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

Jacqueline Kalms (1) , Andrea Schmidt (1) , Stefan Frielingsdorf (2) , Tillmann Utesch (2) , Guillaume Gotthard (3) , David von Stetten (3) , Peter van Der Linden (3) , Antoine Royant (4, 3) , Maria Andrea Mroginski (2) , Philippe Carpentier (5, 3) , Oliver Lenz (2) , Patrick Scheerer (6)

1 BIH - Berlin Institute of Health
2 Institut für Chemie und Biochemie [Berlin]
3 ESRF - European Synchrotron Radiation Facility
4 IBS - UMR 5075 - Institut de biologie structurale
5 LCBM - UMR 5249 - Laboratoire de Chimie et Biologie des Métaux
6 Charite, AG Prot Xray Crystallog, Inst Med Phys & Biophys CC2, D-10117 Berlin, Germany

[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 O2 tolerance relies on a special [4Fe3S] cluster that is capable of releasing two electrons upon O-2 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 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.

Mots clés: oxygen-tolerant [NiFe] hydrogenase; X-ray crystallography; crystal derivatization; iron–sulfur cluster; metalloproteins; ACTIVE-SITE; STRUCTURE VALIDATION; CRYSTAL-STRUCTURE; AMMONIA CHANNEL; GAS-DIFFUSION; EUTROPHA H16; CCP4 SUITE; ACCESS; PROTEINS; O-2

Domaines: Sciences du Vivant [q-bio]

Jacqueline Kalms, Andrea Schmidt, Stefan Frielingsdorf, Tillmann Utesch, Guillaume Gotthard, et al.. Tracking the route of molecular oxygen in O-2-tolerant membrane-bound [NiFe] hydrogenase. Proceedings of the National Academy of Sciences of the United States of America, 2018, 115 (10), pp.E2229-E2237. ⟨10.1073/pnas.1712267115⟩. ⟨hal-01726163⟩