Glutamate Gated Proton-Coupled Electron Transfer Activity of a [NiFe]-Hydrogenase.

[NiFe] hydrogenases are metalloenzymes that catalyze the reversible oxidation of H. While electron transfer to and from the active site is understood to occur through iron-sulfur clusters, the mechanism of proton transfer is still debated. Two mechanisms for proton exchange with the active site have been proposed that involve distinct and conserved ionizable amino acid residues, one a glutamate, and the other an arginine. To examine the potential role of the conserved glutamate on active site acid-base chemistry, we mutated the putative proton donor E to Q in the soluble hydrogenase I from Pyrococcus furiosus using site directed mutagenesis. FTIR spectroscopy, sensitive to the CO and CN ligands of the active site, reveals catalytically active species generated upon reduction with H, including absorption features consistent with the Ni-C intermediate. Time-resolved IR spectroscopy, which probes active site dynamics after hydride photolysis from Ni-C, indicates the EQ mutation does not interfere with the hydride photolysis process generating known intermediates Ni-I and Ni-I. Strikingly, the EQ mutation disrupts obligatory proton-coupled electron transfer from the Ni-I state, thereby preventing formation of Ni-S. These results directly establish E as a proton donor/acceptor in the Ni-S ↔ Ni-C equilibrium.