Abstract
Diverse aerobic bacteria use atmospheric H2 as an energy source for growth and survival1. This globally significant process regulates the composition of the atmosphere, enhances soil biodiversity and drives primary production in extreme environments2,3. Atmospheric H2 oxidation is attributed to uncharacterized members of the [NiFe] hydrogenase superfamily4,5. However, it remains unresolved how these enzymes overcome the extraordinary catalytic challenge of oxidizing picomolar levels of H2 amid ambient levels of the catalytic poison O2 and how the derived electrons are transferred to the respiratory chain1. 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 H2 to the hydrogenation of the respiratory electron carrier menaquinone. Huc uses narrow hydrophobic gas channels to selectively bind atmospheric H2 at the expense of O2, and 3 [3Fe-4S] clusters modulate the properties of the enzyme so that atmospheric H2 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 H2 oxidation, uncover a mode of energy coupling dependent on long-range quinone transport, and pave the way for the development of catalysts that oxidize H2 in ambient air.
![<div class='caption-title'>a, A stereo view of the Huc [NiFe] cluster under ambient air. b, The structure of the three HucS [3Fe–4S] clusters. c, High-temperature (30K) EPR spectra of Huc in the as-isolated oxidized state (Huc-Air) and the H2-reduced state (Huc-H2). The isotropic signal at g = 2.03 (g is the Landé factor) is associated with the oxidized [3Fe–4S]+ clusters, which disappears in Huc-H2 owing to reduction of the clusters. No signal attributable to reduced [4Fe–4S]+ clusters is discernible in the H2-treated form of Huc. d, FTIR spectra of oxidized Huc isolated in ambient air, showing that the [NiFe] active site of the enzyme adopts an oxidized state consistent with an Ni-B state. e, The Huc [NiFe] active site, showing the relative distances between the Ni-B state hydroxyl ligand and the Ni and Fe ions of the cluster. f, The HucSL dimer, showing the location and width of the hydrophobic gas channels that provide substrate access to the [NiFe] active site. g, Left, plot of distance versus time, showing the closest distance between H2 and the Huc [NiFe] active site during molecular dynamics simulations of HucSL in the presence of H2. Right, the location of H2 molecules in a representative subset of simulation frames when H2 molecules are in the closest proximity to the active site. h, Similar to g, but shows location of O2 relative to the Huc [NiFe] site in simulations of HucSL performed in the presence of O2. i, The proximity of HucL d-His166 to the proximal and medial [3Fe–4S] clusters and the location of the unknown ligand. Cryo-EM density potential maps in a,b,i are shown as a transparent blue surface at 5σ.</div>](https://europepmc.org/articles/PMC10017518/bin/41586_2023_5781_Fig3_HTML.jpg "<div class='caption-title'>a, A stereo view of the Huc [NiFe] cluster under ambient air. b, The structure of the three HucS [3Fe–4S] clusters. c, High-temperature (30K) EPR spectra of Huc in the as-isolated oxidized state (Huc-Air) and the H2-reduced state (Huc-H2). The isotropic signal at g = 2.03 (g is the Landé factor) is associated with the oxidized [3Fe–4S]+ clusters, which disappears in Huc-H2 owing to reduction of the clusters. No signal attributable to reduced [4Fe–4S]+ clusters is discernible in the H2-treated form of Huc. d, FTIR spectra of oxidized Huc isolated in ambient air, showing that the [NiFe] active site of the enzyme adopts an oxidized state consistent with an Ni-B state. e, The Huc [NiFe] active site, showing the relative distances between the Ni-B state hydroxyl ligand and the Ni and Fe ions of the cluster. f, The HucSL dimer, showing the location and width of the hydrophobic gas channels that provide substrate access to the [NiFe] active site. g, Left, plot of distance versus time, showing the closest distance between H2 and the Huc [NiFe] active site during molecular dynamics simulations of HucSL in the presence of H2. Right, the location of H2 molecules in a representative subset of simulation frames when H2 molecules are in the closest proximity to the active site. h, Similar to g, but shows location of O2 relative to the Huc [NiFe] site in simulations of HucSL performed in the presence of O2. i, The proximity of HucL d-His166 to the proximal and medial [3Fe–4S] clusters and the location of the unknown ligand. Cryo-EM density potential maps in a,b,i are shown as a transparent blue surface at 5σ.</div>")
PMID: 