Significance Tracking the route of substrates, intermediates, and inhibitors in proteins is fundamental in understanding their specific function. However, following the route of gases like molecular oxygen within enzymes has always been challenging. In protein X-ray crystallography, gases can be mimicked using krypton or xenon (with a higher electron count); however, these have a different physical behavior compared to true substrates/inhibitors. In our crystal structure of the O 2 -tolerant membrane-bound [NiFe] hydrogenase (MBH) from Ralstonia eutropha , we were able to show the direct path of molecular oxygen between the enzyme exterior and the active site with the “soak-and-freeze” derivatization method. This technique might be useful to detect O 2 traveling routes in many other enzymes.