A strong H-bond between a cysteine and the catalytic center of a [NiFe]-hydrogenase

Infrared spectroscopy at cryogenic temperatures was used to monitor protonation changes on an H+-accepting, nickel-coordinating active site cysteine of the H2/H+-cycling membrane-bound [NiFe]-hydrogenase from Cupriavidus necator. Surprisingly, we identified another cysteine in the outer coordination sphere forming a strong H-bond with a cysteine thiolate coordinating both nickel and iron of the catalytic center.