Tracking the route of molecular oxygen in O(2)-tolerant membrane-bound [NiFe] hydrogenase

[NiFe] hydrogenases catalyze the reversible splitting of H(2) 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 O(2), a small subgroup, including the membrane-bound [NiFe] hydrogenase (MBH) of Ralstonia eutropha, is able to overcome aerobic inactivation by catalytic reduction of O(2) to water. This O(2) tolerance relies on a special [4Fe3S] cluster that is capable of releasing two electrons upon O(2) attack. Here, the O(2) 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 O(2) 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 O(2) concentrations used for MBH crystal derivatization. The examination of the O(2)-derivatized data furthermore uncovered two unexpected structural alterations at the [4Fe3S] cluster, which might be related to the O(2) tolerance of the enzyme.