Structural and spectroscopic characterization of CO inhibition of [NiFe]-hydrogenase from Citrobacter sp. S-77.

Hydrogenases catalyze the reversible oxidation of H 2 . Carbon monoxide (CO) is known to be a competitive inhibitor of O 2 -sensitive [NiFe]-hydrogenases. Although the activities of some O 2 -tolerant [NiFe]-hydrogenases are unaffected by CO, the partially O 2 -tolerant [NiFe]-hydrogenase from Citrobacter sp. S-77 (S77-HYB) is inhibited by CO. In this work, the CO-bound state of S77-HYB was characterized by activity assays, spectroscopic techniques and X-ray crystallography. Electron paramagnetic resonance spectroscopy showed a diamagnetic Ni 2+ state, and Fourier-transform infrared spectroscopy revealed the stretching vibration of the exogenous CO ligand. The crystal structure determined at 1.77 Å resolution revealed that CO binds weakly to the nickel ion in the Ni-Fe active site of S77-HYB. These results suggest a positive correlation between O 2 and CO tolerance in [NiFe]-hydrogenases.