Cysteine SH and Glutamate COOH Contributions to [NiFe] Hydrogenase Proton Transfer Revealed by Highly Sensitive FTIR Spectroscopy

Abstract

A [NiFe] hydrogenase (H₂ ase) is a proton-coupled electron transfer enzyme that catalyses reversible H₂ oxidation; however, its fundamental proton transfer pathway remains unknown. Herein, we observed the protonation of Cys546-SH and Glu34-COOH near the Ni-Fe site with high-sensitivity infrared difference spectra by utilizing Ni-C-to-Ni-L and Ni-C-to-Ni-SI_a photoconversions. Protonated Cys546-SH in the Ni-L state was verified by the observed SH stretching frequency (2505 cm^-1 ), whereas Cys546 was deprotonated in the Ni-C and Ni-SI_a states. Glu34-COOH was double H-bonded in the Ni-L state, as determined by the COOH stretching frequency (1700 cm^-1 ), and single H-bonded in the Ni-C and Ni-SI_a states. Additionally, a stretching mode of an ordered water molecule was observed in the Ni-L and Ni-C states. These results elucidate the organized proton transfer pathway during the catalytic reaction of a [NiFe] H₂ ase, which is regulated by the H-bond network of Cys546, Glu34, and an ordered water molecule.