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

Abstract

[NiFe] hydrogenases are metalloenzymes that catalyze the reversible oxidation of H₂. 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 E₁₇ 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 H₂, including absorption features consistent with the Ni_a-C intermediate. Time-resolved IR spectroscopy, which probes active site dynamics after hydride photolysis from Ni_a-C, indicates the E₁₇Q mutation does not interfere with the hydride photolysis process generating known intermediates Ni_a-I¹ and Ni_a-I². Strikingly, the E₁₇Q mutation disrupts obligatory proton-coupled electron transfer from the Ni_a-I¹ state, thereby preventing formation of Ni_a-S. These results directly establish E₁₇ as a proton donor/acceptor in the Ni_a-S ↔ Ni_a-C equilibrium.