Glutamate Gated Proton-Coupled Electron Transfer Activity of a [NiFe]-Hydrogenase.
[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 E17to 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 Nia-C intermediate. Time-resolved IR spectroscopy, which probes active site dynamics after hydride photolysis from Nia-C, indicates the E17Q mutation does not interfere with the hydride photolysis process generating known intermediates Nia-I1and Nia-I2. Strikingly, the E17Q mutation disrupts obligatory proton-coupled electron transfer from the Nia-I1state, thereby preventing formation of Nia-S. These results directly establish E17as a proton donor/acceptor in the Nia-S ↔ Nia-C equilibrium.
Chemistry Department, Emory University , Atlanta, Georgia 30322, United States.
Brandon L
BL
Greene
Chemistry Department, Emory University , Atlanta, Georgia 30322, United States.
Gregory E
GE
Vansuch
Department of Biochemistry, University of Georgia , Athens, Georgia 30602, United States.
Chang-Hao
CH
Wu
Department of Biochemistry, University of Georgia , Athens, Georgia 30602, United States.
Michael W W
MW
Adams
Chemistry Department, Emory University , Atlanta, Georgia 30322, United States.
R Brian
RB
Dyer
D020134:Catalytic Domain; D004579:Electron Transport; D018698:Glutamic Acid / Q000378:metabolism*; D006864:Hydrogenase / Q000737:chemistry / Q000378:metabolism*; D008958:Models, Molecular; D011522:Protons; D020027:Pyrococcus furiosus / Q000201:enzymology
D011522:Protons; D018698:Glutamic Acid; C043296:nickel-iron hydrogenase; D006864:Hydrogenase