Atmospheric hydrogen (H₂) supports the growth and persistence of diverse aerobic microorganisms through specialised [NiFe]-hydrogenases, metalloenzymes that catalyse the reversible oxidation of trace H₂ in the presence of ambient oxygen (O₂). These enzymes are key biological sinks in the global hydrogen cycle, influence the composition of our atmosphere, and provide valuable insights for the development of robust and sustainable H₂ catalysts. However, the molecular mechanisms that enable efficient H₂ capture and O₂ tolerance remain poorly understood, as only a few O₂-adapted H₂-uptake hydrogenases have been biochemically and structurally characterized.
Here, we expand the structural and functional repertoire of O₂-adapted [NiFe]-hydrogenases by investigating two systems: the high-affinity group 1h hydrogenase from Mycobacterium smegmatis (MsHhy) and the uncharacterized Sulfolobales clade 2 (Sul2) hydrogenase from the thermoacidophilic archaeon Metallosphaera sedula.
MsHhy was purified by affinity chromatography and its cryo-EM structure was determined at a resolution of 2.1 Å. The enzyme forms a dimer of heterodimers and shows high structural similarity and conserved cofactor coordination with the actinobacterial-type hydrogenase from Cupriavidus necator. This includes a proximal [4Fe–4S] cluster coordinated by three cysteines and one aspartate - a motif that may enhance H₂ affinity and contribute to O₂ resistance. Modelling indicates that MsHhy is anchored to the cell membrane via two peptides to couple electron transfer to the aerobic respiratory chain, supporting long-term persistence and survival of M. smegmatis.
The Sul2 enzyme is a membrane-spanning complex consisting of an extracellular [NiFe]-hydrogenase, an integral cytochrome-containing protein, and a cytoplasmic iron–sulfur protein. It is hypothesised that this complex forms an electron-transfer relay that oxidises H₂ and channels electrons to membrane quinones and low-potential acceptors, thereby supporting respiration and biosynthesis. Sul2 is the most abundant hydrogenase in M. sedula under aerobic autotrophic growth and was natively purified. The enzyme remains catalytically active in the presence of oxygen and after long-term storage at -80°C, establishing Sul2 as a promising thermostable catalyst.
This work provides new insights into H₂-uptake hydrogenases that are resistant to O₂ inhibition, helping to identify candidates with superior catalytic performance for clean hydrogen-based processes.