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

Abstract

A [ NiFe] hydrogenase (H 2 ase) is ap roton-coupled electron transfer enzyme that catalyses reversible H 2 oxidation; however,i ts fundamental proton transfer pathway remains unknown. Herein, we observed the protonation of Cys546-SH and Glu34-COOH near the Ni-Fes ite with high-sensitivity infrared difference spectra by utilizing Ni-C-to-Ni-L and Ni-Cto-Ni-SI a photoconversions.P rotonated Cys546-SH in the Ni-Ls tate was verified by the observed SH stretching frequency (2505 cm À1 ), whereas Cys546 was deprotonated in the Ni-C and Ni-SI a states.G lu34-COOH was double H-bonded in the Ni-L state,a sd etermined by the COOH stretching frequency (1700 cm À1 ), and single H-bonded in the Ni-C and Ni-SI a states.A dditionally,astretching mode of an ordered water molecule was observed in the Ni-L and Ni-C states.T hese results elucidate the organized proton transfer pathway during the catalytic reaction of a[ NiFe] H 2 ase,w hich is regulated by the H-bond network of Cys546, Glu34, and an ordered water molecule.Supportinginformation and the ORCID identification number(s) for the author(s) of this article can be found under: https://doi.

Selective cysteine-to-selenocysteine changes in a [NiFe]-hydrogenase confirm a special position for catalysis and oxygen tolerance

“…A wealth of evidence currently supports the role of E28 in transferring at least one catalytic proton in [NiFe]hydrogenases (SI Appendix, Fig. S3), the immediate relay partner being the Cys-S located here at position 576 (19,50). Our results now show that these two residues operate in close harmony during catalysis, disruptions to either residue producing the same effect on the catalytic potential profile (see SI Appendix, Fig.…”

Metal–ligand cooperativity in the soluble hydrogenase-1 fromPyrococcus furiosus

“…3 -C hydride under cryogenic conditions to trap the Ni a + -L photoproducts. 23,36,63 However, the Ni a + -L tautomer has been readily observed under ambient conditions in some [NiFe]-hydrogenases, 20,64 and the pK a and bond strength of the Ni a 3+ -C hydride has been hypothesized to vary through subtle electronic and/or structural changes to partially explain such observations. 18,65 A shi of tautomeric equilibrium to favour base protonation over metal-hydride formation has been demonstrated in a cationic [FeFe] mimic and iron-diphosphine complex via ion-pairing of the protonated base with anionic species such as BF 4 À .…”

“…1b. This assumes Pf SH1 exhibits the same type of terminal cysteine protonation observed in Dv MF 23 ). However, the data do not appear to be a simple monoprotic acid-base equilibrium.…”

“…1b. [19][20][21][22][23][24][25][26] Various aspects of the primary and secondary coordination sphere ne tune the electronic structure 25,[27][28][29][30][31] and further aid catalysis by stabilizing redox/protonation states necessary for turnover 32 and facilitating electrostatic and dynamic processes for efficient substrate processing. 10,17,[33][34][35] Directly relevant to protons and hydrides, the primary sphere stores one proton and the two electrons of H 2 as a bridging hydride in the Ni a 3+ -C and Ni a 2+ -SR states.…”

“…1b), and cryogenic FTIR spectroscopy with the Desulfovibrio vulgaris Miyazaki (Dv MF) enzyme demonstrated C418 ‡ is protonated in the Ni a + -L2 state. 23 An electron transfer from Ni a + -L1 to an iron-sulphur cluster and a proton transfer to the secondary sphere forms Ni a 2+ -S. 16,19,21 The closest ionizable residue to C418 is the highly conserved glutamic acid E17 (Fig. 1a 1b.…”