Microbial hydrogen (H₂) 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 H₂-producing Asgard archaeon cultures. We report an ultraminimal hydrogenase in DPANN archaea that binds the catalytic H-cluster and produces H₂. Moreover, we identify and characterize remarkable hybrid complexes formed through the fusion of [FeFe] and [NiFe] hydrogenases in ten other archaeal orders. Phylogenetic analysis and structural modeling suggest a deep evolutionary history of hybrid hydrogenases. These findings reveal new metabolic adaptations of archaea, streamlined H₂ catalysts for biotechnological development, and a surprisingly intertwined evolutionary history between the two major H₂-metabolizing enzymes.

微生物氢 (H ₂ ) 循环支撑着不同缺氧生态系统的多样性和功能。在三个进化上不同的氢化酶超家族中，[FeFe]氢化酶被认为仅限于细菌和真核生物。在这里，我们通过对现有和新基因组的分析与广泛的生化实验相结合，证明厌氧古菌编码多样化、活跃且古老的[FeFe]氢化酶谱系。 [FeFe]氢化酶由九个古菌门的基因组编码，并由产生 H ₂ 的 Asgard 古菌培养物表达。我们报道了 DPANN 古细菌中的一种超最小氢化酶，它结合催化 H 簇并产生 H ₂ 。此外，我们还鉴定并表征了其他 10 种古菌目中通过 [FeFe] 和 [NiFe] 氢化酶融合而形成的显着杂合复合物。系统发育分析和结构建模表明杂合氢化酶具有深厚的进化历史。这些发现揭示了古细菌新的代谢适应、生物技术发展的简化的 H ₂ 催化剂，以及两种主要的 H ₂ 代谢酶之间令人惊讶地交织在一起的进化历史。