biostudies-literatureGreening CEuropean Research Council3357-3372.e19https://www.ebi.ac.uk/biostudies/studies/S-EPMC11216029biostudies-literatureUnknown187(13)Microbial hydrogen (H<sub>2</sub>) cycling underpins the diversity and functionality of diverse anoxic ecosystems. Among the three evolutionarily distinct hydrogenase superfamilies responsible, [FeFe] hydrogenases were thought to be restricted to bacteria and eukaryotes. Here, we show that anaerobic archaea encode diverse, active, and ancient lineages of [FeFe] hydrogenases through combining analysis of existing and new genomes with extensive biochemical experiments. [FeFe] hydrogenases are encoded by genomes of nine archaeal phyla and expressed by H<sub>2</sub>-producing Asgard archaeon cultures. We report an ultraminimal hydrogenase in DPANN archaea that binds the catalytic H-cluster and produces H<sub>2</sub>. Moreover, we identify and characterize remarkable hybrid complexes formed thrCellMinimal and hybrid hydrogenases are active from archaea.PMC11216029803768947317101078476695192Banfield JFValentin Alvarado LESenger MMullen SLand HSchoelmerich MSong JCabotaje PRMao JSpang AWest-Roberts JGrinter RGreening CStairs CWKlamke MAPonce-Toledo RILappan RRodrigues-Oliveira TMilton MSchleper CLeung PMBerggren GfalseMinimal and hybrid hydrogenases are active from archaea.Microbial hydrogen (H<sub>2</sub>) cycling underpins the diversity and functionality of diverse anoxic ecosystems. Among the three evolutionarily distinct hydrogenase superfamilies responsible, [FeFe] hydrogenases were thought to be restricted to bacteria and eukaryotes. Here, we show that anaerobic archaea encode diverse, active, and ancient lineages of [FeFe] hydrogenases through combining analysis of existing and new genomes with extensive biochemical experiments. [FeFe] hydrogenases are encoded by genomes of nine archaeal phyla and expressed by H<sub>2</sub>-producing Asgard archaeon cultures. We report an ultraminimal hydrogenase in DPANN archaea that binds the catalytic H-cluster and produces H<sub>2</sub>. Moreover, we identify and characterize remarkable hybrid complexes formed thr2024-01-01T00:00:00Z2024 Jun2026-05-21T03:17:47.098Z2025-04-04T12:20:35.154ZS-EPMC112160293886601810.1016/j.cell.2024.05.032