Tracking the route of molecular oxygen in O₂-tolerant membrane-bound [NiFe] hydrogenase

[NiFe] hydrogenases catalyze the reversible splitting of H₂ into protons and electrons at a deeply buried active site. The catalytic center can be accessed by gas molecules through a hydrophobic tunnel network. While most [NiFe] hydrogenases are inactivated by O₂, a small subgroup, including the membrane-bound [NiFe] hydrogenase (MBH) of Ralstonia eutropha, is able to overcome aerobic inactivation by catalytic reduction of O₂ to water. This O₂ tolerance relies on a special [4Fe3S] cluster that is capable of releasing two electrons upon O₂ attack. Here, the O₂ accessibility of the MBH gas tunnel network has been probed experimentally using a "soak-and-freeze" derivatization method, accompanied by protein X-ray crystallography and computational studies. This combined approach revealed several sites of O₂ molecules within a hydrophobic tunnel network leading, via two tunnel entrances, to the catalytic center of MBH. The corresponding site occupancies were related to the O₂ concentrations used for MBH crystal derivatization. The examination of the O₂-derivatized data furthermore uncovered two unexpected structural alterations at the [4Fe3S] cluster, which might be related to the O₂ tolerance of the enzyme.