes. We show that hydrogenase function can be predicted by primary sequence alone using an expanded classification scheme (comprising 29 [NiFe], 8 [FeFe], and 1 [Fe] hydrogenase classes) that defines 11 new classes with distinct biological functions. Using this scheme, we built a web tool that rapidly and reliably classifies hydrogenase primary sequences using a combination of k-nearest neighbors’ algorithms and CDD referencing. Demonstrating its capacity, the tool reliably predicted hydrogenase content and function in 12 newly-sequenced bacteria, archaea, and eukaryotes. HydDB provides the capacity to browse the amino acid sequences of 3248 annotated hydrogenase catalytic subunits and also contains a detailed repository of physiological, biochemical, and structural information about the 38 hydrogenase classes defined here. The database and classifier are freely and publicly available at http://services.birc.au.dk/hyddb/